BACKGROUND
inorganic phosphate  is an essential macronutrient for plant growth and development.
despite its high abundance in the rhizosphere, bioavailability of pi is typically limited because its majority is bound in organic compounds or complexed with metal ions such as ca , fe or al   <cit> .
thus, plants evolved strategies to enhance pi acquisition and to conserve or remobilize pi from internal sources to adapt to pi limiting conditions.
previous efforts elucidated some of these adaptive responses, including the identification of high-affinity pi transport systems, the characterization of diverse metabolic bypass reactions, the reutilization of pi from phospholipids, and many more  <cit> .
most of the pi starvation response  genes involved in these systemic adjustments are regulated by the myb transcription factor phr <dig>  .
dynamic redesign of the root system architecture  provides another strategy to maintain cellular pi supply.
in arabidopsis, low external pi availability is locally sensed by the growing root tip, which causes reduction of cell elongation and meristematic activity at the site of pi depletion.
the resultant inhibition of root growth is accompanied by accelerated formation of root hairs and development of lateral roots to increase the absorptive surface for topsoil exploration  <cit> .
the development of a densely branched and/or shallow root systems increases pi starvation tolerance in several plant species, including agronomically important crops such as barley, lupin, soybean or common bean  <cit> .
several arabidopsis mutants with altered pi dependent root growth responses have been described .
however, for most of the underlying genes only little information is available how they affect pi sensing and root growth modulation.
lpr <dig> , its closely related paralog lpr <dig>  and pdr <dig>  have been identified as central players in local root pi sensing  <cit> .
pdr <dig>  which codes for the single p5-type atpase of unknown substrate-specificity , and lpr <dig>  encoding a multicopper oxidase, are expressed in overlapping domains of the root apical meristem .
lpr <dig> and pdr <dig> interact genetically and are required for meristem maintenance and cell elongation in pi-deprived roots.
importantly, the lpr1lpr <dig> mutation impedes local root growth inhibition under pi limitation and suppresses the hypersensitive short-root phenotype of pdr <dig> plants, indicating that they act in the same pathway  <cit> .
previous work revealed that external fe availability modifies local pi sensing  <cit> .
a number of studies observed that pi-starved arabidopsis and rice plants accumulate elevated levels of fe in the root and the shoot , which has been suggested as a proactive strategy to mobilize pi from insoluble fe complexes  <cit> .
fe participates in the formation of reactive oxygen species  and it has been proposed that fe toxicity causes local root growth inhibition  <cit> .
we recently provided evidence for apoplastic lpr <dig> ferroxidase activity and uncovered a major role of the lpr1-pdr <dig> module for root tip-specific deposition of fe3+ in cell walls  of the ram and elongation zone  during pi limitation  <cit> .
we further showed that fe accumulation in the ram is massively enhanced in pi-starved pdr <dig> roots, but suppressed in the insensitive lpr1lpr <dig> line.
fe deposition coincides with sites of ros generation and triggers cw thickening and callose accumulation, which interferes with cell-to-cell communication, ram maintenance, and cell elongation.
in recent years, a set of transcriptome profiling studies provided significant insights into the transcriptional changes upon pi deficiency in arabidopsis .
in addition, a complementary transcriptome and proteome study highlighted the convergence of mrna and protein expression profiles on lipid remodeling and glucose metabolism upon pi-deprivation  <cit> .
in this study, we performed comparative transcriptome and proteome expression profiling on roots of pi-replete and pi-starved wild-type , pdr <dig>  and lpr1lpr <dig> plants in combination with a set of physiological and cell biological experiments.
our analysis emphasizes the importance of root fe uptake and redistribution under pi limitation.
we highlight the potential role of so far unknown players in the regulation of pi-dependent fe-redistribution and demonstrate that apoplastic but not intracellular fe accumulation triggers pi-dependent root growth modulation.
consistently, we observed regulation of several cw modifying enzymes, which correlates with an increased deposition of pectin at sites of fe accumulation.
the potential role of pectin in pi-dependent root fe storage and pi mobilization is discussed.
RESULTS
differential gene expression correlates with genotype-specific pi sensitivity
for transcriptome analysis, wild-type, pdr <dig> and lpr1lpr <dig> seedlings were germinated on + pi agar  and transferred to + pi or –pi medium for 20 h, a period during which pi limitation alters global gene expression  <cit>  as well as root meristem activity  <cit> .
rna was extracted from roots of three biological replicates and prepared for hybridization with ath <dig> affymetrix chips.
data were analyzed using arraystar  and further processed .
hierarchical clustering  confirmed high homogeneity within each replicate set because the biological replicates clustered together for each genotype and pi condition .
it also revealed a clear separation between + pi and –pi samples for the wild-type and the hypersensitive pdr <dig> mutant , but less pronounced differences for the insensitive lpr1lpr <dig> line .
pairwise comparisons using a fold-change cutoff value of ≥  <dig>  for increased and of ≤ <dig>  for decreased transcript levels  revealed  <dig> differentially expressed genes across all genotypes and the two growth conditions.
low pi exposure altered the expression of  <dig>   <dig>  and  <dig> genes in pdr <dig>  wild-type, and lpr1lpr <dig> roots, respectively .
thus, the genotype-specific sensitivity of root growth inhibition in response to pi depletion positively correlates with the number of differentially regulated genes.fig.
<dig> statistical analysis of comparative gene expression analysis.
a hierarchical clustering of all ath <dig> datasets, including wild-type , pdr <dig> and lpr1lpr <dig> samples, the two growth regimes , and three biological replicates.
distances in the dendrogram illustrate the degree of relationship between samples.
note the short distance between biological replicate sets  compared to the relatively long distance between + pi and –pi conditions.
b number of up- and downregulated genes  in wild-type, pdr <dig> and lpr1lpr <dig> roots upon transfer from + pi to –pi , and in the three genotypes under + pi  or –pi  conditions.
c venn diagrams illustrating the number of differentially regulated genes in pairwise comparisons for all three genotypes under both growth regimes
identification of genotype-independent pi-responsive genes
we generated venn diagrams to illustrate the distribution of differentially expressed genes between the three genotypes .
wild-type shared a subset of  <dig> and  <dig> pi-responsive genes with pdr <dig> and lpr1lpr <dig>  respectively, and all three lines had in common a core set of  <dig> genes .
hierarchical clustering of this core set revealed similar expression changes in all genotypes in response to –pi with high positive correlation .
the core set comprises two partially overlapping groups that consist of at least  <dig> psr and  <dig> metal-responsive genes .
members of the first group  are known targets of the pi-regulated myb transcription factor phr <dig> , suggesting that the systemic response to pi deficiency is maintained in pdr <dig> and lpr1lpr <dig> mutants.table  <dig> pi-dependent transcriptional changes of commonly regulated genes
shown is the fold change expression  of all  <dig> pi-responsive genes that are regulated in each of the tested genotypes .
grey and white boxes denote genes that are significantly suppressed or induced, respectively .
all genes were interrogated for published responsiveness to pi-starvation and/or metal-ions.
references are indicated in superscript numbers and listed in additional file 3: table s2
in the second group, fe-related genes are overrepresented  and comprise the majority of repressed genes .
the most strongly suppressed gene in all three genotypes  codes for irt <dig>  the major feedback-regulated fe-uptake system in arabidopsis  <cit> .
many irt <dig> co-regulated genes  are induced under fe deficiency .
interestingly,  <dig> of the top  <dig> co-regulated genes are repressed in pi-starved roots irrespective of the genotype .
intriguingly, pi-replete pdr <dig> roots show higher expression of at least  <dig> fe-related genes , including a group of transcription factors  known to promote fe-uptake under fe deficiency .
the remaining fe-related genes of this group are similarly induced in all three genotypes and encode the fe storage protein ferritin <dig>  and various fe-responsive metal transporters thought to be involved in transition metal detoxification and homeostasis .table  <dig> pi-dependent regulation of the top  <dig> genes co-regulated with irt <dig>
shown is the fold change expression in wild type, pdr <dig> and lpr1lpr <dig> after transfer to –pi or the fold change expression of pi-replete pdr <dig> and lpr1lpr <dig> plants compared to the wild-type.
red and green boxes denote genes that are significantly suppressed or induced
pi depletion alters expression of cell wall-related genes
we identified  <dig> pi-responsive genes that are shared between the wild-type and the hypersensitive pdr <dig> mutant, but not with the insensitive lpr1lpr <dig> line .
surprisingly, only  <dig> genes of unknown functions in pi starvation response were significantly deregulated in pdr <dig> compared with the wild-type , whereas the remaining genes showed a high positive correlation  between both genotypes .
go term analysis revealed high overrepresentation of gene products associated with the extracellular region .
an extended analysis for enriched go terms within a group of  <dig> genes , which are either regulated by –pi in one or more genotypes or are differentially expressed in at least one of the lines in + pi , confirmed overrepresentation of genes  annotated to encode extracellular proteins .
in this group, we identified a subset of  <dig> genes with putative functions in cw remodeling .
a similar number of genes were differentially expressed in pdr <dig>  and wild-type  but only one-third  in lpr1lpr <dig> roots.
as noted for fe-related genes, many cw-modifying genes  were deregulated in pi-replete pdr <dig> roots.
within the subset of  <dig> genes,  <dig> encoded proteins could be assigned a potential function in pectin modification, predominantly pectin methylesterification.
in addition, we noted several expansins and xyloglucan endotransglycosylases  as well as a set of carbohydrate hydrolyzing enzymes.
intriguingly, all these proteins are predicted to regulate cw extensibility  <cit> .table  <dig> pi-dependent regulation of cell wall modifying enzymes
shown is the fold change expression of selected cw modifying enzymes in wild-type, pdr <dig> and lpr1lpr <dig> after transfer to –pi or the fold change expression of pi-replete pdr <dig> and lpr1lpr <dig> plants compared to the wild-type.
candidates were selected from a set of regulated genes annotated to be localized in the extracellular region .
red and green boxes denote significantly suppressed or induced  genes.
pme, pectin methyl esterase; exp, expansin; exl, expansin-like; xth, xyloglucan endotransglucosylase/hydrolyse
go term analysis also revealed overrepresentation of genes encoding tetrapyrrole- and heme-binding proteins  with oxidoreductase activity  .
this group codes for  <dig> peroxidases and most of those  belong to the  <dig> member-family of class iii peroxidases  , which are extracellular enzymes with partly antagonistic functions in ros formation and cw dynamics  <cit> .
while pi-responsive expression of  <dig> ciii prx-encoding genes was similar between wild-type and pdr <dig> roots,  <dig> genes were regulated independently in each line under low pi, and only three ciii prx genes responded significantly to pi limitation in lpr1lpr <dig> plants .
again,  <dig> ciii prx genes were deregulated in pdr <dig> under + pi.
thus, peroxidases may be an important link between ros formation and cw remodeling upon pi starvation.
proteomics supports regulation of pi-responsive genes in pdr <dig> and lpr1lpr <dig> mutants
genotype-specific changes in the root proteome upon pi deficiency were monitored in an unlabeled approach using a fast scanning high resolution accurate mass  lc-ms system.
three biological and three technical replicates were measured for each genotype under + pi and –pi conditions  yielding  <dig> , <dig> ms/ms spectra .
<dig>  spectra could be annotated to a peptide sequence  with a global false discovery rate  threshold of  <dig>  %.
these psms were used to identify  <dig> protein groups , each with at least one unique peptide and a global fdr threshold of 1 % .
protein abundance was inferred based on peptide abundance determined by peptide ion signal peak integration using the progenesis software.
pairwise comparison of all genotypes under both growth regimes revealed  <dig> differentially regulated proteins .
based on this list, we identified  <dig> proteins that were either pi-responsive in at least one genotype or which were already deregulated in one of the mutant lines grown on pi-replete conditions  .
multidimensional scaling  analysis of anova filtered  samples revealed low variance between biological replicates but significant differences between genotypes and pi conditions .
the levels of  <dig> proteins were increased or decreased in the wild-type upon pi depletion .
as expected, the highest number of proteins  were regulated in hypersensitive pdr <dig> mutant, probably reflecting changes in root morphology.
we also identified a high number of pi-responsive proteins  in the insensitive lpr1lpr <dig> line.
of these,  <dig> proteins were unique to lpr1lpr <dig> , indicating that the adjustment of protein expression might contribute to the decreased pi responsiveness.
both mutant lines showed differential regulation of more than  <dig> proteins under pi-replete conditions.
this relatively high value is reminiscent of what we observed in the transcript dataset, supporting the assumption that pdr <dig> and lpr proteins may also regulate pi independent processes.fig.
<dig> statistics of comparative protein expression analysis.
a multidimensional scaling  analysis of all biological replicate samples.
b illustration of the number of up- and downregulated proteins  in wild-type, pdr <dig> and lpr1lpr <dig> roots upon transfer from + pi to –pi , and the number of differentially regulated proteins in pairwise comparisons of the three genotypes under + pi  or –pi  conditions.
c, d, and e venn diagrams illustrating the number of regulated proteins in pairwise comparisons for all three genotypes under both growth regimes
venn diagrams identified a group of  <dig> proteins that were similarly regulated in all lines upon pi depletion .
notably,  <dig> of these proteins were positively correlated with our transcript data, showing induction on both mrna and protein level .
two members of this group were fer <dig> and the pectin modifying enzyme polygalacturonase inhibiting protein <dig>   <cit> , which further indicates that changes in fe distribution and cw modification are associated with the response to low pi.table  <dig> pi-dependent protein/mrna regulation
shown is the fold change expression of the  <dig> proteins  that are pi-responsive in all lines  or the fold change expression of pi-replete pdr <dig> and lpr1lpr <dig> plants compared to the wild-type.
protein expression is compared to transcript changes .
red and green boxes denote genes that are significantly suppressed or induced
correlation of proteome and transcriptome analysis
next, we performed go term analysis to identify groups of proteins involved in genotype-specific pi responsiveness.
most proteins have assigned metabolic functions in wild-type and lpr1lpr <dig>  probably reflecting processes related to pi recycling and mobilization.
strikingly, in + pi condition and upon transfer to –pi, the pdr <dig> line showed a significant regulation of proteins assigned as response to metal ion  and oxidoreductase activity .
a closer examination revealed repression of  <dig> peroxidases in pdr <dig> in + pi and induction of  <dig> peroxidases in –pi condition.
within the group of repressed proteins we identified  <dig> ciii prxs of which  <dig> enzymes were regulated at the transcript level.
only one and six pi-responsive ciii prx were detected in wild-type and lpr1lpr <dig> root extracts, respectively .
to compare the proteome and transcriptome data sets, we plotted all significantly regulated proteins  against their cognate transcript.
for those differentially expressed proteins, the percentage of detected transcripts was  <dig>  % for wild-type ,  <dig>  % for pdr <dig>  and  <dig>  % for lpr1lpr <dig>  roots.
we observed only a low, but highly significant, positive correlation of transcript and protein abundance for all three genotypes  .
we generated a list of significantly altered transcripts, which we compared to the list of significantly altered proteins .
we identified  <dig> cognate genes for wild-type,  <dig> for lp1lpr <dig> and  <dig> for pdr <dig>  the correlation coefficient markedly increased when we plotted these genes against their cognate proteins .fig.
<dig> comparative analysis of transcriptome and proteome data and spcca analysis.
a, b, c, and d correlation between transcript and protein fold-changes upon pi-deficiency.
a for each genotype, all significantly regulated proteins  were plotted against its cognate transcript, if present on the ath <dig> chip.
b, c, and d significantly  regulated protein/mrna pairs were plotted against each other.
scatter plots show  <dig> protein/mrna pairs regulated in wild-type upon pi-deficiency,  <dig> pairs identified for pdr <dig>  and  <dig> pairs identified in lpr1lrp <dig>  e correlation  values for each pairwise comparison.
asterisks indicate significance for each correlation analysis .
f, g canonical variables of the spcca analysis representing a subset of transcripts/proteins which showed maximum correlation with the illustrated patterns that were generated by the spcca algorithm.
we identified the  <dig> genes, including fer <dig> and pgip <dig>  that were co-regulated on mrna and protein level across all genotypes in response to pi depletion .
in wild-type, we noticed induction of ppa <dig> , a candidate for pi mobilization, and pck <dig> , which is involved in metabolic adjustment to pi deprivation  <cit> .
we further identified two hemicellulose modifying enzymes, xth <dig> and xth <dig> , which were slightly decreased in low pi.
interestingly, both enzymes were previously shown to be regulated by siz <dig>  <cit> , a sumo e3-ligase involved in pi dependent root growth remodeling  <cit> .
go term analysis of the  <dig> mrna/protein pairs altered in pdr <dig> revealed an overrepresentation of metabolic processes.
the second most significant term  is consistent with altered metal homeostasis in pdr <dig> plants  <cit> .
for example, we noticed induction of fer <dig> and proteins potentially involved in detoxification of metal ion-induced ros formation, including several glutathione-s-transferases  .
we also identified f6’h <dig> , which is involved in coumarin biosynthesis and fe-mobilization in alkaline soils .
our datasets revealed anti-correlation of f6’h <dig> expression in pdr <dig>  showing elevated protein but decreased transcript levels in –pi and an inverse relation in + pi , which indicates stringent regulation of f6’h <dig> expression in pdr <dig>  in addition, protein level of ccoaomt <dig> , which converts caffeoyl-coa to feruloyl-coa, the substrate of f6’h <dig>  <cit> , was also elevated in pdr <dig> .
thus, coumarin-mediated mobilization of fe may be involved in pi dependent fe accumulation.
integrative spcca analysis supports pi-dependent metal redistribution
we integrated the two –omics approaches to uncover relationships that are supported by both individual datasets.
we performed a supervised penalized canonical correlation analysis , which searches for correlations between a set of transcripts and proteins  <cit> .
the experimental design was integrated into the analysis to allow for biological interpretation of the derived canonical variables.
the experimental factors  were provided as a binary matrix of design vectors that uniquely characterize each sample .
the supervised correlation approach seeks a linear combination of a feature subset from each -omics dataset that correlates maximally with a subset of experimental design factors.
to maximize stringency, only varying transcripts and proteins were considered for spcca.
for transcriptomics, we choose a list of  <dig> anova filtered genes  and for proteomics a list of  <dig> proteins .
our analysis revealed distinct canonical variables , each representing a specific pattern correlating with a subset of proteins and/or transcripts.
the first two cvs revealed structured patterns , while a third cv was disordered and therefore not further examined .
the first cv mainly represented genes/transcripts  that were differentially expressed in pdr <dig> compared to wild-type and lpr1lpr <dig> independently of pi status .
we examined the top 100 g/t in this variable and found several fe-related candidates , such as fe chelate reductase  <dig>   <cit>  and myb <dig>  which is required for growth in fe deficiency  <cit> .
myb <dig> and myb <dig> mediate fe-dependent induction of nicotianamine synthase  <dig>   <cit> , which is also present in this group.
nas proteins synthesize nicotianamine, a fe-chelator essential for fe-remobilization in the root  <cit> .
we further identified a member of the aluminum activated malate transporter  family.
it is of note that almt <dig> is most highly induced in all three genotypes during pi depletion .
the second cv mainly represented g/t that were similarly expressed in pi-replete pdr <dig> and lpr1lpr <dig> roots but slightly differed from the wild-type.
in contrast to the first cv, the majority of these g/t were pi responsive in all genotypes.
as expected, we found several known pi acquisition g/t, including spx <dig>  cax <dig>  the phosphate transporter pt <dig>  and the pi starvation-inducible inorganic pyrophosphatase  <dig> .
many other g/t are implicated in metal homeostasis, e.g., the fe/zn transporters irt <dig> and irt <dig>  the ni transporter ireg <dig>  the zn/cd transporter hma <dig> or the na transporter ysl <dig>  further supporting our observation that metal homeostasis is strictly controlled in all genotypes upon pi starvation.
root growth inhibition in low pi is independent of general fe uptake and cellular storage
we previously reported that lpr1-dependent fe accumulation and distribution in root tips controls ram activity in response to low pi  <cit> .
our comparative transcriptomics and proteomics analysis of entire roots revealed pi-responsive expression of fe-related genes, notably fer <dig> and irt <dig> , which correlated with fe overload in pi-starved roots of the three genotypes under study  <cit>  .
to further investigate the role of fe during the local response of roots to pi availability, we analyzed the impact of fer <dig> and irt <dig> loss-of-function mutants on fe-distribution and root growth inhibition upon pi deprivation.
ferritins are located in plastids and can be visualized by perls/dab fe staining as dot-like structures in root cells of wild-type plants, which are not detectable in fer1-3- <dig> roots lacking fer expression  <cit> .
using semi-thin sections from perls/dab-stained wild-type roots, we observed similar dot-like structures in pi-replete root tips, which strongly increased in number and staining intensity upon transfer to –pi medium.
these punctuate structures are associated with the symplast and are clearly distinctive from apoplastic fe staining .
we next performed root growth assays using the fer1-3- <dig> triple and fer1-2-3- <dig> quadruple mutant.
primary root growth rates of the fer mutants were indistinguishable from the wild-type on both + pi or –pi medium .
thus, ferritins do not affect the local root growth response to –pi.fig.
<dig> root growth in fer and irt <dig> mutant plants and phenotypes of frd <dig> roots.
a 4-days-old seedlings were transferred from + pi to + pi or –pi medium for up to 6 days.
daily increase in root growth was measured and illustrated in segmented boxes within the bar graph .
standard error was calculated from the average total root growth.
b total increase in root length after transfer from + pi to either + pi or –pi medium t-test; p <  <dig>  .
c, d, and e fe staining and root growth assays of wild-type and frd3- <dig> seedlings.
4-days-old plants were transferred from + pi to + pi or –pi medium for up to 6 days.
c perls staining in different root segments 20 h after transfer to + pi or –pi medium.
upper and middle panels show mature root segments.
the lower panels show the ram and early differentiation zone.
scale bar 200 μm.
d fe  and aniline blue  callose staining of root tips and differentiated root segments 6 days after transfer to –pi medium.
scale bar 100 μm.
e root growth of wild-type and frd3- <dig> seedlings within 6 days after transfer to pi-depleted medium.
the bar graph shows the daily increase in root growth, illustrated in segmented boxes.
standard error was calculated from the average total root growth.
*** t-test; p =  <dig> − <dig> .
overview images show the root growth after 3 days and 6 days on –pi medium .
arrows indicate the position of the root tip, directly after transfer to –pi , as well as 3 days and 6 days after transfer.
scale bar 1000 μm.
similarly, we performed perls/dab fe-staining to examine fe distribution in wild type and irt <dig> roots.
compared with pi-replete wild-type seedlings, the irt <dig> mutant showed more intense fe staining on the root surface of the mature root zone , which is in accordance with impaired fe uptake from the rhizosphere.
however, both lines displayed similar fe staining in the ram and ez, which is consistent with predominant irt <dig> expression in the differentiation zone  <cit>  and confirms our previous study  <cit> .
under pi depletion, fe staining increased strongly and comparably in all segments of wild-type and irt <dig> roots, indicating that fe accumulation and distribution in root tips is independent of irt <dig>  we generated homozygous pdr2irt <dig> double and lpr1lpr2irt <dig> triple mutants and monitored primary root growth on + pi and –pi agar.
as expected, the irt <dig> mutation did not affect the pi-dependent root growth response of pdr <dig> and lpr1lpr <dig> plants , indicating irt1-independent fe accumulation in the root tip in response to low pi.
apoplastic fe redistribution modifies pi-dependent root growth adaptation
long distance apoplastic fe transport and distribution in symplastically disconnected tissues are mediated by the citrate exporter ferric redictase defective  <dig>   <cit> .
intriguingly, a previous study reported that frd <dig> plants display a hypersensitive short-root phenotype when grown on –pi medium  <cit> .
to examine a potential role of frd <dig> for mediating pi-dependent fe distribution via fe-citrate complexes, we performed perls fe-staining  on wild-type and frd <dig> roots.
as previously reported , pi-replete frd <dig> roots overaccumulated fe in the vascular tissue .
within 20 h after transfer to –pi, wild-type plants accumulated fe in the outer cell layers, whereas frd <dig> roots showed enhanced fe staining in the vasculature, particularly in differentiated root segments.
importantly, only minor differences were noted in the root tip, where fe accumulation was slightly increased in frd <dig> ; however, extended growth on –pi  progressively increased this difference, finally causing massive overaccumulation of fe within the ez and early differentiation zone of frd <dig> roots .
we previously showed that pi-dependent fe accumulation correlates with callose formation at the sites of fe deposition   <cit> .
after transfer to –pi , callose deposition at sites of fe accumulation and resultant root growth inhibition were similar for wild-type and frd <dig> plants .
however, extended exposure  caused callose overproduction in frd <dig> roots which correlated with an enhanced growth inhibition .
based on our observations, we assumed that mobilization of apoplastic fe-citrate complexes might be involved in the pi dependent modulation of root growth.
to test this, we transferred wild-type plants from + pi conditions to + pi or –pi medium, supplemented with citrate, which was previously shown to restore fe mobilization on frd <dig> mutants  <cit>  and monitored their growth behavior.
indeed, addition of 100–250 μm citrate promotes root growth within the first two days after transfer to –pi.
however, this effect was transient and external supply of citrate eventually suppressed root growth on low pi .
pi deprivation modifies pectins at fe accumulation sites
our comparative expression profiling pointed to a role for pectin-modifying enzymes.
therefore, we studied pi-dependent changes in the pectineous cw by using ruthenium red , an inorganic dye that stains unesterified pectins  <cit> .
roots of wild-type, pdr <dig> and lpr1lpr <dig> showed a similar rr staining pattern on + pi medium.
one day after transfer to –pi, we observed a strong increase in rr staining intensity in wild-type root tips, particularly within the differentiating ez .
compared with wild-type, pdr <dig> seedlings showed a more intense staining in this region while the rr staining in the lpr1lpr <dig> mutant was unaltered.
interestingly, the site of enhanced pectin staining correlated well with the site of low pi induced fe deposition in wild-type and pdr <dig> roots .fig.
<dig> fe and pectin staining of roots after seedling transfer from + pi agar to + pi or –pi medium for up to 2 days.
a perls/dab fe staining  and ruthenium red  staining of non-methylesterified pectins .
shown are overview images of perls/dab and rr stained roots as well as detail images of the elongation/early differentiation zone.
scale bar 100 μm.
b hydroxylamine ferric chloride  staining of methylesterified pectins.
shown are overview images of the root  as well as detail images of the root meristem  and the stem cell niche .
differences in coloration between overview and detail result from the use of different imaging devices.
arrows point to the scn and cortical cell layer in the transition zone, asterisks label the quiescent center cells in the scn.
c details of the perls/dab, rr and hfc staining in pi-depleted cortical cells of col plants and stem cells of pdr <dig> plants.
scale bar 20 μm
we also visualized the distribution of methyl-esterified pectin by using the hydroxylamine ferric chloride  reagent, which specifically reacts with methyl esters of pectin and results in a yellow to red coloration .
only weak staining was evident in roots on + pi  and transfer to –pi did not significantly change the staining pattern in the differentiating ez.
however, higher magnification images revealed increased staining in the ram of wild-type, with the highest intensity in the quiescent center  and the cortical cell layer at the transition zone, which demarcates the border between the ram and ez.
in contrast, pdr <dig> seedlings showed enhanced staining in the ram, particularly within the qc region, but no distinct labeling of the cortical cell layer.
no differences in pectin staining were detected in lpr1lpr <dig> roots after transfer to –pi medium.
high magnification images of rr- and hfc-stained roots revealed simultaneous accumulation of acidic and methyl-esterified pectin in the meristem of the two sensitive lines.
in particular, after transfer to –pi, strong hfc and rr staining was evident in the cortex cell layer of wild-type and in the qc region of pdr <dig> roots , which co-localized with major sites of fe deposition.
discussion
plant adaptation to pi limitation depends on coordinated transcriptional and translational regulation of gene expression .
while comparative transcriptome analysis proved to be a viable approach to distinguish between local and systemic regulation in pi-starved plants  <cit> , only little information is available on the regulation of genes and proteins associated with the pi-dependent adaption of root system architecture.
previous work revealed that pdr <dig> and lpr genes act together in the local response to pi availability by regulating cell type-specific deposition of fe and callose in the root tip  <cit> .
here, we took advantage of the contrasting pi-dependent root phenotype of pdr <dig> and lpr1lpr <dig> plants to investigate the associated changes in steady-state transcript and protein levels in a comparative approach.
genotype independent regulation of several psr genes demonstrated the validity of our experiments and revealed that pdr <dig> and lpr1lpr <dig> mutants are likely not affected in the systemic response to pi limitation .
further analysis of our dataset revealed a number of candidate genes that are possibly involved in the pi-dependent regulation of fe storage and fe redistribution as well as in the modulation of cw dynamics and/or ros formation within the root.
pi depletion modulates root fe distribution
our study revealed genotype-independent repression of numerous fe-responsive and irt1-coregulated genes upon transfer to pi limitation, which likely reflects feedback regulation as a consequence of elevated fe accumulation in pi-starved differentiated roots.
on the other hand, de-repression of fe-related genes in pi-replete pdr <dig> plants may sensitize fe overaccumulation in limiting pi  <cit>  .
fer <dig> and related ferritins are plastid-localized fe storage proteins protecting cells from fe-mediated oxidative stress  <cit> .
using perls/dab fe staining, reyt et al.
<cit>  recently reported dot-like structures in root cells of wild-type plants that likely display ferritin-bound fe because they are absent in fer1-3- <dig> roots  <cit> .
a previous study showed that fer <dig> is induced by phr <dig> in low pi independent of external fe  <cit> , indicating that fer <dig> may play a role in pi-dependent fe distribution.
our comparative analysis revealed induction of fer <dig> expression on mrna and protein level in all three lines .
detection of fe accumulation in dot-like structures supports the notion of intracellular fe storage under pi limitation, possibly as ferritin fe .
importantly, pi-dependent root growth was not affected by loss of ferritins  or loss of irt <dig> in pdr <dig>  and lpr  mutants , indicating that pi dependent root growth modulation is independent of intracellular fe accumulation.
our data are consistent with a recent study reporting indistinguishable primary root growth of fer1-3- <dig> and wild-type plants on high fe  <cit> .
fe mobilization from the rhizosphere is facilitated by chelators such as carboxylates  and coumarins, and apoplastic long distance fe trafficking is mediated by fe-citrate complexes .
frd <dig> exports citrate and the frd <dig> mutant is defective in apoplastic fe translocation, causing fe hyperaccumulation in root stele tissues .
importantly, frd <dig> mutants show a hypersensitive short root phenotype in low pi  <cit>  and we demonstrated fe overaccumulation in pi-deprived frd <dig> roots , which indicates that citrate secretion is required for proper fe-distribution under pi limitation.
interestingly, citrate application transiently promoted frd <dig> root growth in low pi , indicating that the pi-dependent short root phenotype of frd <dig> is likely a consequence of altered fe redistribution in the growing root.
transcript analysis and spcca  revealed regulation of almt genes, including a strong pi-dependent induction of almt <dig> , which was previously shown to exude malate into the rhizosphere as a strategy to cope with aluminum toxicity  <cit> .
earlier studies revealed phr1-dependent accumulation of malate and citrate in pi-depleted plants  <cit> .
interestingly, exudation of both carboxylates into the rhizosphere was shown to facilitate mobilization of pi and fe in several plant species that do not form mycorrhiza  <cit> .
we also noticed deregulation of coumarin biosynthesis-related genes, f6’h <dig> and ccoaomt <dig>  in pdr <dig> roots .
several studies showed that coumarins  are exuded into the rhizosphere to mobilize fe in alkaline soils .
a recent report showed that esculetin accumulates in roots of pi-starved wild-type plants but was suppressed in the phr <dig> mutant, which lacks the induction of psr genes upon pi deficiency  <cit> .
moreover, using a non-targeted approach to identify metabolites from pi-starved arabidopsis root exudates, we recently confirmed pi-dependent regulation of coumarin secretion  <cit> .
thus, our analysis implicates additional fe-chelators in the regulation of pi-dependent fe accumulation and/or distribution in roots.
pi depletion modulates root pectins
inhibition of root cell elongation, formation of root hairs and induction of lateral roots are the most robust local responses to pi deficiency  <cit> , which all require extensive reorganization of the cw.
our analysis revealed pi-dependent regulation of cw-modifying enzymes, particularly in the sensitive wild-type and pdr <dig> plants and to a lesser extent in lpr1lpr <dig> roots .
consistent with a previous transcriptome study  <cit> , we identified several putative pectin esterases and esterase inhibitors.
pectins are secreted into the apoplast in a highly methylesterified state.
in the cw, pectin methylesterases  may remove methyl groups, generating free carboxylate functions on the surface of pectin polymers.
crosslinking of these carboxylate-groups by ca2+ reduces cw extensibility and regulates cell expansion  <cit> .
our experiments revealed low pi-induced accumulation of non-methylated pectin, specifically within the ez of wild-type and pdr <dig> roots , which might contribute to rapid inhibition of cell elongation in these lines.
in addition, there is growing evidence that plants exchange ca2+ ions for other divalent and trivalent metal ions to prevent metal uptake and ros formation  <cit> .
gessa et al.
<cit>  showed in vitro fe3+ binding to carboxylate groups on polygalacturonic acids , and two studies in arabidopsis and rice demonstrated the ability of pga to mobilize pi from fepo <dig> complexes and clay  <cit> .
interestingly, a decrease in pectins in the arabidopsis qua1- <dig> mutant causes a hypersensitive short root phenotype upon pi depletion  <cit> .
here, we show that accumulation of pectin in the root meristem coincides with the sites of fe accumulation .
local pectin deposition might be a strategy to mobilize pi from fe-phosphate complexes.
the data support our previous observations of cw thickening and callose deposition at sites of fe accumulation in the root tip  <cit> .
a recent study of the arabidopsis flower transcriptome revealed deregulation of pgip <dig> and other cw-modifying enzymes in the ferritin1-3- <dig> triple mutant  <cit> .
pgip <dig> is a member of the leucine-rich repeat  protein superfamily and inhibits fungal and bacterial polygalacturonases, which cleave non-methylated pectin residues in infected tissues  <cit> .
it further regulates germination by inhibiting the breakdown of seed coat pectins  <cit> .
intriguingly, our analysis revealed co-regulation of pgip <dig> and fer <dig> on transcript and protein level in all lines upon pi-depletion , further indicating a potential link between the pi-dependent regulation of fe distribution and the modification of pectin in the cw.
peroxidases may modulate ros formation and cell wall dynamics
we identified  <dig> ciii prxs  that were regulated on the mrna and/or protein level, either in response to pi depletion  or as a consequence of the pdr <dig> and lpr mutations .
interestingly, the majority of ciii prx mrnas/proteins  were deregulated in pdr <dig> in pi replete conditions.
ciii prxs are involved in superoxide formation by transferring electrons from nadh to o <dig> as well as in the fe catalyzed generation of hydroxyl radicals  <cit> .
ros formation is likely responsible for the cleavage of cw polysaccharides to promote cell expansion.
on the other hand, oxidation of monolignols by ciii prxs is the predominant mechanism of monolignol polymerization  which rigidifies the cw and degrades h2o <dig>  <cit> .
the potential role of ciii prxs for modulating ros levels and cw dynamics and their strong deregulation in pdr <dig> mutants points to a function in local root growth adaptation.
a comprehensive analysis of available transcriptome and proteome data revealed that most ciii prxs are mainly expressed in the root  <cit> .
two of those, prx <dig> and prx <dig>  bind to ca2+ polygalacturonates and mediate root growth in arabidopsis  <cit> .
a more recent study demonstrated that prx <dig> and prx <dig> knock-down lines exhibited reduced ros and callose formation upon treatment with microbe-associated molecular patterns , implicating a direct role of these gene products in ros formation  <cit> .
using specific ros indicators, we recently demonstrated the formation of apoplastic ros at the site of –pi induced fe deposition  <cit> .
the underlying mechanism remains elusive but ciii prxs may constitute a missing link between pi dependent ros formation and cw remodeling.
comparative transcriptome and proteome analysis allows in-depth dissection of gene expression
our comparative transcriptome and proteome analysis revealed a highly significant but relatively low positive correlation for the abundance of psr proteins and their cognate transcripts in all three genotypes tested .
the majority of mrna/protein pairs in our dataset showed discordant changes, which has been previously observed and discussed in arabidopsis and other organisms like mice and humans and which is likely explained by  translational regulation and/or a temporal delay between the regulation of transcript and protein abundance.
in addition, technical limitations in the efficiency of protein identification  may restrict the detection of proteins relative to their cognate transcripts .
correlation values significantly increased when gene activity was subcategorized.
for example, we observed a strong positive correlation between protein and mrna abundance when we focused on proteins that were pi-responsive in all genotypes .
similarly, we found an enhanced positive correlation when we compared only significantly regulated genes with their cognate proteins .
moreover, our observations suggest that the integration of transcriptome and proteome datasets can be used as a valuable complementary approach.
for example, we identified  <dig> and  <dig> ciii prx, regulated on the transcript and/or protein level, respectively.
only  <dig> of those showed correlative expression changes in both datasets .
however, the integration of both approaches revealed regulation of  <dig> ciii prx, suggesting that the majority of ciii prx are involved in the response to pi deprivation.
we demonstrate that spcca is a useful tool to integrate all experimental factors in our investigation, including the proteome and transcriptome data, pi-status and genotype in order to elucidate unknown correlations in this multidimensional dataset.
interestingly, the first two cvs of our spcca indicated a prominent role of genes and proteins that were differentially regulated in pi-replete pdr <dig> seedlings .
indeed, detailed analysis of our datasets revealed that the majority of pi-responsive genes was not significantly deregulated in pdr <dig>  compared to the wild type .
on the other hand, several fe-related genes, ciii prx and pectin modifying enzymes were differentially regulated in pi-replete pdr <dig> plants , indicating that conditional hypersensitivity in pdr <dig> might be a cause of constitutive de-repression or sensitization of these genes/proteins.
p5-type atpases are orphan, membrane localized er proteins with unknown substrate specificity  <cit> .
mutant studies on yeast spf <dig> and arabidopsis mia/pdr <dig> strongly suggest a function in er quality control, protein folding and regulation of secretory processes .
hyperaccumulation of pectin and callose in the cw of pi-depleted pdr <dig> roots  <cit>   support a function of pdr <dig> in regulating er-dependent secretion.
CONCLUSIONS
we performed complementary transcriptomics and proteomics approaches to monitor changes in steady-state transcript and protein levels upon pi deprivation of arabidopsis wild-type, pdr <dig> and lpr1lpr <dig> roots.
our analysis reveals a set of genes and proteins that are involved in the regulation of fe homeostasis, cell wall remodeling and ros formation.
we observed increased fer <dig> and decreased irt <dig> expression in all genotypes, which are consistent with intracellular fe accumulation and feed-back inhibited fe uptake in pi-depleted roots, respectively.
analysis of fer1-3- <dig>  fer1-2-3- <dig> and irt <dig> mutants demonstrates that cellular fe uptake and fe storage in ferritin are not involved in pi-dependent modulation of root growth.
we provide evidence for the importance of apoplastic fe redistribution to maintain root growth upon pi-depletion and for a role of frd <dig> in this process.
our data further reveal pi-dependent regulation of cell wall-modifying enzyme expression and changes in the deposition of pectins in pi-deprived roots.
the high correlation between sites of fe deposition and enhanced pectin accumulation suggests that pectins might be involved in fe binding and/or pi mobilization from fe-p complexes.

 inorganic phosphate is an essential macronutrient for plant growth and development. despite its high abundance in the rhizosphere, bioavailability of pi is typically limited because its majority is bound in organic compounds or complexed with metal ions such as ca, fe or al. thus, plants evolved strategies to enhance pi acquisition and to conserve or remobilize pi from internal sources to adapt to pi limiting conditions. previous efforts elucidated some of these adaptive responses, including the identification of high-affinity pi transport systems, the characterization of diverse metabolic bypass reactions, the reutilization of pi from phospholipids, and many more. most of the pi starvation response genes involved in these systemic adjustments are regulated by the myb transcription factor phr. dynamic redesign of the root system architecture provides another strategy to maintain cellular pi supply. in arabidopsis, low external pi availability is locally sensed by the growing root tip, which causes reduction of cell elongation and meristematic activity at the site of pi depletion. the resultant inhibition of root growth is accompanied by accelerated formation of root hairs and development of lateral roots to increase the absorptive surface for topsoil exploration. the development of a densely branched and/or shallow root systems increases pi starvation tolerance in several plant species, including agronomically important crops such as barley, lupin, soybean or common bean. several arabidopsis mutants with altered pi dependent root growth responses have been described. however, for most of the underlying genes only little information is available how they affect pi sensing and root growth modulation. lpr, its closely related paralog lpr and pdr have been identified as central players in local root pi sensing. pdr which codes for the single p5-type atpase of unknown substrate-specificity, and lpr encoding a multicopper oxidase, are expressed in overlapping domains of the root apical meristem. lpr and pdr interact genetically and are required for meristem maintenance and cell elongation in pi-deprived roots. importantly, the lpr1lpr mutation impedes local root growth inhibition under pi limitation and suppresses the hypersensitive short-root phenotype of pdr plants, indicating that they act in the same pathway. previous work revealed that external fe availability modifies local pi sensing. a number of studies observed that pi-starved arabidopsis and rice plants accumulate elevated levels of fe in the root and the shoot, which has been suggested as a proactive strategy to mobilize pi from insoluble fe complexes. fe participates in the formation of reactive oxygen species and it has been proposed that fe toxicity causes local root growth inhibition. we recently provided evidence for apoplastic lpr ferroxidase activity and uncovered a major role of the lpr1-pdr module for root tip-specific deposition of fe3+ in cell walls of the ram and elongation zone during pi limitation. we further showed that fe accumulation in the ram is massively enhanced in pi-starved pdr roots, but suppressed in the insensitive lpr1lpr line. fe deposition coincides with sites of ros generation and triggers cw thickening and callose accumulation, which interferes with cell-to-cell communication, ram maintenance, and cell elongation. in recent years, a set of transcriptome profiling studies provided significant insights into the transcriptional changes upon pi deficiency in arabidopsis. in addition, a complementary transcriptome and proteome study highlighted the convergence of mrna and protein expression profiles on lipid remodeling and glucose metabolism upon pi-deprivation. in this study, we performed comparative transcriptome and proteome expression profiling on roots of pi-replete and pi-starved wild-type, pdr and lpr1lpr plants in combination with a set of physiological and cell biological experiments. our analysis emphasizes the importance of root fe uptake and redistribution under pi limitation. we highlight the potential role of so far unknown players in the regulation of pi-dependent fe-redistribution and demonstrate that apoplastic but not intracellular fe accumulation triggers pi-dependent root growth modulation. consistently, we observed regulation of several cw modifying enzymes, which correlates with an increased deposition of pectin at sites of fe accumulation. the potential role of pectin in pi-dependent root fe storage and pi mobilization is discussed.  differential gene expression correlates with genotype-specific pi sensitivity for transcriptome analysis, wild-type, pdr and lpr1lpr seedlings were germinated on + pi agar and transferred to + pi or –pi medium for 20 h, a period during which pi limitation alters global gene expression as well as root meristem activity. rna was extracted from roots of three biological replicates and prepared for hybridization with ath affymetrix chips. data were analyzed using arraystar and further processed. hierarchical clustering confirmed high homogeneity within each replicate set because the biological replicates clustered together for each genotype and pi condition. it also revealed a clear separation between + pi and –pi samples for the wild-type and the hypersensitive pdr mutant, but less pronounced differences for the insensitive lpr1lpr line. pairwise comparisons using a fold-change cutoff value of ≥  for increased and of ≤ for decreased transcript levels revealed differentially expressed genes across all genotypes and the two growth conditions. low pi exposure altered the expression of and genes in pdr wild-type, and lpr1lpr roots, respectively. thus, the genotype-specific sensitivity of root growth inhibition in response to pi depletion positively correlates with the number of differentially regulated genes.fig. statistical analysis of comparative gene expression analysis. a hierarchical clustering of all ath datasets, including wild-type, pdr and lpr1lpr samples, the two growth regimes, and three biological replicates. distances in the dendrogram illustrate the degree of relationship between samples. note the short distance between biological replicate sets compared to the relatively long distance between + pi and –pi conditions. b number of up- and downregulated genes in wild-type, pdr and lpr1lpr roots upon transfer from + pi to –pi, and in the three genotypes under + pi or –pi conditions. c venn diagrams illustrating the number of differentially regulated genes in pairwise comparisons for all three genotypes under both growth regimes identification of genotype-independent pi-responsive genes we generated venn diagrams to illustrate the distribution of differentially expressed genes between the three genotypes. wild-type shared a subset of and pi-responsive genes with pdr and lpr1lpr respectively, and all three lines had in common a core set of genes. hierarchical clustering of this core set revealed similar expression changes in all genotypes in response to –pi with high positive correlation. the core set comprises two partially overlapping groups that consist of at least psr and metal-responsive genes. members of the first group are known targets of the pi-regulated myb transcription factor phr, suggesting that the systemic response to pi deficiency is maintained in pdr and lpr1lpr mutants.table pi-dependent transcriptional changes of commonly regulated genes shown is the fold change expression of all pi-responsive genes that are regulated in each of the tested genotypes. grey and white boxes denote genes that are significantly suppressed or induced, respectively. all genes were interrogated for published responsiveness to pi-starvation and/or metal-ions. references are indicated in superscript numbers and listed in additional file 3: table s2 in the second group, fe-related genes are overrepresented and comprise the majority of repressed genes. the most strongly suppressed gene in all three genotypes codes for irt the major feedback-regulated fe-uptake system in arabidopsis. many irt co-regulated genes are induced under fe deficiency. interestingly, of the top co-regulated genes are repressed in pi-starved roots irrespective of the genotype. intriguingly, pi-replete pdr roots show higher expression of at least fe-related genes, including a group of transcription factors known to promote fe-uptake under fe deficiency. the remaining fe-related genes of this group are similarly induced in all three genotypes and encode the fe storage protein ferritin and various fe-responsive metal transporters thought to be involved in transition metal detoxification and homeostasis.table pi-dependent regulation of the top genes co-regulated with irt shown is the fold change expression in wild type, pdr and lpr1lpr after transfer to –pi or the fold change expression of pi-replete pdr and lpr1lpr plants compared to the wild-type. red and green boxes denote genes that are significantly suppressed or induced pi depletion alters expression of cell wall-related genes we identified pi-responsive genes that are shared between the wild-type and the hypersensitive pdr mutant, but not with the insensitive lpr1lpr line. surprisingly, only genes of unknown functions in pi starvation response were significantly deregulated in pdr compared with the wild-type, whereas the remaining genes showed a high positive correlation between both genotypes. go term analysis revealed high overrepresentation of gene products associated with the extracellular region. an extended analysis for enriched go terms within a group of genes, which are either regulated by –pi in one or more genotypes or are differentially expressed in at least one of the lines in + pi, confirmed overrepresentation of genes annotated to encode extracellular proteins. in this group, we identified a subset of genes with putative functions in cw remodeling. a similar number of genes were differentially expressed in pdr and wild-type but only one-third in lpr1lpr roots. as noted for fe-related genes, many cw-modifying genes were deregulated in pi-replete pdr roots. within the subset of genes, encoded proteins could be assigned a potential function in pectin modification, predominantly pectin methylesterification. in addition, we noted several expansins and xyloglucan endotransglycosylases as well as a set of carbohydrate hydrolyzing enzymes. intriguingly, all these proteins are predicted to regulate cw extensibility.table pi-dependent regulation of cell wall modifying enzymes shown is the fold change expression of selected cw modifying enzymes in wild-type, pdr and lpr1lpr after transfer to –pi or the fold change expression of pi-replete pdr and lpr1lpr plants compared to the wild-type. candidates were selected from a set of regulated genes annotated to be localized in the extracellular region. red and green boxes denote significantly suppressed or induced genes. pme, pectin methyl esterase; exp, expansin; exl, expansin-like; xth, xyloglucan endotransglucosylase/hydrolyse go term analysis also revealed overrepresentation of genes encoding tetrapyrrole- and heme-binding proteins with oxidoreductase activity. this group codes for peroxidases and most of those belong to the member-family of class iii peroxidases, which are extracellular enzymes with partly antagonistic functions in ros formation and cw dynamics. while pi-responsive expression of ciii prx-encoding genes was similar between wild-type and pdr roots, genes were regulated independently in each line under low pi, and only three ciii prx genes responded significantly to pi limitation in lpr1lpr plants. again, ciii prx genes were deregulated in pdr under + pi. thus, peroxidases may be an important link between ros formation and cw remodeling upon pi starvation. proteomics supports regulation of pi-responsive genes in pdr and lpr1lpr mutants genotype-specific changes in the root proteome upon pi deficiency were monitored in an unlabeled approach using a fast scanning high resolution accurate mass lc-ms system. three biological and three technical replicates were measured for each genotype under + pi and –pi conditions yielding, ms/ms spectra. spectra could be annotated to a peptide sequence with a global false discovery rate threshold of  %. these psms were used to identify protein groups, each with at least one unique peptide and a global fdr threshold of 1 %. protein abundance was inferred based on peptide abundance determined by peptide ion signal peak integration using the progenesis software. pairwise comparison of all genotypes under both growth regimes revealed differentially regulated proteins. based on this list, we identified proteins that were either pi-responsive in at least one genotype or which were already deregulated in one of the mutant lines grown on pi-replete conditions. multidimensional scaling analysis of anova filtered samples revealed low variance between biological replicates but significant differences between genotypes and pi conditions. the levels of proteins were increased or decreased in the wild-type upon pi depletion. as expected, the highest number of proteins were regulated in hypersensitive pdr mutant, probably reflecting changes in root morphology. we also identified a high number of pi-responsive proteins in the insensitive lpr1lpr line. of these, proteins were unique to lpr1lpr, indicating that the adjustment of protein expression might contribute to the decreased pi responsiveness. both mutant lines showed differential regulation of more than proteins under pi-replete conditions. this relatively high value is reminiscent of what we observed in the transcript dataset, supporting the assumption that pdr and lpr proteins may also regulate pi independent processes.fig. statistics of comparative protein expression analysis. a multidimensional scaling analysis of all biological replicate samples. b illustration of the number of up- and downregulated proteins in wild-type, pdr and lpr1lpr roots upon transfer from + pi to –pi, and the number of differentially regulated proteins in pairwise comparisons of the three genotypes under + pi or –pi conditions. c, d, and e venn diagrams illustrating the number of regulated proteins in pairwise comparisons for all three genotypes under both growth regimes venn diagrams identified a group of proteins that were similarly regulated in all lines upon pi depletion. notably, of these proteins were positively correlated with our transcript data, showing induction on both mrna and protein level. two members of this group were fer and the pectin modifying enzyme polygalacturonase inhibiting protein, which further indicates that changes in fe distribution and cw modification are associated with the response to low pi.table pi-dependent protein/mrna regulation shown is the fold change expression of the proteins that are pi-responsive in all lines or the fold change expression of pi-replete pdr and lpr1lpr plants compared to the wild-type. protein expression is compared to transcript changes. red and green boxes denote genes that are significantly suppressed or induced correlation of proteome and transcriptome analysis next, we performed go term analysis to identify groups of proteins involved in genotype-specific pi responsiveness. most proteins have assigned metabolic functions in wild-type and lpr1lpr probably reflecting processes related to pi recycling and mobilization. strikingly, in + pi condition and upon transfer to –pi, the pdr line showed a significant regulation of proteins assigned as response to metal ion and oxidoreductase activity. a closer examination revealed repression of peroxidases in pdr in + pi and induction of peroxidases in –pi condition. within the group of repressed proteins we identified ciii prxs of which enzymes were regulated at the transcript level. only one and six pi-responsive ciii prx were detected in wild-type and lpr1lpr root extracts, respectively. to compare the proteome and transcriptome data sets, we plotted all significantly regulated proteins against their cognate transcript. for those differentially expressed proteins, the percentage of detected transcripts was  % for wild-type,  % for pdr and  % for lpr1lpr roots. we observed only a low, but highly significant, positive correlation of transcript and protein abundance for all three genotypes. we generated a list of significantly altered transcripts, which we compared to the list of significantly altered proteins. we identified cognate genes for wild-type, for lp1lpr and for pdr the correlation coefficient markedly increased when we plotted these genes against their cognate proteins.fig. comparative analysis of transcriptome and proteome data and spcca analysis. a, b, c, and d correlation between transcript and protein fold-changes upon pi-deficiency. a for each genotype, all significantly regulated proteins were plotted against its cognate transcript, if present on the ath chip. b, c, and d significantly regulated protein/mrna pairs were plotted against each other. scatter plots show protein/mrna pairs regulated in wild-type upon pi-deficiency, pairs identified for pdr and pairs identified in lpr1lrp e correlation values for each pairwise comparison. asterisks indicate significance for each correlation analysis. f, g canonical variables of the spcca analysis representing a subset of transcripts/proteins which showed maximum correlation with the illustrated patterns that were generated by the spcca algorithm. we identified the genes, including fer and pgip that were co-regulated on mrna and protein level across all genotypes in response to pi depletion. in wild-type, we noticed induction of ppa, a candidate for pi mobilization, and pck, which is involved in metabolic adjustment to pi deprivation. we further identified two hemicellulose modifying enzymes, xth and xth, which were slightly decreased in low pi. interestingly, both enzymes were previously shown to be regulated by siz, a sumo e3-ligase involved in pi dependent root growth remodeling. go term analysis of the mrna/protein pairs altered in pdr revealed an overrepresentation of metabolic processes. the second most significant term is consistent with altered metal homeostasis in pdr plants. for example, we noticed induction of fer and proteins potentially involved in detoxification of metal ion-induced ros formation, including several glutathione-s-transferases. we also identified f6’h, which is involved in coumarin biosynthesis and fe-mobilization in alkaline soils. our datasets revealed anti-correlation of f6’h expression in pdr showing elevated protein but decreased transcript levels in –pi and an inverse relation in + pi, which indicates stringent regulation of f6’h expression in pdr in addition, protein level of ccoaomt, which converts caffeoyl-coa to feruloyl-coa, the substrate of f6’h, was also elevated in pdr. thus, coumarin-mediated mobilization of fe may be involved in pi dependent fe accumulation. integrative spcca analysis supports pi-dependent metal redistribution we integrated the two –omics approaches to uncover relationships that are supported by both individual datasets. we performed a supervised penalized canonical correlation analysis, which searches for correlations between a set of transcripts and proteins. the experimental design was integrated into the analysis to allow for biological interpretation of the derived canonical variables. the experimental factors were provided as a binary matrix of design vectors that uniquely characterize each sample. the supervised correlation approach seeks a linear combination of a feature subset from each -omics dataset that correlates maximally with a subset of experimental design factors. to maximize stringency, only varying transcripts and proteins were considered for spcca. for transcriptomics, we choose a list of anova filtered genes and for proteomics a list of proteins. our analysis revealed distinct canonical variables, each representing a specific pattern correlating with a subset of proteins and/or transcripts. the first two cvs revealed structured patterns, while a third cv was disordered and therefore not further examined. the first cv mainly represented genes/transcripts that were differentially expressed in pdr compared to wild-type and lpr1lpr independently of pi status. we examined the top 100 g/t in this variable and found several fe-related candidates, such as fe chelate reductase and myb which is required for growth in fe deficiency. myb and myb mediate fe-dependent induction of nicotianamine synthase, which is also present in this group. nas proteins synthesize nicotianamine, a fe-chelator essential for fe-remobilization in the root. we further identified a member of the aluminum activated malate transporter family. it is of note that almt is most highly induced in all three genotypes during pi depletion. the second cv mainly represented g/t that were similarly expressed in pi-replete pdr and lpr1lpr roots but slightly differed from the wild-type. in contrast to the first cv, the majority of these g/t were pi responsive in all genotypes. as expected, we found several known pi acquisition g/t, including spx cax the phosphate transporter pt and the pi starvation-inducible inorganic pyrophosphatase. many other g/t are implicated in metal homeostasis, e.g., the fe/zn transporters irt and irt the ni transporter ireg the zn/cd transporter hma or the na transporter ysl further supporting our observation that metal homeostasis is strictly controlled in all genotypes upon pi starvation. root growth inhibition in low pi is independent of general fe uptake and cellular storage we previously reported that lpr1-dependent fe accumulation and distribution in root tips controls ram activity in response to low pi. our comparative transcriptomics and proteomics analysis of entire roots revealed pi-responsive expression of fe-related genes, notably fer and irt, which correlated with fe overload in pi-starved roots of the three genotypes under study. to further investigate the role of fe during the local response of roots to pi availability, we analyzed the impact of fer and irt loss-of-function mutants on fe-distribution and root growth inhibition upon pi deprivation. ferritins are located in plastids and can be visualized by perls/dab fe staining as dot-like structures in root cells of wild-type plants, which are not detectable in fer1-3- roots lacking fer expression. using semi-thin sections from perls/dab-stained wild-type roots, we observed similar dot-like structures in pi-replete root tips, which strongly increased in number and staining intensity upon transfer to –pi medium. these punctuate structures are associated with the symplast and are clearly distinctive from apoplastic fe staining. we next performed root growth assays using the fer1-3- triple and fer1-2-3- quadruple mutant. primary root growth rates of the fer mutants were indistinguishable from the wild-type on both + pi or –pi medium. thus, ferritins do not affect the local root growth response to –pi.fig. root growth in fer and irt mutant plants and phenotypes of frd roots. a 4-days-old seedlings were transferred from + pi to + pi or –pi medium for up to 6 days. daily increase in root growth was measured and illustrated in segmented boxes within the bar graph. standard error was calculated from the average total root growth. b total increase in root length after transfer from + pi to either + pi or –pi medium t-test; p < . c, d, and e fe staining and root growth assays of wild-type and frd3- seedlings. 4-days-old plants were transferred from + pi to + pi or –pi medium for up to 6 days. c perls staining in different root segments 20 h after transfer to + pi or –pi medium. upper and middle panels show mature root segments. the lower panels show the ram and early differentiation zone. scale bar 200 μm. d fe and aniline blue callose staining of root tips and differentiated root segments 6 days after transfer to –pi medium. scale bar 100 μm. e root growth of wild-type and frd3- seedlings within 6 days after transfer to pi-depleted medium. the bar graph shows the daily increase in root growth, illustrated in segmented boxes. standard error was calculated from the average total root growth. *** t-test; p =  −. overview images show the root growth after 3 days and 6 days on –pi medium. arrows indicate the position of the root tip, directly after transfer to –pi, as well as 3 days and 6 days after transfer. scale bar 1000 μm. similarly, we performed perls/dab fe-staining to examine fe distribution in wild type and irt roots. compared with pi-replete wild-type seedlings, the irt mutant showed more intense fe staining on the root surface of the mature root zone, which is in accordance with impaired fe uptake from the rhizosphere. however, both lines displayed similar fe staining in the ram and ez, which is consistent with predominant irt expression in the differentiation zone and confirms our previous study. under pi depletion, fe staining increased strongly and comparably in all segments of wild-type and irt roots, indicating that fe accumulation and distribution in root tips is independent of irt we generated homozygous pdr2irt double and lpr1lpr2irt triple mutants and monitored primary root growth on + pi and –pi agar. as expected, the irt mutation did not affect the pi-dependent root growth response of pdr and lpr1lpr plants, indicating irt1-independent fe accumulation in the root tip in response to low pi. apoplastic fe redistribution modifies pi-dependent root growth adaptation long distance apoplastic fe transport and distribution in symplastically disconnected tissues are mediated by the citrate exporter ferric redictase defective. intriguingly, a previous study reported that frd plants display a hypersensitive short-root phenotype when grown on –pi medium. to examine a potential role of frd for mediating pi-dependent fe distribution via fe-citrate complexes, we performed perls fe-staining on wild-type and frd roots. as previously reported, pi-replete frd roots overaccumulated fe in the vascular tissue. within 20 h after transfer to –pi, wild-type plants accumulated fe in the outer cell layers, whereas frd roots showed enhanced fe staining in the vasculature, particularly in differentiated root segments. importantly, only minor differences were noted in the root tip, where fe accumulation was slightly increased in frd; however, extended growth on –pi progressively increased this difference, finally causing massive overaccumulation of fe within the ez and early differentiation zone of frd roots. we previously showed that pi-dependent fe accumulation correlates with callose formation at the sites of fe deposition. after transfer to –pi, callose deposition at sites of fe accumulation and resultant root growth inhibition were similar for wild-type and frd plants. however, extended exposure caused callose overproduction in frd roots which correlated with an enhanced growth inhibition. based on our observations, we assumed that mobilization of apoplastic fe-citrate complexes might be involved in the pi dependent modulation of root growth. to test this, we transferred wild-type plants from + pi conditions to + pi or –pi medium, supplemented with citrate, which was previously shown to restore fe mobilization on frd mutants and monitored their growth behavior. indeed, addition of 100–250 μm citrate promotes root growth within the first two days after transfer to –pi. however, this effect was transient and external supply of citrate eventually suppressed root growth on low pi. pi deprivation modifies pectins at fe accumulation sites our comparative expression profiling pointed to a role for pectin-modifying enzymes. therefore, we studied pi-dependent changes in the pectineous cw by using ruthenium red, an inorganic dye that stains unesterified pectins. roots of wild-type, pdr and lpr1lpr showed a similar rr staining pattern on + pi medium. one day after transfer to –pi, we observed a strong increase in rr staining intensity in wild-type root tips, particularly within the differentiating ez. compared with wild-type, pdr seedlings showed a more intense staining in this region while the rr staining in the lpr1lpr mutant was unaltered. interestingly, the site of enhanced pectin staining correlated well with the site of low pi induced fe deposition in wild-type and pdr roots.fig. fe and pectin staining of roots after seedling transfer from + pi agar to + pi or –pi medium for up to 2 days. a perls/dab fe staining and ruthenium red staining of non-methylesterified pectins. shown are overview images of perls/dab and rr stained roots as well as detail images of the elongation/early differentiation zone. scale bar 100 μm. b hydroxylamine ferric chloride staining of methylesterified pectins. shown are overview images of the root as well as detail images of the root meristem and the stem cell niche. differences in coloration between overview and detail result from the use of different imaging devices. arrows point to the scn and cortical cell layer in the transition zone, asterisks label the quiescent center cells in the scn. c details of the perls/dab, rr and hfc staining in pi-depleted cortical cells of col plants and stem cells of pdr plants. scale bar 20 μm we also visualized the distribution of methyl-esterified pectin by using the hydroxylamine ferric chloride reagent, which specifically reacts with methyl esters of pectin and results in a yellow to red coloration. only weak staining was evident in roots on + pi and transfer to –pi did not significantly change the staining pattern in the differentiating ez. however, higher magnification images revealed increased staining in the ram of wild-type, with the highest intensity in the quiescent center and the cortical cell layer at the transition zone, which demarcates the border between the ram and ez. in contrast, pdr seedlings showed enhanced staining in the ram, particularly within the qc region, but no distinct labeling of the cortical cell layer. no differences in pectin staining were detected in lpr1lpr roots after transfer to –pi medium. high magnification images of rr- and hfc-stained roots revealed simultaneous accumulation of acidic and methyl-esterified pectin in the meristem of the two sensitive lines. in particular, after transfer to –pi, strong hfc and rr staining was evident in the cortex cell layer of wild-type and in the qc region of pdr roots, which co-localized with major sites of fe deposition. discussion plant adaptation to pi limitation depends on coordinated transcriptional and translational regulation of gene expression. while comparative transcriptome analysis proved to be a viable approach to distinguish between local and systemic regulation in pi-starved plants, only little information is available on the regulation of genes and proteins associated with the pi-dependent adaption of root system architecture. previous work revealed that pdr and lpr genes act together in the local response to pi availability by regulating cell type-specific deposition of fe and callose in the root tip. here, we took advantage of the contrasting pi-dependent root phenotype of pdr and lpr1lpr plants to investigate the associated changes in steady-state transcript and protein levels in a comparative approach. genotype independent regulation of several psr genes demonstrated the validity of our experiments and revealed that pdr and lpr1lpr mutants are likely not affected in the systemic response to pi limitation. further analysis of our dataset revealed a number of candidate genes that are possibly involved in the pi-dependent regulation of fe storage and fe redistribution as well as in the modulation of cw dynamics and/or ros formation within the root. pi depletion modulates root fe distribution our study revealed genotype-independent repression of numerous fe-responsive and irt1-coregulated genes upon transfer to pi limitation, which likely reflects feedback regulation as a consequence of elevated fe accumulation in pi-starved differentiated roots. on the other hand, de-repression of fe-related genes in pi-replete pdr plants may sensitize fe overaccumulation in limiting pi. fer and related ferritins are plastid-localized fe storage proteins protecting cells from fe-mediated oxidative stress. using perls/dab fe staining, reyt et al. recently reported dot-like structures in root cells of wild-type plants that likely display ferritin-bound fe because they are absent in fer1-3- roots. a previous study showed that fer is induced by phr in low pi independent of external fe, indicating that fer may play a role in pi-dependent fe distribution. our comparative analysis revealed induction of fer expression on mrna and protein level in all three lines. detection of fe accumulation in dot-like structures supports the notion of intracellular fe storage under pi limitation, possibly as ferritin fe. importantly, pi-dependent root growth was not affected by loss of ferritins or loss of irt in pdr and lpr mutants, indicating that pi dependent root growth modulation is independent of intracellular fe accumulation. our data are consistent with a recent study reporting indistinguishable primary root growth of fer1-3- and wild-type plants on high fe. fe mobilization from the rhizosphere is facilitated by chelators such as carboxylates and coumarins, and apoplastic long distance fe trafficking is mediated by fe-citrate complexes. frd exports citrate and the frd mutant is defective in apoplastic fe translocation, causing fe hyperaccumulation in root stele tissues. importantly, frd mutants show a hypersensitive short root phenotype in low pi and we demonstrated fe overaccumulation in pi-deprived frd roots, which indicates that citrate secretion is required for proper fe-distribution under pi limitation. interestingly, citrate application transiently promoted frd root growth in low pi, indicating that the pi-dependent short root phenotype of frd is likely a consequence of altered fe redistribution in the growing root. transcript analysis and spcca revealed regulation of almt genes, including a strong pi-dependent induction of almt, which was previously shown to exude malate into the rhizosphere as a strategy to cope with aluminum toxicity. earlier studies revealed phr1-dependent accumulation of malate and citrate in pi-depleted plants. interestingly, exudation of both carboxylates into the rhizosphere was shown to facilitate mobilization of pi and fe in several plant species that do not form mycorrhiza. we also noticed deregulation of coumarin biosynthesis-related genes, f6’h and ccoaomt in pdr roots. several studies showed that coumarins are exuded into the rhizosphere to mobilize fe in alkaline soils. a recent report showed that esculetin accumulates in roots of pi-starved wild-type plants but was suppressed in the phr mutant, which lacks the induction of psr genes upon pi deficiency. moreover, using a non-targeted approach to identify metabolites from pi-starved arabidopsis root exudates, we recently confirmed pi-dependent regulation of coumarin secretion. thus, our analysis implicates additional fe-chelators in the regulation of pi-dependent fe accumulation and/or distribution in roots. pi depletion modulates root pectins inhibition of root cell elongation, formation of root hairs and induction of lateral roots are the most robust local responses to pi deficiency, which all require extensive reorganization of the cw. our analysis revealed pi-dependent regulation of cw-modifying enzymes, particularly in the sensitive wild-type and pdr plants and to a lesser extent in lpr1lpr roots. consistent with a previous transcriptome study, we identified several putative pectin esterases and esterase inhibitors. pectins are secreted into the apoplast in a highly methylesterified state. in the cw, pectin methylesterases may remove methyl groups, generating free carboxylate functions on the surface of pectin polymers. crosslinking of these carboxylate-groups by ca2+ reduces cw extensibility and regulates cell expansion. our experiments revealed low pi-induced accumulation of non-methylated pectin, specifically within the ez of wild-type and pdr roots, which might contribute to rapid inhibition of cell elongation in these lines. in addition, there is growing evidence that plants exchange ca2+ ions for other divalent and trivalent metal ions to prevent metal uptake and ros formation. gessa et al. showed in vitro fe3+ binding to carboxylate groups on polygalacturonic acids, and two studies in arabidopsis and rice demonstrated the ability of pga to mobilize pi from fepo complexes and clay. interestingly, a decrease in pectins in the arabidopsis qua1- mutant causes a hypersensitive short root phenotype upon pi depletion. here, we show that accumulation of pectin in the root meristem coincides with the sites of fe accumulation. local pectin deposition might be a strategy to mobilize pi from fe-phosphate complexes. the data support our previous observations of cw thickening and callose deposition at sites of fe accumulation in the root tip. a recent study of the arabidopsis flower transcriptome revealed deregulation of pgip and other cw-modifying enzymes in the ferritin1-3- triple mutant. pgip is a member of the leucine-rich repeat protein superfamily and inhibits fungal and bacterial polygalacturonases, which cleave non-methylated pectin residues in infected tissues. it further regulates germination by inhibiting the breakdown of seed coat pectins. intriguingly, our analysis revealed co-regulation of pgip and fer on transcript and protein level in all lines upon pi-depletion, further indicating a potential link between the pi-dependent regulation of fe distribution and the modification of pectin in the cw. peroxidases may modulate ros formation and cell wall dynamics we identified ciii prxs that were regulated on the mrna and/or protein level, either in response to pi depletion or as a consequence of the pdr and lpr mutations. interestingly, the majority of ciii prx mrnas/proteins were deregulated in pdr in pi replete conditions. ciii prxs are involved in superoxide formation by transferring electrons from nadh to o as well as in the fe catalyzed generation of hydroxyl radicals. ros formation is likely responsible for the cleavage of cw polysaccharides to promote cell expansion. on the other hand, oxidation of monolignols by ciii prxs is the predominant mechanism of monolignol polymerization which rigidifies the cw and degrades h2o. the potential role of ciii prxs for modulating ros levels and cw dynamics and their strong deregulation in pdr mutants points to a function in local root growth adaptation. a comprehensive analysis of available transcriptome and proteome data revealed that most ciii prxs are mainly expressed in the root. two of those, prx and prx bind to ca2+ polygalacturonates and mediate root growth in arabidopsis. a more recent study demonstrated that prx and prx knock-down lines exhibited reduced ros and callose formation upon treatment with microbe-associated molecular patterns, implicating a direct role of these gene products in ros formation. using specific ros indicators, we recently demonstrated the formation of apoplastic ros at the site of –pi induced fe deposition. the underlying mechanism remains elusive but ciii prxs may constitute a missing link between pi dependent ros formation and cw remodeling. comparative transcriptome and proteome analysis allows in-depth dissection of gene expression our comparative transcriptome and proteome analysis revealed a highly significant but relatively low positive correlation for the abundance of psr proteins and their cognate transcripts in all three genotypes tested. the majority of mrna/protein pairs in our dataset showed discordant changes, which has been previously observed and discussed in arabidopsis and other organisms like mice and humans and which is likely explained by translational regulation and/or a temporal delay between the regulation of transcript and protein abundance. in addition, technical limitations in the efficiency of protein identification may restrict the detection of proteins relative to their cognate transcripts. correlation values significantly increased when gene activity was subcategorized. for example, we observed a strong positive correlation between protein and mrna abundance when we focused on proteins that were pi-responsive in all genotypes. similarly, we found an enhanced positive correlation when we compared only significantly regulated genes with their cognate proteins. moreover, our observations suggest that the integration of transcriptome and proteome datasets can be used as a valuable complementary approach. for example, we identified and ciii prx, regulated on the transcript and/or protein level, respectively. only of those showed correlative expression changes in both datasets. however, the integration of both approaches revealed regulation of ciii prx, suggesting that the majority of ciii prx are involved in the response to pi deprivation. we demonstrate that spcca is a useful tool to integrate all experimental factors in our investigation, including the proteome and transcriptome data, pi-status and genotype in order to elucidate unknown correlations in this multidimensional dataset. interestingly, the first two cvs of our spcca indicated a prominent role of genes and proteins that were differentially regulated in pi-replete pdr seedlings. indeed, detailed analysis of our datasets revealed that the majority of pi-responsive genes was not significantly deregulated in pdr compared to the wild type. on the other hand, several fe-related genes, ciii prx and pectin modifying enzymes were differentially regulated in pi-replete pdr plants, indicating that conditional hypersensitivity in pdr might be a cause of constitutive de-repression or sensitization of these genes/proteins. p5-type atpases are orphan, membrane localized er proteins with unknown substrate specificity. mutant studies on yeast spf and arabidopsis mia/pdr strongly suggest a function in er quality control, protein folding and regulation of secretory processes. hyperaccumulation of pectin and callose in the cw of pi-depleted pdr roots support a function of pdr in regulating er-dependent secretion.  we performed complementary transcriptomics and proteomics approaches to monitor changes in steady-state transcript and protein levels upon pi deprivation of arabidopsis wild-type, pdr and lpr1lpr roots. our analysis reveals a set of genes and proteins that are involved in the regulation of fe homeostasis, cell wall remodeling and ros formation. we observed increased fer and decreased irt expression in all genotypes, which are consistent with intracellular fe accumulation and feed-back inhibited fe uptake in pi-depleted roots, respectively. analysis of fer1-3- fer1-2-3- and irt mutants demonstrates that cellular fe uptake and fe storage in ferritin are not involved in pi-dependent modulation of root growth. we provide evidence for the importance of apoplastic fe redistribution to maintain root growth upon pi-depletion and for a role of frd in this process. our data further reveal pi-dependent regulation of cell wall-modifying enzyme expression and changes in the deposition of pectins in pi-deprived roots. the high correlation between sites of fe deposition and enhanced pectin accumulation suggests that pectins might be involved in fe binding and/or pi mobilization from fe-p complexes. 
vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth. we took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response mutant and low phosphate response and double mutant to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. the results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. we observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. our experiments reveal that ferric reductase defective mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. we further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization. arabidopsis thalianaphosphate deficiencyroot growthproteomicstranscriptomicsiron transportcell wallpectininstitutional core funding provided by the state of saxony-anhalt and the federal republic of germanyissue-copyright-statement© the author 2016 we recently demonstrated that the low phosphate response - phosphate deficiency response module mediates apoplastic deposition of ferric iron in the growing root tip during phosphate limitation. we further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.
 plant adaptation to limited phosphate availability comprises a wide range of responses to conserve and remobilize internal phosphate sources and to enhance phosphate acquisition. vigorous restructuring of root system architecture provides a developmental strategy for topsoil exploration and phosphate scavenging. changes in external phosphate availability are locally sensed at root tips and adjust root growth by modulating cell expansion and cell division. the functionally interacting arabidopsis genes, low phosphate response and and phosphate deficiency response, are key components of root phosphate sensing. we recently demonstrated that the low phosphate response - phosphate deficiency response module mediates apoplastic deposition of ferric iron in the growing root tip during phosphate limitation. iron deposition coincides with sites of reactive oxygen species generation and triggers cell wall thickening and callose accumulation, which interfere with cell-to-cell communication and inhibit root growth.  we took advantage of the opposite phosphate-conditional root phenotype of the phosphate deficiency response mutant and low phosphate response and double mutant to investigate the phosphate dependent regulation of gene and protein expression in roots using genome-wide transcriptome and proteome analysis. we observed an overrepresentation of genes and proteins that are involved in the regulation of iron homeostasis, cell wall remodeling and reactive oxygen species formation, and we highlight a number of candidate genes with a potential function in root adaptation to limited phosphate availability. our experiments reveal that ferric reductase defective mediated, apoplastic iron redistribution, but not intracellular iron uptake and iron storage, triggers phosphate-dependent root growth modulation. we further highlight expressional changes of several cell wall-modifying enzymes and provide evidence for adjustment of the pectin network at sites of iron accumulation in the root.  our study reveals new aspects of the elaborate interplay between phosphate starvation responses and changes in iron homeostasis. the results emphasize the importance of apoplastic iron redistribution to mediate phosphate-dependent root growth adjustment and suggest an important role for citrate in phosphate-dependent apoplastic iron transport. we further demonstrate that root growth modulation correlates with an altered expression of cell wall modifying enzymes and changes in the pectin network of the phosphate-deprived root tip, supporting the hypothesis that pectins are involved in iron binding and/or phosphate mobilization. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords arabidopsis thalianaphosphate deficiencyroot growthproteomicstranscriptomicsiron transportcell wallpectininstitutional core funding provided by the state of saxony-anhalt and the federal republic of germanyissue-copyright-statement© the author 2016 