BACKGROUND
nanoparticles  are materials with at least one dimension in the nanoscale .
the high surface-to-volume ratios and unique physicochemical properties of these materials have led to myriad practical applications, resulting in increased production and potential release into the environment.
however, the effect of np exposure on biological systems may differ from what is known about exposure to their bulk counterparts.
fate and transport studies have demonstrated that disposed nps are present in nature at concentrations that might pose a risk  <cit> .
unicellular algae are widely used to test np acute toxicity in the aquatic environment.
at the cellular level, nano-forms of zno <dig>  tio <dig>  or ag can differently modify growth rates, cellular viability, and chlorophyll content compared to their corresponding bulk materials  <cit> .
subcellularly, alterations of organelles like chloroplasts and vacuoles and accumulation of nps near the cell wall and plasma membrane have been observed  <cit> .
at the molecular level, nps increase reactive oxygen species  formation and lipid peroxidation to a higher extent that bulk materials  <cit> .
however, many environmental pollutants can also induce ros formation through an endogenous mechanism to alert for potential cell damage and increase environmental stress tolerance  <cit> .
physiological studies of higher plants have demonstrated that nps up to 40 nm can be taken up by roots and travel symplastically through the vascular system, whereas larger nps accumulate in the apoplastic space.
therefore, some of the damaging effects of nps have been attributed to mechanical damage or clogging of plant structures, like plasmodesmata or stomata, which regulate water flux  <cit> .
in maize tio2- and clay-nps reduced the size of cell-wall pores of roots and inhibited hydraulic conductivity and transpiration  <cit> .
consequently, plant molecular responses to mitigate np-related damage might involve mechanisms to control water-stress, although this possibility has not been tested.
research on crop plants has prompted further questions about the impacts of released nps in agricultural production and the implications for the food chain  <cit> .
uptake, translocation, and generational transmission of carbonaceous nanomaterials have been demonstrated in rice  <cit> .
photothermal and photoacoustic methods have revealed the spatial distribution of carbon nanotubes  in roots, leaves, and fruits of tomato plants  <cit> .
in that work, the authors assayed the addition of four carbon-based materials to the plant growth medium and observed physiological responses for the single  and multiwall  cnts only.
mwcnts over swcnts produced a maximum effect on biomass accumulation, thereof plants fed with mwcnts were chosen for further examination by photothermal/photoacoustic scanning cytometry or microarray analysis.
imaging techniques detected mwcnts in roots but also in the leaves and fruits of the plants exposed to nps, and data were integrated in parallel with tomato microarray analysis to discover that mwcnts induced changes in gene expression of stress- related genes.
a number of differentially-expressed transcripts were identified that corresponded to  <dig> genes with known function, some of which are involved in plant responses to pathogen infection.
this observation suggested that plants sense the penetration of nano-sized materials into their tissues as a biotic stress factor similar to pathogen or herbivore attack.
gene expression analyses of the model plant arabidopsis thaliana have provided new insights into the molecular mechanisms of plant response to nps  <cit> .
these two studies both reported gene expression changes upon long-term exposure to tio2-nps, zno-nps, ag-nps, and fullerene of different sizes.
there were important differences among np doses, germination conditions, and plant developmental stages during exposure.
landa et al.
<cit>  concluded that zno-nps caused the most dramatic changes in gene expression, resulting in the up- and down-regulation of  <dig> and  <dig> genes, respectively, whereas tio2-nps only affected  <dig> and  <dig> genes, respectively, indicating minimal toxicity.
zno-nps and fullerenes up-regulated genes involved in functional responses to abiotic  and biotic stresses, whereas tio2-np exposure differentially regulated  genes involved in both stresses, although data about the significance of these functional representation were not provided.
in the study by kaveh et al.
<cit> , exposure to ag-nps was associated with the down regulation of genes involved in pathogen response, but a significant overlap was observed with the genes responding to bulk material.
importantly, some of the genes regulating hormonal stimuli and stress response were also identified as np-responsive and connected with systemic acquired response , an enhanced immunity in tissues remote from the initial infection site  <cit> .
sar is triggered upon challenge by certain pathogens or, to some extent, by mechanical damage, i.e., as a result of wounding by herbivore insects.
sar results in thickening of the cell wall and other physiological responses that enhance general plant defences in a non-specific way.
here we produced a set of  <dig> comparable transcriptome profiles to monitor early changes in gene expression upon np and stress exposure.
we evaluated a. thaliana response to different types  and sizes  of nps in comparison to biotic and abiotic stress inducers representing the most common environmental challenges for plants.
biotic stress was induced by infection with a necrotizing fungus  or a hemibiotrophic bacterium .
these pathogens trigger plant defence and immune responses through mechanisms involving respectively, phytohormones like salicylic acid , a key modulator of sar, and jasmonic acid/ethylene pathways  <cit> .
abiotic stresses induced by hyper-saline conditions, drought, and mechanical wounding were also assayed.
the effect of abscisic acid , the most studied stress-responsive phytohormone, which mediates stomatal closure and other responses to drought and osmotic stress  <cit> , on the gene expressions and phenotypes of np-exposed plants was also tested.
RESULTS
exposure of plants to pathogens, abiotic stress and nps
since the range of the transcriptional responses to stress depends on the growth conditions and developmental stage of the plant, synchronized hydroponic cultures were used for all exposures.
plantlets were grown in liquid ms medium until rosette leaves  emerged and begun aerial development.
at this stage leaves can be inoculated with microbial pathogens as in the infection models used before to study immune response and roots are well developed to allow np or salt uptake.
plants were infected with a. brassicola  or p. syringae  to study the transcriptional response to biotic stress in  <dig> of the  <dig> conditions that were assayed.
infected plants developed chlorotic leaves or other macroscopic signals of a plant hyper-sensitive immune response by the second day post infection , indicating that mechanisms of defence against biotic stress were already activated.
similarly, physiological responses to saline stress were observed 2 days upon addition of 100 mm nacl  to the medium.
drought  and wounding  stress were induced as described in the methods section to assay a total of  <dig> conditions for abiotic stress that have been connected before with np-induced damage.
nps were added to the cultures at the same time that the other stressors and incubated with the plants for 2 days before collection.
we performed preliminary dose- and size-dependent measurements of the effects of nps on plant biomass under these particular growth conditions.
these experiments narrowed the range of concentrations at which nps had measurable effects on plant growth to  <dig> –25 μg/ml, similar to previous studies  <cit> .
we assayed nps made of  <dig> different materials, ag, tio <dig> and carbonaceous materials , and diameters ranging from  <dig> to 80 nm for ag-nps,  <dig> to 40 nm for tio2-nps and 4–12 nm for cooh-mwcnts.
this made a total of  <dig> different conditions  for np-exposed plants.
bulk materials that release equivalent metallic ions  were added to  <dig> additional series of plants and one more condition was assayed in which plants were supplemented with aba 12 h. before addition of cooh-mwcnts .
combined exposure to nps and aba was performed to determine whether np-induced effects at the transcriptomic level could be prevented or reverted with aba treatment.
this hormone activates physiological responses to drought, salt, and other abiotic stresses through a complex network of transcriptional regulators.
exogenous addition of aba can mimic the endogenous accumulation of the hormone that triggers stress responses in the tissues and increases plant tolerance to saline stress  <cit> .
transcriptome analyses of exposed plants
we examined the changes that occurred in the transcription of  <dig>  genes represented in the at v <dig> microarray.
hybridization signals from the cdna of plants exposed to the  <dig> conditions described above were normalised relative to the values of non-exposed plants .
changes in these normalised gene expression values were further analysed.
principal component analysis  was performed to detect major trends in the expression data.
the first and second components of the analysis, which captured respectively  <dig> % and  <dig> % of the variation, were plotted to visualize similarities among samples .
the scatter-plot shows that all np exposures clustered together, independently of material  or size in the nano scale.
the biotic stresses induced upon pathogen challenge  were discriminated from other conditions to the right side of the plot.
the abiotic stressors  graphed closer to the np exposure samples.
thus, plants exposed to nps show similar transcriptional behaviours, and pathogen challenge, saline or drought stress -in this order- cause major changes in the transcriptome when compared to np exposure.
in support of this, box-plot representation of normalised expression ratios shows that the ranges of expression changes under these stress conditions are spanned .
for example, 90% of the genes in the pst-infected plants had expression ratios between − <dig>  and  <dig> , whereas this range was only − <dig>  to  <dig>  in samples exposed to nps.
pre-treatment with aba in cooh-mwcnt-exposed plants did not decrease the range of expression changes to that of non-exposed plants, suggesting that the addition of this hormone had side effects on the plant transcriptome.figure  <dig> principal component analysis and distribution of normalised expression ratios for the total of genes analyzed.
pca was performed to reduce the dimensions of expression data to  <dig> components.
components  <dig> to  <dig> explained respectively  <dig> %,  <dig> %,  <dig> % and  <dig> % of variation in the data.
the first and second components were used as the x and y axes to plot values from the  <dig> conditions in this study.
ag <dig>  ag <dig>  ag <dig>  ag <dig> and agn represent the addition of ag-nps of 10-, 20-, 40- and 80-nm diameter or agno <dig> bulk material, respectively.
ti <dig>  ti <dig>  ti <dig> and tio the addition of tio2-nps of 10-, 20-, 40-nm diameter and tio <dig> bulk material.
cnt and cnt+ indicate cooh-mwcnts added to non-supplemented or aba-supplemented plants.
box plot of the expression ratios for the  <dig>  genes analysed with the atv <dig> microarrays.
boxes represent 10th and 90th percentiles, with the median  in the middle line of the box.
minimum and maximum values  are shown by the end of the vertical lines  and upper or lower outliers by red diamonds.
identification and functional classification of genes with significant transcriptional responses to nps
microarray data was statistically analysed to identify differentially-expressed genes that responded to all np treatments, irrespectively of particle size or type.
the analysis recovered a set of  <dig> genes with significant  changes in expression .
quartile  representation of the expression changes for this set  showed that most were repressed  in np-treated samples, and median expression ratios were low  compared with those of non-exposed plants.figure  <dig> quartile distribution of the expression ratios for the  <dig> np-responsive genes.
boxes represent q <dig> and q <dig> quartiles and the middle line of the box is the median .
whiskers show the minimum and maximum values  and red diamonds outliers.
to detect genes that were empirically  responding to biotic or abiotic stress, we performed independent analyses of abr, pst, nac, drou, and wou samples to define five sets of biotic- or abiotic-stress-responsive genes under our experimental conditions.
these sets were overlapped with the group of  <dig> np-responsive genes to produce four subsets of  <dig> ,  <dig> ,  <dig> , and  <dig>  differentially-expressed genes, with broad overlap between the abr- and pst-responsive subsets .
thus, most of the  <dig> genes with significant responses to nps were also regulated upon pathogen challenge in our hydroponic growth model.
in contrast, none of the significant wounding-responsive genes were included in the 351-gene set.figure  <dig> distribution of the expression ratios for the  <dig> subsets of stress-responsive genes within the 351-gene set.
venn diagrams represent the  <dig> subsets of  <dig> ,  <dig> ,  <dig>  and  <dig>  stress-responsive genes that were included in the group of  <dig> np-responsive genes, with the number of overlapping genes in each intersection.
distribution of the expression ratios within each subset.
boxes and middle line represent q1-q <dig> quartiles and the median of the distribution.
whiskers show the minimum and maximum values  whereas red diamonds show outliers.
functional in silico classification of the  <dig> genes was achieved via gene ontology  analysis.
a total of  <dig> go terms were significantly  enriched in the three supercategories of biological process, cellular component, and molecular function .
go terms in biological process are shown in figure  <dig>  the most represented term in the 351-gene set is response to stress, which included  <dig> genes from the set  and was significantly  enriched with respect to the total of  <dig>  genes .
additional analyses of some of these functional classes are presented in the following sections.
in cellular component, a significant percentage of the  <dig> genes corresponded to extracellular proteins , whereas none of the other sub-cellular compartments were over-represented .
the most enriched category within molecular function was alkaline phosphatases.figure  <dig> go enrichment analysis of the 351-gene set.
the percentages of genes that are associated to a go-term are shown in the left bars for the set of  <dig> differentially expressed genes and for the total set of  <dig>  genes enrichment scores  are represented by the bar graph in the right.
the number of genes associated to each go term is shown in the right column.
only go-terms in the category of biological process are shown.
some descendant terms  that contain the same genes have been merged and represented as a single category bar for simplicity .
transcriptional responses to nps include repression of pathogen-activated genes and salicylic acid-mediated pathways
abr- and pst-responsive genes in the 141- and 114-gene subset were generally repressed in all np treatments .
we focused on genes previously associated with stress responses by the go annotation and for which more information was available.
hierarchical clustering grouped them into two main branches  which roughly corresponded to genes that were up-  or down-regulated  by one or both biotic stresses.
most of the genes in both branches  were repressed in all np-treated samples  and only  <dig> genes  responded to np treatments with log ratios above  <dig>  although overall these genes were up-regulated more by pathogen challenge than by np exposure.figure  <dig> clustering of the stress-annotated, pathogen-responsive genes in the 351-gene-set.
hierarchical clustering was represented for the  <dig> gene-entities using euclidean distances and following ward’s linkage rule.
branch a <dig> includes genes that are activated upon pathogen challenge and repressed in np-exposed plants.
this branch contains most sar-responsive genes.
branch a <dig> corresponds to genes activated by pathogen challenge and also, but to a lesser extent, by np exposure.
branch b is formed by the genes repressed both by pathogen and np-exposure .
most pathogen-responsive genes that were activated during our abr/pst challenges are involved in early defence signalling and represent main effectors of the plant immune response that triggers sar upon pathogen infection.
such was the case with frk <dig> , an inducible kinase in the pathway that activates the basal immune response upon perception bacterial flagellin  <cit> , and other genes in the enriched go category of sar via sa signalling .
these genes were all significantly repressed by np exposure .
these data suggest that repression of pathogen-induced genes and sa response is a common feature of np exposure.
exposure to nps represses expression of phosphate-starvation and root-development genes
the most regulated genes  in the set of  <dig> differentially-expressed genes were related to the phosphate starvation response .
the enriched go subset included  <dig> genes that are also present in the more general go categories of cellular response to starvation, cellular response to nutrient levels, and cellular response to extracellular stimulus.
purple-acid phosphatases  and other genes in the set that are induced by phosphate starvation  were strongly repressed in all np-treated samples, as confirmed by rt-qpcr .figure  <dig> repression of the phosphate-starvation genes upon np exposure and overlapping go categories.
the  <dig> np-responsive genes that are included in the enriched go category of phosphate starvation response are shown clustered.
overlapping of the enriched categories that contain most phosphate-starvation genes  box-plot representation of q <dig>  q <dig> and median expression ratios of the  <dig> phosphate-starvation genes; whiskers show minimum and maximum values and red diamonds outliers.
transcriptional responses to phosphate starvation have been widely studied  <cit>  and result in important changes to root morphology by promoting the inhibition of primary root growth and the formation of lateral roots, as well as the proliferation of root hairs  <cit> .
all these changes increase the capacity of plants to absorb available phosphate from the soil and constitute a local response to external phosphate starvation  <cit> .
sensing of low phosphate availability also triggers long distance signals to systemic tissues that activate phosphate homeostasis mechanisms via increased phosphate transport, recovery, and recycling by modulating the expressions of high-affinity transporters, secreted phosphatases, and phospholipid catabolism enzymes.
in np-exposed plants, we observed repression of phosphate homeostatic enzymes as well as genes directly involved in root hair development from epidermal trichoblasts.
root-hair-specific genes  and  <dig> other genes with roles in root hair emergence and differentiation  <cit>  constituted the enriched subsets of trichoblast/epidermal cell differentiation and root morphogenesis genes in the 351-gene set .
unlike the phosphate-starvation genes, root hair genes were repressed, rather than activated, during abr/pst stress .
because many of them encode cell-wall modification enzymes that shape the trichoblast, differential expression of this group likely involves structural changes in the roots of plants exposed to nps, which is not necessary implicit in the transcriptional regulation of phosphate homeostatic enzymes.figure  <dig> repression of the root morphogenesis genes upon np-exposure.
the  <dig> np-responsive genes included in the enriched go categories of trichoblast differentiation/root morphogenesis are shown.
box-plot representation of the q <dig>  q <dig> and median expression values for the set.
exposure to nps increases bacterial survival in local and distal leaves of plants infected with pst
we next tested some predictions of the transcriptional reprogramming observed upon np exposure on the phenotypic response of the plant to biotic stress.
a significant number of np-repressed genes are activated upon pathogen infection and are involved in sar, therefore exposure to nps before pathogen challenge might compromise a plant’s ability to respond to infection.
however, if this effect were modulated by sa, exogenous supplementation with the hormone might compensate for it and prevent the np-induced phenotype.
we measured the spread of the hemibiotrophic pathogen pst in the leaves of np-exposed plants.
rosette leaves were inoculated with the bacterium 1 day after addition of nps to the ms growth medium and compared with np-unexposed control plants.
another series of plants were treated with sa before the addition of nps.
bacterial growth in local  and distal  leaves was determined 2 days after inoculation .
the np exposure was associated with increased bacterial growth, both in local and distal leaves of infected plants, compared with the control.
control plants experienced only about half the level of infection in np-exposed plants, consistent with an intact  transcriptional mechanism that can activate pathogen-resistance genes.
exogenous supplementation of sa prevented the bacterial growth increases associated with np treatment in the distal leaves, where systemic signalling by sa enhances resistance to infection.figure  <dig> bacterial growth upon pst infection of np-exposed and non-exposed plants.
mayor rossete leaves of plants were inoculated with a suspension containing  <dig> colony forming units  of the pst strain or with a mock solution  24 h after exposure to nps.cfu were counted in inoculated and distal leaves 2 days after inoculation.
the effect of supplementation with sa 12 h before np addition was measured in another series of plants.
bacterial growth in distal leaves of np-treated plants was significantly increased respect to the control plants.
exposure to nps inhibits root hair development
transcriptional repression of the root development genes predicted an altered root phenotype in plants exposed to nps.
absorption of nps in the root cap and accumulation in the columella has been shown in a. thaliana plants that were germinated and fed with high concentrations of 40 nm agnps  <cit> .
similarly, we observed the accumulation of cooh-mwcnts around the root caps of plants growing under our hydroponic conditions .
shortening of the root tips and other alterations to root morphology have been described in a. thaliana and other plants exposed to some types of nps  <cit> .
however our transcriptome analysis pointed to a specific effect of nps on the epidermal cells that originate root hairs and indicated that rhs genes were repressed by all types of nps tested in our experiments.
root hairs substantially increase the root surface area in contact with the soil, and most of the water and nutrients that enter the plant are absorbed through them.
thus, their development is significantly affected by environment stimuli and stress signals  <cit> .figure  <dig> inhibition of root hair production upon np exposure and effect of sa pre-treatment.
accumulation of nps in the root cap.
microscope photographs of the roots of plants exposed to cooh-mwcnts 4 days upon addition of the nps to the growth medium: black aggregates appear around the root cap.
microphotographs of the lateral roots of plants exposed to ag-nps ; np-exposed roots show a “hairless” phenotype.
.
number of root hairs per cm of root in plants exposed to ag-nps, tio2-nps, and cooh-mwcnts, compared to control  plants.
a significant decrease is observed in all the plants treated with nps.
effect of the pre-treatment with aba, methyl jasmonate  and sa phytohormones before exposure to 20 nm ag-nps; only the addition of sa can prevent the decrease in the number of root hairs/cm observed upon exposure to nps.
pre-treatment with sa prevents the reduction in root hair density upon treatment with tio2-nps and cooh-mwcnts.
we performed detailed microscopic observations to quantify the effects of nps on root morphology under our plant growth conditions.
superficially, np-treated roots resembled some “hair-less” mutants  <cit>  .
quantification confirmed that np-exposure significantly decreased root hair density regardless of the size or type of np .
because hormonal balance regulates root development, and aba has been shown to be locally involved in the readjustment of root morphology under abiotic stress conditions  <cit> , we tested the effects of exogenous supplementation with different hormones ) before the addition of nps.
supplementation with sa prevented the reduction in root hair density caused by np exposure .
further assays with tio <dig> nps and cooh-mwcnts confirmed that supplementation with sa could rescue the phenotypic effects of all types of nps on root hair development .
discussion
nanoparticle exposure had less of an impact on the plant transcriptome than the other stress conditions assayed in this work .
as measured by genome expression changes, biotic stress represented the major challenge for these plants, whereas saline or drought stresses have lesser effects, although still greater than that of np exposure.
the plant–pathogen system or abiotic stresses used here may represent harsher environmental conditions than np exposure.
however none of the stresses assayed here posed severe challenges to plant survival, because our seedlings  completed their vegetative life cycles, bolted, and eventually flowered 1– <dig> weeks after exposure.
exposure to nps under these conditions induced significant changes in gene expression as well as measurable phenotypic differences , so their effects at these doses cannot be considered negligible.
therefore, compared with other environmental challenges, a. thaliana has the genetic resources to withstand np-induced stress, although to a great extent the response will depend on the concentrations and activities of the nanomaterials in the plant environment  <cit> .
more interestingly, in our analysis, all tested types and sizes of nanoparticles plotted in the same area in the pca, supporting the idea of common regulatory mechanisms in the arabidopsis response to nps that could be exploited to prevent negative effects on plant development.
down-regulation of gene expression was an overall response to nps.
the q <dig> values for the  <dig> differentially-expressed genes were below zero in all the np-treated samples .
overall, this down-regulation affected all the gene subsets in the go categories represented in the figure  <dig>  except for cellular response to hypoxia .
the subsets of  <dig>   <dig>  and  <dig> genes that responded to biotic or saline stress were also down-regulated in the np samples , and only the  <dig> drought-responsive genes responded to nps with increased expressions.
transcriptional repression is suspected to be an important mechanism to keep stress responses under tight control  <cit> ; early response to nps might involve preventive repression of gene expression to allow genetic checkpoints to be readjusted before further responses are triggered.
alternatively, other mechanisms different from transcriptional repression could be mediating the down-regulation of np-responsive genes.
interestingly, tio2-nps induced the expression of micrornas in tobacco  <cit> ; these molecules are important repressors of mrna processing that function in plant responses to environmental stress  <cit> .
a significant number of np-responsive genes were involved in the biotic stress response in our study, in agreement with data from other authors that identified differentially-expressed genes upon long-term exposure to some types of nps  <cit> .
we assayed nps of different materials and sizes and look for common patterns in the transcriptional response to np exposure.
nps induced early differential expression of  <dig> genes that, under similar growth and developmental conditions, were also regulated by pathogen challenge , and we show that the genes up-regulated by pathogens were down-regulated by nps .
in particular, down-regulation affected a significant proportion of genes that are key components of the pathogen-detection pathways that activate sar and sa signalling, like frk <dig> .
the repression of the sar response that we observed 2 days upon np exposure has been corroborated in experiments with arabidopsis exposed for 10 days to ag-nps  <cit> .
the long-term repression of these genes would have important consequences for the plant’s capacity to withstand biotic stress under environmental conditions.
in a phenotypic analysis, we showed that exposure to nps and subsequent infection with phytopathogenic bacteria resulted in increased bacterial colonization of the plant that could be prevented by exogenous application of sa .
in agreement, exogenous sa can prevent transcriptional repression of frk <dig> by nps and up-regulates this gene to the levels of infected plants .
the go classification showed that phosphate-starvation genes were significantly over-represented  in the set of np-responsive genes .
genes in this category were the most strongly repressed by treatment with all types of nps and included genes encoding enzymes like pap phosphatases, as well as non-protein-coding genes such as ips <dig> , that is involved in the complex regulation of phosphate-starvation responses through microrna production  <cit> .
consistent with this finding, other non-overlapping genes in the go categories of np-regulated genes were physiologically connected to the phosphate-starvation response.
this was the case not only for the genes related to root hair development but also the over-represented categories of anthocyanin and flavonoid biosynthesis .
anthocyanins accumulate in the roots of phosphate-starved plants and are often used as metabolic markers of phosphate starvation, whereas low nitrogen induces roots to accumulate their precursors, flavonoids  <cit> .
phosphate starvation and other stress signals shape the plant root and can induce root hairs to proliferate to increase nutrient and water absorption.
root hairs are formed by the differentiation of old epidermal cells that originate from the apical meristematic region.
these epidermal cells become either root hair cells or non-hair cells depending on whether they are located over the intercellular space between two underlying cortical cells or over a single cell.
transcriptomic studies have revealed the temporal patterns of gene expression during the development of hair and not-hair cell lines and dissected the complex regulatory network involved in epidermis cell differentiation  <cit> .
these studies define a group of  <dig> hair-cell genes and  <dig> non-hair-cell genes within the “core” of  <dig> root epidermal genes that were regulated in opposite directions in “hairy” and “hair-less” phenotypic mutants.
we observed consistent, np-induced, down-regulation of  <dig> of the  <dig> “core” root epidermal genes in the group of  <dig> differentially-expressed genes, and all were hair-cell genes.
moreover, q <dig> values in the box-plot representations of the  <dig> hair and  <dig> non-hair genes show that down-regulation was specific of hair-cell genes .
our phenotypic observations demonstrated a significant decrease in root hair densities with all the np types assayed .
other works have shown that exposure to nps affects root development, although the transcriptional mechanisms involved were not elucidated.
ultrasmall tio2-nps accumulate in the roots, but not shoots, of arabidopsis seedlings  <cit>  and cause isotropic growth of root epidermal cells and swelling of root tips as soon as 36 h after np addition  <cit> .
this effect has been attributed to the disruption of microtubular networks by nps, thus increasing the 26s proteasome workload to degrade the depolymerised tubulin.
however, our transcriptome analysis did not suggest proteasome alterations.
similar to the increased sensibility of leaves to pst infection, the hairless-like root phenotype induced by nps could be prevented by supplementation with sa but not other hormones like aba or mj .
pre-treatment or “priming” of plants with hormones may improve resistance to future exposure to environmental stress  <cit>  and, under our conditions of np-induced stress, sa seemed to have a priming effect to reduce pathogen proliferation and the root hair-less phenotype.
genes up-regulated by np exposure are included in the go category of cellular response to hypoxia and in the subset of drought-responsive genes .
the first group includes two peroxidase superfamily proteins expressed in root tissues  and cyp82c <dig> , which modulates jasmonate-induced root growth inhibition and defence gene expression  <cit> .
early induction of peroxidase and superoxide-dismutase genes has been reported both in plant protoplasts and in leaves upon injection of cooh-mwcnts  <cit>  and was connected to programmed cell death of protoplasts.
drought stress in our study affected a number of np-induced genes with previously unknown functions as well as the better characterized mybl <dig>  and col <dig>  transcription factors.
mybl <dig> is a proto-oncogene homologue involved in bes <dig> co-repression in the brassinosteroid signalling pathway  <cit>  and was more recently identified as a dehydration stress memory gene regulated by multiple signalling pathways, including the aba, jasmonic acid, and sa pathways  <cit> .
col <dig> is a member of the constans-like family of flowering regulators involved in the response to water deprivation  <cit> .
interestingly, pre-treatment with aba, as well as with mj or sa, can decrease the transcriptional induction of these genes by cooh-mwcnts, and in particular, sa can by-pass np-induced activation and regulate their expressions down to the levels of abr infection .
mybl <dig> and col <dig> also respond to abr/pst pathogen challenge and, as members of very complex families of transcription factors, might play important roles in integrating hormonal signalling in the plant response to np-induced stress.
CONCLUSIONS
we evaluated the transcriptome changes of arabidopsis after brief exposure to nps and compared them with those under biotic or abiotic stresses that represent common environmental challenges for plants.
principal component analysis clusters together transcriptomes of plants exposed to nps of different materials and sizes in the nano-scale.
the study of transcriptomic patterns could distinguish the impacts of all tested nps from those of other stressors and defined a set of np-responsive, differentially expressed genes.
np exposure repressed a significant number of genes involved in the response to pathogen challenge and increased bacterial survival during an experimental infection with pst.
the mechanism involved in sar via sa signalling could by-pass np-induced repression and reduce bacterial colonization.
in addition, np-induced repression largely involved genes activated by phosphate-starvation and other conditions that promote root hair development, but supplementation with sa could remediate the resulting root hair-less phenotype.
overall, the basic molecular mechanisms of the early response of arabidopsis to nps were based on transcriptional repression and had a common pattern regardless of the composition of the different nps.

 nanoparticles are materials with at least one dimension in the nanoscale. the high surface-to-volume ratios and unique physicochemical properties of these materials have led to myriad practical applications, resulting in increased production and potential release into the environment. however, the effect of np exposure on biological systems may differ from what is known about exposure to their bulk counterparts. fate and transport studies have demonstrated that disposed nps are present in nature at concentrations that might pose a risk. unicellular algae are widely used to test np acute toxicity in the aquatic environment. at the cellular level, nano-forms of zno tio or ag can differently modify growth rates, cellular viability, and chlorophyll content compared to their corresponding bulk materials. subcellularly, alterations of organelles like chloroplasts and vacuoles and accumulation of nps near the cell wall and plasma membrane have been observed. at the molecular level, nps increase reactive oxygen species formation and lipid peroxidation to a higher extent that bulk materials. however, many environmental pollutants can also induce ros formation through an endogenous mechanism to alert for potential cell damage and increase environmental stress tolerance. physiological studies of higher plants have demonstrated that nps up to 40 nm can be taken up by roots and travel symplastically through the vascular system, whereas larger nps accumulate in the apoplastic space. therefore, some of the damaging effects of nps have been attributed to mechanical damage or clogging of plant structures, like plasmodesmata or stomata, which regulate water flux. in maize tio2- and clay-nps reduced the size of cell-wall pores of roots and inhibited hydraulic conductivity and transpiration. consequently, plant molecular responses to mitigate np-related damage might involve mechanisms to control water-stress, although this possibility has not been tested. research on crop plants has prompted further questions about the impacts of released nps in agricultural production and the implications for the food chain. uptake, translocation, and generational transmission of carbonaceous nanomaterials have been demonstrated in rice. photothermal and photoacoustic methods have revealed the spatial distribution of carbon nanotubes in roots, leaves, and fruits of tomato plants. in that work, the authors assayed the addition of four carbon-based materials to the plant growth medium and observed physiological responses for the single and multiwall cnts only. mwcnts over swcnts produced a maximum effect on biomass accumulation, thereof plants fed with mwcnts were chosen for further examination by photothermal/photoacoustic scanning cytometry or microarray analysis. imaging techniques detected mwcnts in roots but also in the leaves and fruits of the plants exposed to nps, and data were integrated in parallel with tomato microarray analysis to discover that mwcnts induced changes in gene expression of stress- related genes. a number of differentially-expressed transcripts were identified that corresponded to genes with known function, some of which are involved in plant responses to pathogen infection. this observation suggested that plants sense the penetration of nano-sized materials into their tissues as a biotic stress factor similar to pathogen or herbivore attack. gene expression analyses of the model plant arabidopsis thaliana have provided new insights into the molecular mechanisms of plant response to nps. these two studies both reported gene expression changes upon long-term exposure to tio2-nps, zno-nps, ag-nps, and fullerene of different sizes. there were important differences among np doses, germination conditions, and plant developmental stages during exposure. landa et al. concluded that zno-nps caused the most dramatic changes in gene expression, resulting in the up- and down-regulation of and genes, respectively, whereas tio2-nps only affected and genes, respectively, indicating minimal toxicity. zno-nps and fullerenes up-regulated genes involved in functional responses to abiotic and biotic stresses, whereas tio2-np exposure differentially regulated genes involved in both stresses, although data about the significance of these functional representation were not provided. in the study by kaveh et al., exposure to ag-nps was associated with the down regulation of genes involved in pathogen response, but a significant overlap was observed with the genes responding to bulk material. importantly, some of the genes regulating hormonal stimuli and stress response were also identified as np-responsive and connected with systemic acquired response, an enhanced immunity in tissues remote from the initial infection site. sar is triggered upon challenge by certain pathogens or, to some extent, by mechanical damage, i.e., as a result of wounding by herbivore insects. sar results in thickening of the cell wall and other physiological responses that enhance general plant defences in a non-specific way. here we produced a set of comparable transcriptome profiles to monitor early changes in gene expression upon np and stress exposure. we evaluated a. thaliana response to different types and sizes of nps in comparison to biotic and abiotic stress inducers representing the most common environmental challenges for plants. biotic stress was induced by infection with a necrotizing fungus or a hemibiotrophic bacterium. these pathogens trigger plant defence and immune responses through mechanisms involving respectively, phytohormones like salicylic acid, a key modulator of sar, and jasmonic acid/ethylene pathways. abiotic stresses induced by hyper-saline conditions, drought, and mechanical wounding were also assayed. the effect of abscisic acid, the most studied stress-responsive phytohormone, which mediates stomatal closure and other responses to drought and osmotic stress, on the gene expressions and phenotypes of np-exposed plants was also tested.  exposure of plants to pathogens, abiotic stress and nps since the range of the transcriptional responses to stress depends on the growth conditions and developmental stage of the plant, synchronized hydroponic cultures were used for all exposures. plantlets were grown in liquid ms medium until rosette leaves emerged and begun aerial development. at this stage leaves can be inoculated with microbial pathogens as in the infection models used before to study immune response and roots are well developed to allow np or salt uptake. plants were infected with a. brassicola or p. syringae to study the transcriptional response to biotic stress in of the conditions that were assayed. infected plants developed chlorotic leaves or other macroscopic signals of a plant hyper-sensitive immune response by the second day post infection, indicating that mechanisms of defence against biotic stress were already activated. similarly, physiological responses to saline stress were observed 2 days upon addition of 100 mm nacl to the medium. drought and wounding stress were induced as described in the methods section to assay a total of conditions for abiotic stress that have been connected before with np-induced damage. nps were added to the cultures at the same time that the other stressors and incubated with the plants for 2 days before collection. we performed preliminary dose- and size-dependent measurements of the effects of nps on plant biomass under these particular growth conditions. these experiments narrowed the range of concentrations at which nps had measurable effects on plant growth to –25 μg/ml, similar to previous studies. we assayed nps made of different materials, ag, tio and carbonaceous materials, and diameters ranging from to 80 nm for ag-nps, to 40 nm for tio2-nps and 4–12 nm for cooh-mwcnts. this made a total of different conditions for np-exposed plants. bulk materials that release equivalent metallic ions were added to additional series of plants and one more condition was assayed in which plants were supplemented with aba 12 h. before addition of cooh-mwcnts. combined exposure to nps and aba was performed to determine whether np-induced effects at the transcriptomic level could be prevented or reverted with aba treatment. this hormone activates physiological responses to drought, salt, and other abiotic stresses through a complex network of transcriptional regulators. exogenous addition of aba can mimic the endogenous accumulation of the hormone that triggers stress responses in the tissues and increases plant tolerance to saline stress. transcriptome analyses of exposed plants we examined the changes that occurred in the transcription of genes represented in the at v microarray. hybridization signals from the cdna of plants exposed to the conditions described above were normalised relative to the values of non-exposed plants. changes in these normalised gene expression values were further analysed. principal component analysis was performed to detect major trends in the expression data. the first and second components of the analysis, which captured respectively % and % of the variation, were plotted to visualize similarities among samples. the scatter-plot shows that all np exposures clustered together, independently of material or size in the nano scale. the biotic stresses induced upon pathogen challenge were discriminated from other conditions to the right side of the plot. the abiotic stressors graphed closer to the np exposure samples. thus, plants exposed to nps show similar transcriptional behaviours, and pathogen challenge, saline or drought stress -in this order- cause major changes in the transcriptome when compared to np exposure. in support of this, box-plot representation of normalised expression ratios shows that the ranges of expression changes under these stress conditions are spanned. for example, 90% of the genes in the pst-infected plants had expression ratios between − and, whereas this range was only − to in samples exposed to nps. pre-treatment with aba in cooh-mwcnt-exposed plants did not decrease the range of expression changes to that of non-exposed plants, suggesting that the addition of this hormone had side effects on the plant transcriptome.figure principal component analysis and distribution of normalised expression ratios for the total of genes analyzed. pca was performed to reduce the dimensions of expression data to components. components to explained respectively %, %, % and % of variation in the data. the first and second components were used as the x and y axes to plot values from the conditions in this study. ag ag ag ag and agn represent the addition of ag-nps of 10-, 20-, 40- and 80-nm diameter or agno bulk material, respectively. ti ti ti and tio the addition of tio2-nps of 10-, 20-, 40-nm diameter and tio bulk material. cnt and cnt+ indicate cooh-mwcnts added to non-supplemented or aba-supplemented plants. box plot of the expression ratios for the genes analysed with the atv microarrays. boxes represent 10th and 90th percentiles, with the median in the middle line of the box. minimum and maximum values are shown by the end of the vertical lines and upper or lower outliers by red diamonds. identification and functional classification of genes with significant transcriptional responses to nps microarray data was statistically analysed to identify differentially-expressed genes that responded to all np treatments, irrespectively of particle size or type. the analysis recovered a set of genes with significant changes in expression. quartile representation of the expression changes for this set showed that most were repressed in np-treated samples, and median expression ratios were low compared with those of non-exposed plants.figure quartile distribution of the expression ratios for the np-responsive genes. boxes represent q and q quartiles and the middle line of the box is the median. whiskers show the minimum and maximum values and red diamonds outliers. to detect genes that were empirically responding to biotic or abiotic stress, we performed independent analyses of abr, pst, nac, drou, and wou samples to define five sets of biotic- or abiotic-stress-responsive genes under our experimental conditions. these sets were overlapped with the group of np-responsive genes to produce four subsets of,,, and differentially-expressed genes, with broad overlap between the abr- and pst-responsive subsets. thus, most of the genes with significant responses to nps were also regulated upon pathogen challenge in our hydroponic growth model. in contrast, none of the significant wounding-responsive genes were included in the 351-gene set.figure distribution of the expression ratios for the subsets of stress-responsive genes within the 351-gene set. venn diagrams represent the subsets of,, and stress-responsive genes that were included in the group of np-responsive genes, with the number of overlapping genes in each intersection. distribution of the expression ratios within each subset. boxes and middle line represent q1-q quartiles and the median of the distribution. whiskers show the minimum and maximum values whereas red diamonds show outliers. functional in silico classification of the genes was achieved via gene ontology analysis. a total of go terms were significantly enriched in the three supercategories of biological process, cellular component, and molecular function. go terms in biological process are shown in figure  the most represented term in the 351-gene set is response to stress, which included genes from the set and was significantly enriched with respect to the total of genes. additional analyses of some of these functional classes are presented in the following sections. in cellular component, a significant percentage of the genes corresponded to extracellular proteins, whereas none of the other sub-cellular compartments were over-represented. the most enriched category within molecular function was alkaline phosphatases.figure go enrichment analysis of the 351-gene set. the percentages of genes that are associated to a go-term are shown in the left bars for the set of differentially expressed genes and for the total set of genes enrichment scores are represented by the bar graph in the right. the number of genes associated to each go term is shown in the right column. only go-terms in the category of biological process are shown. some descendant terms that contain the same genes have been merged and represented as a single category bar for simplicity. transcriptional responses to nps include repression of pathogen-activated genes and salicylic acid-mediated pathways abr- and pst-responsive genes in the 141- and 114-gene subset were generally repressed in all np treatments. we focused on genes previously associated with stress responses by the go annotation and for which more information was available. hierarchical clustering grouped them into two main branches which roughly corresponded to genes that were up- or down-regulated by one or both biotic stresses. most of the genes in both branches were repressed in all np-treated samples and only genes responded to np treatments with log ratios above although overall these genes were up-regulated more by pathogen challenge than by np exposure.figure clustering of the stress-annotated, pathogen-responsive genes in the 351-gene-set. hierarchical clustering was represented for the gene-entities using euclidean distances and following ward’s linkage rule. branch a includes genes that are activated upon pathogen challenge and repressed in np-exposed plants. this branch contains most sar-responsive genes. branch a corresponds to genes activated by pathogen challenge and also, but to a lesser extent, by np exposure. branch b is formed by the genes repressed both by pathogen and np-exposure. most pathogen-responsive genes that were activated during our abr/pst challenges are involved in early defence signalling and represent main effectors of the plant immune response that triggers sar upon pathogen infection. such was the case with frk, an inducible kinase in the pathway that activates the basal immune response upon perception bacterial flagellin, and other genes in the enriched go category of sar via sa signalling. these genes were all significantly repressed by np exposure. these data suggest that repression of pathogen-induced genes and sa response is a common feature of np exposure. exposure to nps represses expression of phosphate-starvation and root-development genes the most regulated genes in the set of differentially-expressed genes were related to the phosphate starvation response. the enriched go subset included genes that are also present in the more general go categories of cellular response to starvation, cellular response to nutrient levels, and cellular response to extracellular stimulus. purple-acid phosphatases and other genes in the set that are induced by phosphate starvation were strongly repressed in all np-treated samples, as confirmed by rt-qpcr.figure repression of the phosphate-starvation genes upon np exposure and overlapping go categories. the np-responsive genes that are included in the enriched go category of phosphate starvation response are shown clustered. overlapping of the enriched categories that contain most phosphate-starvation genes box-plot representation of q q and median expression ratios of the phosphate-starvation genes; whiskers show minimum and maximum values and red diamonds outliers. transcriptional responses to phosphate starvation have been widely studied and result in important changes to root morphology by promoting the inhibition of primary root growth and the formation of lateral roots, as well as the proliferation of root hairs. all these changes increase the capacity of plants to absorb available phosphate from the soil and constitute a local response to external phosphate starvation. sensing of low phosphate availability also triggers long distance signals to systemic tissues that activate phosphate homeostasis mechanisms via increased phosphate transport, recovery, and recycling by modulating the expressions of high-affinity transporters, secreted phosphatases, and phospholipid catabolism enzymes. in np-exposed plants, we observed repression of phosphate homeostatic enzymes as well as genes directly involved in root hair development from epidermal trichoblasts. root-hair-specific genes and other genes with roles in root hair emergence and differentiation constituted the enriched subsets of trichoblast/epidermal cell differentiation and root morphogenesis genes in the 351-gene set. unlike the phosphate-starvation genes, root hair genes were repressed, rather than activated, during abr/pst stress. because many of them encode cell-wall modification enzymes that shape the trichoblast, differential expression of this group likely involves structural changes in the roots of plants exposed to nps, which is not necessary implicit in the transcriptional regulation of phosphate homeostatic enzymes.figure repression of the root morphogenesis genes upon np-exposure. the np-responsive genes included in the enriched go categories of trichoblast differentiation/root morphogenesis are shown. box-plot representation of the q q and median expression values for the set. exposure to nps increases bacterial survival in local and distal leaves of plants infected with pst we next tested some predictions of the transcriptional reprogramming observed upon np exposure on the phenotypic response of the plant to biotic stress. a significant number of np-repressed genes are activated upon pathogen infection and are involved in sar, therefore exposure to nps before pathogen challenge might compromise a plant’s ability to respond to infection. however, if this effect were modulated by sa, exogenous supplementation with the hormone might compensate for it and prevent the np-induced phenotype. we measured the spread of the hemibiotrophic pathogen pst in the leaves of np-exposed plants. rosette leaves were inoculated with the bacterium 1 day after addition of nps to the ms growth medium and compared with np-unexposed control plants. another series of plants were treated with sa before the addition of nps. bacterial growth in local and distal leaves was determined 2 days after inoculation. the np exposure was associated with increased bacterial growth, both in local and distal leaves of infected plants, compared with the control. control plants experienced only about half the level of infection in np-exposed plants, consistent with an intact transcriptional mechanism that can activate pathogen-resistance genes. exogenous supplementation of sa prevented the bacterial growth increases associated with np treatment in the distal leaves, where systemic signalling by sa enhances resistance to infection.figure bacterial growth upon pst infection of np-exposed and non-exposed plants. mayor rossete leaves of plants were inoculated with a suspension containing colony forming units of the pst strain or with a mock solution 24 h after exposure to nps.cfu were counted in inoculated and distal leaves 2 days after inoculation. the effect of supplementation with sa 12 h before np addition was measured in another series of plants. bacterial growth in distal leaves of np-treated plants was significantly increased respect to the control plants. exposure to nps inhibits root hair development transcriptional repression of the root development genes predicted an altered root phenotype in plants exposed to nps. absorption of nps in the root cap and accumulation in the columella has been shown in a. thaliana plants that were germinated and fed with high concentrations of 40 nm agnps. similarly, we observed the accumulation of cooh-mwcnts around the root caps of plants growing under our hydroponic conditions. shortening of the root tips and other alterations to root morphology have been described in a. thaliana and other plants exposed to some types of nps. however our transcriptome analysis pointed to a specific effect of nps on the epidermal cells that originate root hairs and indicated that rhs genes were repressed by all types of nps tested in our experiments. root hairs substantially increase the root surface area in contact with the soil, and most of the water and nutrients that enter the plant are absorbed through them. thus, their development is significantly affected by environment stimuli and stress signals.figure inhibition of root hair production upon np exposure and effect of sa pre-treatment. accumulation of nps in the root cap. microscope photographs of the roots of plants exposed to cooh-mwcnts 4 days upon addition of the nps to the growth medium: black aggregates appear around the root cap. microphotographs of the lateral roots of plants exposed to ag-nps; np-exposed roots show a “hairless” phenotype.. number of root hairs per cm of root in plants exposed to ag-nps, tio2-nps, and cooh-mwcnts, compared to control plants. a significant decrease is observed in all the plants treated with nps. effect of the pre-treatment with aba, methyl jasmonate and sa phytohormones before exposure to 20 nm ag-nps; only the addition of sa can prevent the decrease in the number of root hairs/cm observed upon exposure to nps. pre-treatment with sa prevents the reduction in root hair density upon treatment with tio2-nps and cooh-mwcnts. we performed detailed microscopic observations to quantify the effects of nps on root morphology under our plant growth conditions. superficially, np-treated roots resembled some “hair-less” mutants. quantification confirmed that np-exposure significantly decreased root hair density regardless of the size or type of np. because hormonal balance regulates root development, and aba has been shown to be locally involved in the readjustment of root morphology under abiotic stress conditions, we tested the effects of exogenous supplementation with different hormones ) before the addition of nps. supplementation with sa prevented the reduction in root hair density caused by np exposure. further assays with tio nps and cooh-mwcnts confirmed that supplementation with sa could rescue the phenotypic effects of all types of nps on root hair development. discussion nanoparticle exposure had less of an impact on the plant transcriptome than the other stress conditions assayed in this work. as measured by genome expression changes, biotic stress represented the major challenge for these plants, whereas saline or drought stresses have lesser effects, although still greater than that of np exposure. the plant–pathogen system or abiotic stresses used here may represent harsher environmental conditions than np exposure. however none of the stresses assayed here posed severe challenges to plant survival, because our seedlings completed their vegetative life cycles, bolted, and eventually flowered 1– weeks after exposure. exposure to nps under these conditions induced significant changes in gene expression as well as measurable phenotypic differences, so their effects at these doses cannot be considered negligible. therefore, compared with other environmental challenges, a. thaliana has the genetic resources to withstand np-induced stress, although to a great extent the response will depend on the concentrations and activities of the nanomaterials in the plant environment. more interestingly, in our analysis, all tested types and sizes of nanoparticles plotted in the same area in the pca, supporting the idea of common regulatory mechanisms in the arabidopsis response to nps that could be exploited to prevent negative effects on plant development. down-regulation of gene expression was an overall response to nps. the q values for the differentially-expressed genes were below zero in all the np-treated samples. overall, this down-regulation affected all the gene subsets in the go categories represented in the figure  except for cellular response to hypoxia. the subsets of and genes that responded to biotic or saline stress were also down-regulated in the np samples, and only the drought-responsive genes responded to nps with increased expressions. transcriptional repression is suspected to be an important mechanism to keep stress responses under tight control; early response to nps might involve preventive repression of gene expression to allow genetic checkpoints to be readjusted before further responses are triggered. alternatively, other mechanisms different from transcriptional repression could be mediating the down-regulation of np-responsive genes. interestingly, tio2-nps induced the expression of micrornas in tobacco; these molecules are important repressors of mrna processing that function in plant responses to environmental stress. a significant number of np-responsive genes were involved in the biotic stress response in our study, in agreement with data from other authors that identified differentially-expressed genes upon long-term exposure to some types of nps. we assayed nps of different materials and sizes and look for common patterns in the transcriptional response to np exposure. nps induced early differential expression of genes that, under similar growth and developmental conditions, were also regulated by pathogen challenge, and we show that the genes up-regulated by pathogens were down-regulated by nps. in particular, down-regulation affected a significant proportion of genes that are key components of the pathogen-detection pathways that activate sar and sa signalling, like frk. the repression of the sar response that we observed 2 days upon np exposure has been corroborated in experiments with arabidopsis exposed for 10 days to ag-nps. the long-term repression of these genes would have important consequences for the plant’s capacity to withstand biotic stress under environmental conditions. in a phenotypic analysis, we showed that exposure to nps and subsequent infection with phytopathogenic bacteria resulted in increased bacterial colonization of the plant that could be prevented by exogenous application of sa. in agreement, exogenous sa can prevent transcriptional repression of frk by nps and up-regulates this gene to the levels of infected plants. the go classification showed that phosphate-starvation genes were significantly over-represented in the set of np-responsive genes. genes in this category were the most strongly repressed by treatment with all types of nps and included genes encoding enzymes like pap phosphatases, as well as non-protein-coding genes such as ips, that is involved in the complex regulation of phosphate-starvation responses through microrna production. consistent with this finding, other non-overlapping genes in the go categories of np-regulated genes were physiologically connected to the phosphate-starvation response. this was the case not only for the genes related to root hair development but also the over-represented categories of anthocyanin and flavonoid biosynthesis. anthocyanins accumulate in the roots of phosphate-starved plants and are often used as metabolic markers of phosphate starvation, whereas low nitrogen induces roots to accumulate their precursors, flavonoids. phosphate starvation and other stress signals shape the plant root and can induce root hairs to proliferate to increase nutrient and water absorption. root hairs are formed by the differentiation of old epidermal cells that originate from the apical meristematic region. these epidermal cells become either root hair cells or non-hair cells depending on whether they are located over the intercellular space between two underlying cortical cells or over a single cell. transcriptomic studies have revealed the temporal patterns of gene expression during the development of hair and not-hair cell lines and dissected the complex regulatory network involved in epidermis cell differentiation. these studies define a group of hair-cell genes and non-hair-cell genes within the “core” of root epidermal genes that were regulated in opposite directions in “hairy” and “hair-less” phenotypic mutants. we observed consistent, np-induced, down-regulation of of the “core” root epidermal genes in the group of differentially-expressed genes, and all were hair-cell genes. moreover, q values in the box-plot representations of the hair and non-hair genes show that down-regulation was specific of hair-cell genes. our phenotypic observations demonstrated a significant decrease in root hair densities with all the np types assayed. other works have shown that exposure to nps affects root development, although the transcriptional mechanisms involved were not elucidated. ultrasmall tio2-nps accumulate in the roots, but not shoots, of arabidopsis seedlings and cause isotropic growth of root epidermal cells and swelling of root tips as soon as 36 h after np addition. this effect has been attributed to the disruption of microtubular networks by nps, thus increasing the 26s proteasome workload to degrade the depolymerised tubulin. however, our transcriptome analysis did not suggest proteasome alterations. similar to the increased sensibility of leaves to pst infection, the hairless-like root phenotype induced by nps could be prevented by supplementation with sa but not other hormones like aba or mj. pre-treatment or “priming” of plants with hormones may improve resistance to future exposure to environmental stress and, under our conditions of np-induced stress, sa seemed to have a priming effect to reduce pathogen proliferation and the root hair-less phenotype. genes up-regulated by np exposure are included in the go category of cellular response to hypoxia and in the subset of drought-responsive genes. the first group includes two peroxidase superfamily proteins expressed in root tissues and cyp82c, which modulates jasmonate-induced root growth inhibition and defence gene expression. early induction of peroxidase and superoxide-dismutase genes has been reported both in plant protoplasts and in leaves upon injection of cooh-mwcnts and was connected to programmed cell death of protoplasts. drought stress in our study affected a number of np-induced genes with previously unknown functions as well as the better characterized mybl and col transcription factors. mybl is a proto-oncogene homologue involved in bes co-repression in the brassinosteroid signalling pathway and was more recently identified as a dehydration stress memory gene regulated by multiple signalling pathways, including the aba, jasmonic acid, and sa pathways. col is a member of the constans-like family of flowering regulators involved in the response to water deprivation. interestingly, pre-treatment with aba, as well as with mj or sa, can decrease the transcriptional induction of these genes by cooh-mwcnts, and in particular, sa can by-pass np-induced activation and regulate their expressions down to the levels of abr infection. mybl and col also respond to abr/pst pathogen challenge and, as members of very complex families of transcription factors, might play important roles in integrating hormonal signalling in the plant response to np-induced stress.  we evaluated the transcriptome changes of arabidopsis after brief exposure to nps and compared them with those under biotic or abiotic stresses that represent common environmental challenges for plants. principal component analysis clusters together transcriptomes of plants exposed to nps of different materials and sizes in the nano-scale. the study of transcriptomic patterns could distinguish the impacts of all tested nps from those of other stressors and defined a set of np-responsive, differentially expressed genes. np exposure repressed a significant number of genes involved in the response to pathogen challenge and increased bacterial survival during an experimental infection with pst. the mechanism involved in sar via sa signalling could by-pass np-induced repression and reduce bacterial colonization. in addition, np-induced repression largely involved genes activated by phosphate-starvation and other conditions that promote root hair development, but supplementation with sa could remediate the resulting root hair-less phenotype. overall, the basic molecular mechanisms of the early response of arabidopsis to nps were based on transcriptional repression and had a common pattern regardless of the composition of the different nps. 
nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. the exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria. this study integrates the effect of nanoparticles on gene expression with plant responses to major sources of environmental stress and paves the way to remediate the impact of these potentially damaging compounds through hormonal priming. inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. arabidopsis plants were exposed to tio2-nanoparticles, ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic or abiotic stresses. however, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed. changes in gene expression profiles and plant phenotypic responses were evaluated. transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers.
 the impact of nano-scaled materials on photosynthetic organisms needs to be evaluated. plants represent the largest interface between the environment and biosphere, so understanding how nanoparticles affect them is especially relevant for environmental assessments. nanotoxicology studies in plants allude to quantum size effects and other properties specific of the nano-stage to explain increased toxicity respect to bulk compounds. however, gene expression profiles after exposure to nanoparticles and other sources of environmental stress have not been compared and the impact on plant defence has not been analysed.  arabidopsis plants were exposed to tio2-nanoparticles, ag-nanoparticles, and multi-walled carbon nanotubes as well as different sources of biotic or abiotic stresses. changes in gene expression profiles and plant phenotypic responses were evaluated. transcriptome analysis shows similarity of expression patterns for all plants exposed to nanoparticles and a low impact on gene expression compared to other stress inducers. nanoparticle exposure repressed transcriptional responses to microbial pathogens, resulting in increased bacterial colonization during an experimental infection. inhibition of root hair development and transcriptional patterns characteristic of phosphate starvation response were also observed. the exogenous addition of salicylic acid prevented some nano-specific transcriptional and phenotypic effects, including the reduction in root hair formation and the colonization of distal leaves by bacteria.  this study integrates the effect of nanoparticles on gene expression with plant responses to major sources of environmental stress and paves the way to remediate the impact of these potentially damaging compounds through hormonal priming. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords nanoparticlesnanotoxycologyarabidopsisdefencetranscriptomestresssystemic acquired responseissue-copyright-statement© the author 2015 