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The identification of transcription factor (TF) target genes is central in biology. A popular approach is based on the location by pattern matching of potential cis-regulatory elements (CREs). During the last few years, tools integrating next-generation sequencing data have been developed to improve the performance of pattern matching. However, such tools have not yet been comprehensively evaluated in plants. Hence, we developed a new streamlined method aiming at predicting CREs and target genes of plant TFs in specific organs or conditions. Our approach implements a supervised machine learning strategy, which allows decision rule models to be learnt using TF ChIP-chip/seq experimental data. Different layers of genomic features were integrated in predictive models: the position on the gene, the DNA sequence conservation, the chromatin state and various CRE footprints. Among the tested features, the chromatin features were crucial for improving the accuracy of the method. Furthermore, we evaluated the transferability of predictive models across TFs, organs and species. Finally, we validated our method by correctly inferring the target genes of key TFs controlling metabolite biosynthesis at the organ level in Arabidopsis. We developed a tool—Wimtrap—to reproduce our approach in plant species and conditions/organs for which ChIP-chip/seq data are available. Wimtrap is a user-friendly R package that supports an R Shiny web interface and is provided with pre-built models that can be used to quickly get predictions of CREs and TF gene targets in different organs or conditions in Arabidopsis thaliana, Solanum lycopersicum, Oryza sativa and Zea mays.

The circadian clock coordinates the physiological responses of a biological system to day and night rhythms through complex loops of transcriptional/translational regulation. It can respond to external stimuli and adjust generated circadian oscillations accordingly to maintain an endogenous period close to 24 h. However, the interaction between nutritional status and circadian rhythms in plants is poorly understood. Magnesium (Mg) is essential for numerous biological processes in plants, and its homeostasis is crucial to maintain optimal development and growth. Magnesium deficiency in young Arabidopsis thaliana seedlings increased the period of circadian oscillations of the CIRCADIAN CLOCK-ASSOCIATED 1 (CCA1) promoter (pCCA1:LUC) activity and dampened their amplitude under constant light in a dose-dependent manner. Although the circadian period increase caused by Mg deficiency was light dependent, it did not depend on active photosynthesis. Mathematical modeling of the Mg input into the circadian clock reproduced the experimental increase of the circadian period and suggested that Mg is likely to affect global transcription/translation levels rather than a single component of the circadian oscillator. Upon addition of a low dose of cycloheximide to perturb translation, the circadian period increased further under Mg deficiency, which was rescued when sufficient Mg was supplied, supporting the model’s prediction. These findings suggest that sufficient Mg supply is required to support proper timekeeping in plants.

info:eu-repo/semantics/published

Essential trace metals function as structural components or cofactors in many proteins involved in a wide range of physiological processes in plants. Hence, trace metal deficiency can significantly hamper plant growth and development. On the other hand, excess concentrations of trace metals can also induce phytotoxicity, for example via an enhanced production of reactive oxygen species. Besides their roles in plant growth under favourable environmental conditions, trace metals also contribute to plant responses to biotic and abiotic stresses. Heat is a stress factor that will become more prevalent due to increasing climate change and is known to negatively affect crop yield and quality, posing a severe threat to food security for future generations. Gaining insight into heat stress responses is essential to develop strategies to optimize plant growth and quality under unfavourable temperatures. In this context, trace metals deserve particular attention as they contribute to defence responses and are important determinants of plant nutritional value. Here, we provide an overview of heat-induced effects on plant trace metal homeostasis and the involvement of trace metals and trace metal-dependent enzymes in plant responses to heat stress. Furthermore, avenues for future research on the interactions between heat stress and trace metals are discussed.

International audience; Metallophytes constitute powerful models for the study of metal homeostasis, adaptation to extreme environments and the evolution of naturally selected traits. Arabidopsis halleri is a pseudometallophyte which shows constitutive zinc/cadmium (Zn/Cd) tolerance and Zn hyperaccumulation but high intraspecific variability in Cd accumulation. To examine the molecular basis of the variation in metal tolerance and accumulation, ionome, transcriptome and cell wall glycan array profiles were compared in two genetically close A. halleri populations from metalliferous and nonmetalliferous sites in Northern Italy. The metallicolous population displayed increased tolerance to and reduced hyperaccumulation of Zn, and limited accumulation of Cd, as well as altered metal homeostasis, compared to the nonmetallicolous population. This correlated well with the differential expression of transporter genes involved in trace metal entry and in Cd/Zn vacuolar sequestration in roots. Many cell wall-related genes were also more highly expressed in roots of the metallicolous population. Glycan array and histological staining analyses demonstrated that there were major differences between the two populations in terms of the accumulation of specific root pectin and hemicellulose epitopes. Our results support the idea that both specific cell wall components and regulation of transporter genes play a role in limiting accumulation of metals in A. halleri at contaminated sites.

The circadian clock coordinates the physiological response of a biological system to day and night rhythms through complex loops of transcriptional/ translational regulation. It can respond to external stimuli and adjust generated circadian oscillations accordingly to keep an endogenous period close to 24 h. To date, the interaction between nutritional status and circadian rhythms in plants is poorly understood. Magnesium (Mg) is essential for numerous biological processes in plants and its homeostasis is crucial to maintain optimal development and growth. Magnesium deficiency in young Arabidopsis thaliana seedlings increased the circadian period of pCCA1:LUC oscillations and dampened its amplitude in constant light in a dose-dependent manner. Although circadian period increase by Mg deficiency was light dependent, it did not depend on active photosynthesis. Mathematical modelling of the Mg input to the circadian clock reproduced the experimental increase of the circadian period and suggested that Mg is likely to affect global transcription/translation levels rather than a single component of the circadian oscillator. The model prediction was supported by a synergistic interaction between Mg deficiency and cyclohexamide, an inhibitor of translation. These findings suggest that proper Mg supply is required to support proper timekeeping in plants.One sentence summaryMagnesium maintains the circadian period in Arabidopsis seedlings and interferes with the circadian oscillator most likely through translational mechanisms.

Pseudometallophytes are commonly used to study the evolution of metal tolerance and accumulation traits in plants. Within the Arabidopsis genus, the adaptation of Arabidopsis halleri to metalliferous soils has been widely studied, which is not the case for the closely related species Arabidopsis arenosa. We performed an in-depth physiological comparison between the A. halleri and A. arenosa populations from the same polluted site, together with the geographically close non-metallicolous (NM) populations of both species. The ionomes, growth, photosynthetic parameters and pigment content were characterized in the plants that were growing on their native site and in a hydroponic culture under Cd treatments. In situ, the metallicolous (M) populations of both species hyperaccumulated Cd and Zn. The NM population of A. halleri hyperaccumulated Cd and Zn while the NM A. arenosa did not. In the hydroponic experiments, the NM populations of both species accumulated more Cd in their shoots than the M populations. Our research suggests that the two Arabidopsis species evolved different strategies of adaptation to extreme metallic environments that involve fine regulation of metal homeostasis, adjustment of the photosynthetic apparatus and accumulation of flavonols and anthocyanins., SCOPUS: ar.j, info:eu-repo/semantics/published

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., SCOPUS: ar.j, info:eu-repo/semantics/published

Advances on the ecology and evolution of adaptation to metal toxicity are based on studying metallophytes that are not restricted to soils strongly enriched in trace elements. The evolution of Cu and Co tolerance and accumulation, which principally occurs among the Copperbelt of Central Africa, is poorly known. In this paper, we studied Cu and Co tolerance and accumulation in a facultative metallophyte occupying a very broad ecological niche in southeastern Democratic Republic of Congo: Anisopappus chinensis (Asteraceae). The population variation in Cu and Co tolerance and accumulation was experimentally investigated using four metallicolous and two non-metallicolous populations from contrasted habitats. Surprisingly, Cu tolerance was poorly expressed in metallicolous populations grown in hydroponics, suggesting that specific rhizosphere processes may account for the ability to grow without toxicity symptoms under high Cu concentration on metalliferous soils. Population variation in Co tolerance and accumulation was demonstrated, which positively correlated to the concentration of Co in the native soil. Increased tolerance seems to have evolved in populations originating from Co-enriched soils. Foliar Co accumulation above 300 mg kg−1 dry weight with increased translocation was observed in the most tolerant populations, possibly making such populations promising materials to test for Co-phytomining applications.

The molecular analysis of metal hyperaccumulation in species such as Arabidopsis halleri offers the chance to gain insights into metal homeostasis and into the evolution of adaptation to extreme habitats. A prerequisite of metal hyperaccumulation is metal hypertolerance. Genetic analysis of a backcross population derived from Arabidopsis lyrata × A. halleri crosses revealed three quantitative trait loci for Cd hypertolerance. A candidate gene for Cdtol2 is AhCAX1, encoding a vacuolar Ca2+ /H+ antiporter. We developed a method for the transformation of vegetatively propagated A. halleri plants and generated AhCAX1-silenced lines. Upon Cd2+ exposure, several-fold higher accumulation of reactive oxygen species (ROS) was detectable in roots of AhCAX1-silenced plants. In accordance with the dependence of Cdtol2 on external Ca2+ concentration, this phenotype was exclusively observed in low Ca2+ conditions. The effects of external Ca2+ on Cd accumulation cannot explain the phenotype as they were not influenced by the genotype. Our data strongly support the hypothesis that higher expression of CAX1 in A. halleri relative to other Arabidopsis species represents a Cd hypertolerance factor. We propose a function of AhCAX1 in preventing a positive feedback loop of Cd-elicited ROS production triggering further Ca2+ -dependent ROS accumulation.

The circadian clock is an endogenous 24 hour rhythm that helps organisms anticipate and adapt to daily and seasonal variations in environment, such as the day/night cycle or changing temperatures. The plant clock is a complex network of transcription factors that regulate each other, forming interlocked feedback loops. Most of its components are light-regulated in some way, making the system highly sensitive to changes in light conditions. Here, we explore the mechanisms by which the plant clock adapts to changing day length. We first present some experimental data illustrating the variety of behaviors found in seedlings exposed to external day/night cycles different from 24h. We then use a mathematical model to characterize the response of the clock to a wide range of external cycle lengths and photoperiods. We show the existence of several domains of periodic entrainment with different ratios between the external cycle length and the period of the clock, and the presence of quasiperiodic and chaotic behaviors outside of the entrainment range. We simulate knockout mutants with impaired clock function and theoretical variants with diminished light sensitivity to highlight the role of a complex network and multiple light inputs in keeping the clock entrained over a wide range of conditions.

The nitrogen (N) utilization efficiency in hydroponically grown durum wheat was dissected during nitrate depletion at post-flowering stage. Feeding plants with low nitrate dose after anthesis lowered photosynthetic activity, triggered senescence, but induced N remobilization from vegetative parts. At harvest, tissue N concentration was 50% and 80% less important in leaves and culms, respectively, but similar in grains of N-deficient plants compared with control ones.

Hyperaccumulation and hypertolerance of Trace Metal Elements (TME) like Cd and Zn are highly variable in pseudo-metallophytes species. In this study we compared the impact of high Cd or Zn concentration on the photosynthetic apparatus of the Arabidopsis arenosa and Arabidopsis halleri pseudo-metallophytes growing on the same contaminated site in Piekary Slaskie in southern Poland. Plants were grown in hydroponic culture for 6 weeks, and then treated with 1.0 mM Cd or 5.0 mM Zn for 5 days. Chlorophyll a fluorescence and pigment content were measured after 0, 1, 2, 3, 4, and 5 days in plants grown in control and exposed to Cd or Zn treatments. Moreover, the effect of TME excess on the level of oxidative stress and gas-exchange parameters were investigated. In both plant species, exposure to high Cd or Zn induced a decrease in chlorophyll and an increase in anthocyanin contents in leaves compared to the control condition. After 5 days Cd treatment, energy absorbance, trapped energy flux and the percentage of active reaction centers decreased in both species. However, the dissipated energy flux in the leaves of A. arenosa was smaller than in A. halleri. Zn treatment had more toxic effect than Cd on electron transport in A. halleri compared with A. arenosa. A. arenosa plants treated with Zn excess did not react as strongly as in the Cd treatment and a decrease only in electron transport flux and percentage of active reaction centers compared with control was observed. The two species showed contrasting Cd and Zn accumulation. Cd concentration was almost 3-fold higher in A. arenosa leaves than in A. halleri. On the opposite, A. halleri leaves contained 3-fold higher Zn concentration than A. arenosa. In short, our results showed that the two Arabidopsis metallicolous populations are resistant to high Cd or Zn concentration, however, the photosynthetic apparatus responded differently to the toxic effects., Narodowe Centrum Nauki

The mechanisms underlying the response and adaptation of plants to excess of trace elements are not fully described. Here, we analyzed the importance of protein lysine methylation for plants to cope with cadmium. We analyzed the effect of cadmium on lysine-methylated proteins and protein lysine methyltransferases (KMTs) in two cadmium-sensitive species,Arabidopsis thalianaandA. lyrata, and in three populations ofA. halleriwith 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 is regulated by cadmium. Also, we showed that nine out of 23A. thalianamutants interrupted inKMTgenes 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, the other was interrupted in aKMTgene 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.

Hyperaccumulation and hypertolerance of Trace Metal Elements (TME) like Cd and Zn are highly variable in pseudo-metallophytes species. In this study we compared the impact of high Cd or Zn concentration on the photosynthetic apparatus of the Arabidopsis arenosa and Arabidopsis halleri pseudo-metallophytes growing on the same contaminated site in Piekary Slaskie in southern Poland. Plants were grown in hydroponic culture for 6 weeks, and then treated with 1.0 mM Cd or 5.0 mM Zn for 5 days. Chlorophyll a fluorescence and pigment content were measured after 0, 1, 2, 3, 4, and 5 days in plants grown in control and exposed to Cd or Zn treatments. Moreover, the effect of TME excess on the level of oxidative stress and gas-exchange parameters were investigated. In both plant species, exposure to high Cd or Zn induced a decrease in chlorophyll and an increase in anthocyanin contents in leaves compared to the control condition. After 5 days Cd treatment, energy absorbance, trapped energy flux and the percentage of active reaction centers decreased in both species. However, the dissipated energy flux in the leaves of A. arenosa was smaller than in A. halleri. Zn treatment had more toxic effect than Cd on electron transport in A. halleri compared with A. arenosa. A. arenosa plants treated with Zn excess did not react as strongly as in the Cd treatment and a decrease only in electron transport flux and percentage of active reaction centers compared with control was observed. The two species showed contrasting Cd and Zn accumulation. Cd concentration was almost 3-fold higher in A. arenosa leaves than in A. halleri. On the opposite, A. halleri leaves contained 3-fold higher Zn concentration than A. arenosa. In short, our results showed that the two Arabidopsis metallicolous populations are resistant to high Cd or Zn concentration, however, the photosynthetic apparatus responded differently to the toxic effects.

Copper (Cu) rich soils derived from rocks of the Katangan Copperbelt in the south-eastern Democratic Republic of Congo (DRC) support a rich diversity of metallophytes including 550 heavy metal tolerant; 24 broad Cu soil endemic; and 33 strict Cu soil endemic plant species. The majority of the plant species occur on prominent Cu hills scattered along the copperbelt. Heavy metal mining on the Katangan Copperbelt has resulted in extensive degradation and destruction of the Cu hill ecosystems. As a result, approximately 80 % of the strict Cu endemic plant species are classified as threatened according to IUCN criteria and represent a conservation priority. Little is known about the soil Cu tolerance optimum of the Cu endemic plant species. The purpose of this study was to quantify the soil Cu concentration (Cu edaphic niche) of four Cu endemic plant species to inform soil propagation conditions and microhabitat site selection for planting of the species in Cu hill ecosystem restoration. The soil Cu concentration tolerance of Cu endemic plant species was studied including Crotalaria cobalticola (CRCO); Gladiolus ledoctei (GLLE); Diplolophium marthozianum (DIMA); and Triumfetta welwitschii var. rogersii (TRWE-RO). The in situ natural habitat distributions of the Cu endemic plant species with respect to soil Cu concentration (Cu edaphic niche) was calculated by means of a generalised additive model. Additionally, the seedling emergence and growth of the four Cu endemic plant species in three soil Cu concentrations was tested ex situ and the results were compared to that of the natural habitat soil Cu concentration optimum (Cu edaphic niche). CRCO exhibited greater performance on the highest soil Cu concentration, consistent with its calculated Cu edaphic niche occurring at the highest soil Cu concentrations. In contrast, both DIMA and TRWE-RO exhibited greatest performance at the lowest soil Cu concentration, despite the calculated Cu edaphic niche occurring at moderate soil Cu concentrations. GLLE exhibited equal performances in the entire range of soil Cu concentrations. These results suggest that CRCO evolved via the edaphic specialization model where it is most competitive in Cu hill habitat with the highest soil Cu concentration. In comparison, DIMA and TRWE-RO appear to have evolved via the endemism refuge model, which indicates that the species were excluded into (i.e., took refuge in) the lower plant competition Cu hill habitat due to their inability to effectively compete with higher plant competition on normal soils. The soil Cu edaphic niche determined for the four species will be useful in conservation activities including informing soil propagation conditions and microhabitat site selection for planting of the species in Cu hill ecosystem restoration.

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 and 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. 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 twenty 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, in particular by manipulating features like the biomass production and the timing of senescence upon nitrogen limitation., SCOPUS: ar.j, info:eu-repo/semantics/published

Cobalt (Co) tolerance and accumulation were studied in three Haumaniastrum species: the obligate metallophyte (i.e. the strict endemic) Haumaniastrum robertii, its facultative metallophyte congener, H. katangense, and a species from nonmetalliferous soils, H. villosum. They were grown hydroponically and their growth during Co exposure was monitored. The two metallophytes did not grow in the absence of supplements, i.e., H. katangense required high concentrations of copper (Cu) to develop and H. robertii growth was conditional with regard to both Cu and Co. This discrepancy in metal requirement for growth originates from differences in ecological niches between the facultative and the absolute metallophytes. The beneficial effect of Co on plant growth was observed for all three species of Haumaniastrum. In the non-metallophyte, Co was beneficial at low concentrations and became toxic above 20 μM. In the two metallophytes the positive effect of Co was conditional on supplementation of Cu, and no toxicity was observed in the range of tested Co concentrations (3–150 μM). Co was essential to the growth of the absolute metallophyte. Mineral bulk analysis of plant parts by ICP-MS showed that Co accumulated more and was more mobile than Cu in all species investigated. This was confirmed by LA-ICP-MS mapping of root and leaf tissues, showing localisation of the two trace elements. H. katangense hyperaccumulated Co when exposed to 6 μM and H. robertii when exposed to 20 μM. None of the species studied hyperaccumulated Cu. Furthermore, Co and Cu did not occur in the same root tissues in any of the three Haumaniastrum species studied. In leaves of H. villosum and H. robertii, Cu and Co accumulated in different tissues while in those of H. katangense, Cu and Co were found to co-localize in the mesophyll cells. In summary our study supports the existence of effective Co tolerance mechanisms in the two Haumaniastrum metallophytes, mechanisms which are at least partially different from those for Cu and involve fitness costs.

Ca2+ signals in plant cells are important for adaptive responses to environmental stresses. Here, we report that the Arabidopsis CATION/Ca2+ EXCHANGER2 (CCX2), encoding a putative cation/Ca2+ exchanger that localizes to the endoplasmic reticulum (ER), is strongly induced by salt and osmotic stresses. Compared with the WT, AtCCX2 loss-of-function mutant was less tolerant to osmotic stress and displayed the most noteworthy phenotypes (less root/shoot growth) during salt stress. Conversely, AtCCX2 gain-of-function mutants were more tolerant to osmotic stress. In addition, AtCCX2 partially suppresses the Ca2+ sensitivity of K667 yeast triple mutant, characterized by Ca2+ uptake deficiency. Remarkably, Cameleon Ca2+ sensors revealed that the absence of AtCCX2 activity results in decreased cytosolic and increased ER Ca2+ concentrations in comparison with both WT and the gain-of-function mutants. This was observed in both salt and nonsalt osmotic stress conditions. It appears that AtCCX2 is directly involved in the control of Ca2+ fluxes between the ER and the cytosol, which plays a key role in the ability of plants to cope with osmotic stresses. To our knowledge, Atccx2 is unique as a plant mutant to show a measured alteration in ER Ca2+ concentrations. In this study, we identified the ER-localized AtCCX2 as a pivotal player in the regulation of ER Ca2+ dynamics that heavily influence plant growth upon salt and osmotic stress.

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.

Zinc (Zn) hyperaccumulation and hypertolerance are highly variable traits in Arabidopsis halleri. Metallicolous populations have evolved from nearby nonmetallicolous populations in multiple independent adaptation events. To determine whether these events resulted in similar or divergent adaptive strategies to high soil Zn concentrations, we compared two A. halleri metallicolous populations from distant genetic units in Europe (Poland (PL22) and Italy (I16)). The ionomic (Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)) and transcriptomic (RNA sequencing (RNA-Seq)) responses to growth at 5 and 150 μM Zn were analyzed in root and shoot tissues to examine the contribution of the geographic origin and treatment to variation among populations. These analyses were enabled by the generation of a reference A. halleri transcriptome assembly. The genetic unit accounted for the largest variation in the gene expression profile, whereas the two populations had contrasting Zn accumulation phenotypes and shared little common response to the Zn treatment. The PL22 population displayed an iron deficiency response at high Zn in roots and shoots, which may account for higher Zn accumulation. By contrast, I16, originating from a highly Zn-contaminated soil, strongly responded to control conditions. Our data suggest that distinct mechanisms support adaptation to high Zn in soils among A. halleri metallicolous populations.

Given the lack of a nervous system in plants, signaling molecules, such as Ca2+, are of vital importance for perceiving environmental stimuli. However, little information on the genes that control this network is available. Our demonstration that Arabidopsis CCX2, localized to the endoplasmic reticulum, modulates osmotic stress responses through the regulation of cytosolic and endoplasmic reticulum Ca2+ concentrations is one more step toward identifying all the players involved in plant stress responses. The elucidation of these pathways will help to meet the necessary requirements to face global challenges, such as high salinity on the world’s surface, including arable land. This work sheds light on an as yet uncharacterized and key player that tunes intracellular Ca2+ homeostasis, with particular significance for plants under salt stress.

This review synthesizes contemporary understanding of copper–cobalt (Cu–Co) tolerance and accumulation in plants. Accumulation of foliar Cu and Co to > 300 μg g−1 is exceptionally rare globally, and known principally from the Copperbelt of Central Africa. Cobalt accumulation is also observed in a limited number of nickel (Ni) hyperaccumulator plants occurring on ultramafic soils around the world. None of the putative Cu or Co hyperaccumulator plants appears to comply with the fundamental principle of hyperaccumulation, as foliar Cu–Co accumulation is strongly dose-dependent. Abnormally high plant tissue Cu concentrations occur only when plants are exposed to high soil Cu with a low root to shoot translocation factor. Most Cu-tolerant plants are Excluders sensu Baker and therefore setting nominal threshold values for Cu hyperaccumulation is not informative. Abnormal accumulation of Co occurs under similar circumstances in the Copperbelt of Central Africa as well as sporadically in Ni hyperaccumulator plants on ultramafic soils; however, Co-tolerant plants behave physiologically as Indicators sensu Baker. Practical application of Cu–Co accumulator plants in phytomining is limited due to their dose-dependent accumulation characteristics, although for Co field trials may be warranted on highly Co-contaminated mineral wastes because of its relatively high metal value.

Crepidorhopalon perennis is an endemic metallophyte restricted to only one site on Cu-rich soils in Katanga. Crepidorhopalon tenuis has a broader niche, from normal to Cu-rich soils, but has high affinity for cupriferous habitats. Both plants have been considered as Cu-Co accumulators. Cu tolerance and accumulation of C. tenuis were studied in axenic conditions in vitro in four metallicolous populations and one non-metallicolous population whereas for C. perennis only one population was investigated. Results showed a Cu tolerance of both C. tenuis and C. perennis. Variation of tolerance among metallicolous and non-metallicolous populations was also observed. The addition of Cu enhanced the growth of some metallicolous populations under sterile conditions, hence confirming the high needs in copper of metallicolous populations. This could represent a cost of tolerance which would explain the high affinity of species for cupriferous habitats. On the other hand, Crepidorhopalon perennis did not show the same features. Its restricted distribution may be explained by a cost of Cu tolerance. Crepidorhopalon perennis and C. tenuis are not Cu hyperaccumulators and seem to behave rather like excluder species., SCOPUS: ar.j, info:eu-repo/semantics/published

Investigation of genetic determinants of Cd tolerance in the Zn/Cd hyperaccumulator Arabidopsis halleri allowed the identification of the vacuolar Ca2+/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 Ca2+ spikes, especially at low external Ca2+ supply. In this study expression of genes involved in ROS homeostasis, cell wall composition, apoplastic pH regulation and Ca2+ 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 Ca2+ 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.

The circadian clock is an endogenous timekeeper that allows organisms to anticipate and adapt to the daily variations of their environment. The plant clock is an intricate network of interlocked feedback loops, in which transcription factors regulate each other to generate oscillations with expression peaks at specific times of the day. Over the last decade, mathematical modeling approaches have been used to understand the inner workings of the clock in the model plant Arabidopsis thaliana. Those efforts have produced a number of models of ever increasing complexity. Here, we present an alternative model that combines a low number of equations and parameters, similar to the very earliest models, with the complex network structure found in more recent ones. This simple model describes the temporal evolution of the abundance of eight clock genes and captures key features of the clock on a qualitative level, namely the entrained and free-running behaviors of the wild type clock, as well as the defects found in knockout mutants (such as altered free-running periods, lack of entrainment, or changes in the expression of other clock genes). Additionally, our model produces complex responses to various light cues, such as extreme photoperiods and non-24h environmental cycles, and can describe the control of hypocotyl growth by the clock. Our model constitutes a useful tool to probe dynamical properties of the clock as well as model more clock-dependent processes., SCOPUS: ar.j, info:eu-repo/semantics/published

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.

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.

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.

The root phenotype of an Arabidopsis (Arabidopsis thaliana) mutant of CHITINASE-LIKE1 (CTL1), called arm (for anion-related root morphology), was previously shown to be conditional on growth on high nitrate, chloride, or sucrose. Mutants grown under restrictive conditions displayed inhibition of primary root growth, radial swelling, proliferation of lateral roots, and increased root hair density. We found here that the spatial pattern of CTL1 expression was mainly in the root and root tips during seedling development and that the protein localized to the cell wall. Fourier-transform infrared microspectroscopy of mutant root tissues indicated differences in spectra assigned to linkages in cellulose and pectin. Indeed, root cell wall polymer composition analysis revealed that the arm mutant contained less crystalline cellulose and reduced methylesterification of pectins. We also explored the implication of growth regulators on the phenotype of the mutant response to the nitrate supply. Exogenous abscisic acid application inhibited more drastically primary root growth in the arm mutant but failed to repress lateral branching compared with the wild type. Cytokinin levels were higher in the arm root, but there were no changes in mitotic activity, suggesting that cytokinin is not directly involved in the mutant phenotype. Ethylene production was higher in arm but inversely proportional to the nitrate concentration in the medium. Interestingly, eto2 and eto3 ethylene overproduction mutants mimicked some of the conditional root characteristics of the arm mutant on high nitrate. Our data suggest that ethylene may be involved in the arm mutant phenotype, albeit indirectly, rather than functioning as a primary signal.

*Unravelling mechanisms that control plant growth as a function of nutrient availability presents a major challenge in plant biology. This study reports the first transcriptome response to long-term (1 wk) magnesium (Mg) depletion and restoration in Arabidopsis thaliana. *Before the outbreak of visual symptoms, genes responding to Mg starvation and restoration were monitored in the roots and young mature leaves and compared with the Mg fully supplied as control. *After 1 wk Mg starvation in roots and leaves, 114 and 2991 genes were identified to be differentially regulated, respectively, which confirmed the later observation that the shoot development was more affected than the root in Arabidopsis. After 24 h of Mg resupply, restoration was effective for the expression of half of the genes altered. We emphasized differences in the expression amplitude of genes associated with the circadian clock predominantly in leaves, a higher expression of genes in the ethylene biosynthetic pathway, in the reactive oxygen species detoxification and in the photoprotection of the photosynthetic apparatus. Some of these observations at the molecular level were verified by metabolite analysis. *The results obtained here will help us to better understand how changes in Mg availability are translated into adaptive responses in the plant.

Cu tolerance and accumulation have been studied in Haumaniastrum katangense, a cuprophyte from Katanga (DR Congo), previously described as a copper hyperaccumulator. Nicotiana plumbaginifolia, a well-known non-tolerant and non-accumulator species, was used as a control. The germination rate of H. katangense was enhanced by copper and fungicide addition, suggesting that fungal pathogens, which restrain germination in normal conditions, are limiting. In hydroponic culture in the Hoagland medium, H. katangense did not grow well, in contrast to N. plumbaginifolia. Better growth was achieved by adding fungicide or higher copper concentrations. The maximal non-effective concentration (NEC) was 12 µM CuSO4 for H. katangense grown in hydroponics, i.e. 24 times greater than Cu concentration in the Hoagland medium. By comparison, copper concentrations greater than 0.5 µM had a negative effect on the growth of N. plumbaginifolia. EC50 (50% effective concentration) in hydroponics was 40 µM CuSO4 for H. katangense and 6 µM CuSO4 for N. plumbaginifolia. EC100 (100% effective concentration) was 100 µM CuSO4 for H. katangense and 15 µM CuSO4 for N. plumbaginifolia. In soil, growth was also stimulated by Cu addition up to 300 mg kg-1 CuSO4. 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. Cu accumulation in the shoot has been measured for N. plumbaginifolia and H. katangense at their respective NEC. Cu allocation in the two species showed a similar response to increasing Cu concentrations, i.e. root/shoot concentration ratio well above 1. In conclusion, H. katangense is highly tolerant to copper and has elevated copper requirement even in the absence of biotic interactions. Its accumulation pattern is typical of an excluder species.

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.

Contents Summary 759 I. Hyperaccumulation: the phenomenon 759 II. Macroevolution of hyperaccumulation 760 III. Microevolution of hyperaccumulation: variation within hyperaccumulator species 760 IV. Genetic analysis of trace metal accumulation and tolerance 761 V. Mechanisms of trace metal accumulation 762 VI. General discussion and research perspectives 769 Acknowledgements 772 References 772 Summary Metal hyperaccumulator plants accumulate and detoxify extraordinarily high concentrations of metal ions in their shoots. Metal hyperaccumulation is a fascinating phenomenon, which has interested scientists for over a century. Hyperaccumulators constitute an exceptional biological material for understanding mechanisms regulating plant metal homeostasis as well as plant adaptation to extreme metallic environments. Our understanding of metal hyperaccumulation physiology has recently increased as a result of the development of molecular tools. This review presents key aspects of our current understanding of plant metal – in particular cadmium (Cd), nickel (Ni) and zinc (Zn) – hyperaccumulation.

Cuprophytes are plants that mostly occur on Cu-rich soil in SC Africa. Crepidorhopalon perennis is endemic of a single site. C. tenuis has a broader niche, from normal to Cu-rich soil. Both have been considered as Cu-Co accumulators. We examined soil factors controlling heavy metal accumulation and plant fitness in natural populations. Plant mass and element concentrations in plants and soil were determined in 153 samples from five populations of C. tenuis on copper soil (CTC), two on normal soil (CTN) and the single population of C. perennis (CP). Soil in Cu-sites had higher concentrations of Ca, Mg, P, Mn, Zn, Cu, Co. Plants from Cu-sites were larger and had higher Cu and Co content, and lower Mg, Mn and Ca. Cu in shoots was influenced positively by Cu and Mn and negatively by Ca in the soil. Co in shoots was influenced positively by Co and negatively by Mn and Fe in the soil. Shoot mass was influenced positively by Cu and Mn (CT) or by Cu and Co (both species pooled) in the soil. The results suggest that C. tenuis and C. perennis are genuinely cuprophilous species. Large variation in metal accumulation in shoots can be accounted for by synergistic and antagonistic interactions among several heavy metals, yielding specific accumulation patterns in different populations.

Arabidopsis halleri is a model plant for Zn and Cd hyperaccumulation. The objective of this study was to determine the relationship between the chemical forms of Cd, its distribution in leaves, and Cd accumulation and tolerance. An interspecific cross was carried out between A. halleri and the non-tolerant and non-hyperaccumulating relative A. lyrata providing progenies segregating for Cd tolerance and accumulation. Cd speciation and distribution were investigated using X-ray absorption spectroscopy and microfocused X-ray fluorescence. In A. lyrata and non-tolerant progenies, Cd was coordinated by S atoms only or with a small contribution of O groups. Interestingly, the proportion of O ligands increased in A. halleri and tolerant progenies, and they were predominant in most of them, while S ligands were still present. Therefore, the binding of Cd with O ligands was associated with Cd tolerance. In A. halleri, Cd was mainly located in the xylem, phloem, and mesophyll tissue, suggesting a reallocation process for Cd within the plant. The distribution of the metal at the cell level was further discussed. In A. lyrata, the vascular bundles were also Cd enriched, but the epidermis was richer in Cd as compared with the mesophyll. Cd was identified in trichomes of both species. This work demonstrated that both Cd speciation and localization were related to the tolerance character of the plant.

The mitotic checkpoint (MC) guards faithful sister chromatid segregation by monitoring the attachment of spindle microtubules to the kinetochores. When chromosome attachment errors are detected, MC delays the metaphase-to-anaphase transition through the inhibition of the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase. In contrast to yeast and mammals, our knowledge on the proteins involved in MC in plants is scarce. Transient synchronization of root tips as well as promoter-reporter gene fusions were performed to analyze temporal and spatial expression of COPPER MODIFIED RESISTANCE1/PATRONUS1 (CMR1/PANS1) in developing Arabidopsis thaliana seedlings. Functional analysis of the gene was carried out, including CYCB1;2 stability in CMR1/PANS1 knockout and overexpressor background as well as metaphase-anaphase chromosome status. CMR1/PANS1 is transcriptionally active during M phase. Its deficiency provokes premature cell cycle exit and in consequence a rapid consumption of the number of meristematic cells in particular under stress conditions that are known to affect spindle microtubules. Root growth impairment is correlated with a failure to delay the onset of anaphase, resulting in anaphase bridges and chromosome missegregation. CMR1/PANS1 overexpression stabilizes the mitotic CYCB1;2 protein. Likely, CMR1/PANS1 coordinates mitotic cell cycle progression by acting as an APC/C inhibitor and plays a key role in growth adaptation to stress.

Highlight A huge variability in Cd tolerance and accumulation exist within A. halleri, and the relationship between tolerance, accumulation, and edaphic type is not straightforward. Cd-induced cell wall modifications suggest various shoot detoxification mechanisms., 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.

Plants dynamically cope with the variability of mineral nutrient distribution in soil by constantly modulating nutrient uptake and shaping root-system architecture. The changes in root morphology in response to major essential elements are largely documented, but little is known about how the root system responds to magnesium (Mg) availability. Thirty-six natural accessions of the model species Arabidopsis thaliana were subjected to an in vitro screen for identifying variation in root system architecture in response to Mg availability. Response of root morphology was observed on 2-dimensional agar plates. Low Mg supply repressed the elongation of the lateral roots more than of the primary root. However, some accessions exhibited higher number and length of lateral roots than the reference Columbia-0. Across all accessions, the root morphological traits did not correlate with tissue Mg concentrations. Interestingly, shoot calcium and root phosphorus concentrations were positively correlated with the number and length of lateral roots, whereas root iron concentration was negatively correlated with the primary root length. The diversity of root phenotypes identified in this report is a useful resource to study the genetic component determining root morphology in response to Mg availability., SCOPUS: ar.j, info:eu-repo/semantics/published

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 calcium2+/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.

Plants constantly sense the changes in their environment; when mineral elements are scarce, they often allocate a greater proportion of their biomass to the root system. This acclimatory response is a consequence of metabolic changes in the shoot and an adjustment of carbohydrate transport to the root. It has long been known that deficiencies of essential macronutrients (nitrogen, phosphorus, potassium and magnesium) result in an accumulation of carbohydrates in leaves and roots, and modify the shoot-to-root biomass ratio. Here, we present an update on the effects of mineral deficiencies on the expression of genes involved in primary metabolism in the shoot, the evidence for increased carbohydrate concentrations and altered biomass allocation between shoot and root, and the consequences of these changes on the growth and morphology of the plant root system.

Cadmium (Cd) tolerance seems to be a constitutive species-level trait in Arabidopsis halleri. In order to identify genes potentially implicated in Cd tolerance, a backcross (BC1) segregating population was produced from crosses between A. halleri ssp. halleri and its closest non-tolerant relative A. lyrata ssp. petraea. The most sensitive and tolerant genotypes of the BC1 were analysed on a transcriptome-wide scale by cDNA-amplified fragment length polymorphism (AFLP). A hundred and thirty-four genes expressed more in the root of tolerant genotypes than in sensitive genotypes were identified. Most of the identified genes showed no regulation in their expression when exposed to Cd in a hydroponic culture medium and belonged to diverse functional classes, including reactive oxygen species (ROS) detoxification, cellular repair, metal sequestration, water transport, signal transduction, transcription regulation, and protein degradation, which are discussed.

Although the symptoms of magnesium deficiency are well documented in plants, the primary physiological effects of low Mg availability remain largely unknown. This paper describes the physiological responses of Mg starvation in Arabidopsis thaliana. Growth characteristics, Mg and sugar concentration, and photochemical performance were measured at regular intervals during the induction of Mg deficiency. These data show that Mg deficiency increased the sugar concentration and altered sucrose export from young source leaves before any noticeable effect on photosynthetic activity was seen. The decline in photosynthetic activity might be elicited by increased leaf sugar concentrations. Transcript levels of Cab2 (encoding a chlorophyll a/b protein) were lower in Mg-deficient plants before any obvious decrease in the chlorophyll concentration. These transcriptional data suggest that the reduction of chlorophyll is a response to sugar levels, rather than a lack of Mg atoms for chelating chlorophyll.