BACKGROUND
ribosomes are large ribonucleoprotein complexes which synthesize the vast majority of cellular peptides.
the structure of ribosomes is essentially the same in prokaryotes and eukaryotes in that they are composed of ribosomal rnas  and ribosomal proteins , organized in a large and a small subunit.
however, ribosomes of eukaryotes exhibit greater structural diversity, reflecting the higher complexity of the molecular mechanism underlying translation in the latter group  <cit> .
in arabidopsis thaliana, cytoplasmic ribosomes are composed of four distinct rrnas  and  <dig> distinct ribosomal proteins   <cit> .
the  <dig> rps are encoded by a total of  <dig> genes; thus, several paralogous genes encode the same rp  <cit> .
these paralogous genes could be redundant or, as indicated for certain rp genes, may be involved in specific plant processes or developmental stages  <cit> .
for example, among the four paralogous genes encoding rpl <dig>  one gene was highly expressed in cotyledons, whereas the other three genes were preferentially expressed in roots, developing anthers and pollen  <cit> .
in addition, it has been reported that a mutation in rpl23aa  lead to impaired growth and developmental abnormalities, suggesting that rpl23aa plays an essential role in fitness traits.
by contrast, knockdown of the closely related gene family member rpl23ab had little effect on the phenotype  <cit> .
similarly, genes encoding rps15a were differentially expressed in arabidopsis, with one paralogous gene being completely transcriptionally quiescent, while the other three were highly expressed in mitotically active regions   <cit> .
ribosomal proteins are essential for protein synthesis and, consequently, play an important role in metabolism, cell division, and growth.
in addition to their housekeeping functions, the phenotypes resulting from mutations in several different rp genes provide strong evidence that rps participate as regulatory components in developmental processes  <cit> .
generally, rp mutants share developmental abnormalities such as reduced shoot growth, reduced cell proliferation and increased nuclear ploidy in leaf cells  <cit> .
for example, a semi-dominant mutation in rpl27ac affected multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, as well as seed set  <cit> .
silencing of rps <dig> disturbed the ratio between the small and large subunits of mitoribosomes, causing an excess of the latter  <cit> .
introducing rps <dig> antisense and rpl23aa rnai constructs resulted in an altered number of cotyledons  <cit> .
also, a dominant missense mutation in rpl10a suppressed stem-elongation in comparison with the wild type  <cit> .
less severe phenotypes were reported for mutations in other rp genes.
pointed first leaf  encodes the small subunit rps <dig>  <cit> .
pfl mutants showed changes in the shape of early vegetative leaves from the spatulate wild-type shape to a pointed, narrow shape.
mutations in the pfl <dig>  gene caused a delay in the transition to flowering and the production of more vegetative leaves than in the wild type  <cit> .
in addition to effects on plant development and growth, rps also took part in the response to stress.
under uv-b stress, rpl <dig> genes were differentially regulated in a dosage- and time-dependent manner; while rpl10c was induced and rpl10b was down-regulated at high uv-b intensity, rpl10a was not responsive to uv-b  <cit> .
another study showed that specific rps changed in abundance in response to sucrose feeding, implying that different rps are incorporated into ribosomes depending on the growth condition  <cit> .
transcripts of rps15af increased following treatment with cytokinin 6-benzylaminopurine  and auxin indole acetic acid , while abscisic acid  treatment decreased transcript abundance.
in addition, transcripts of rps15aa, rps15ad and rps15af showed increased abundance upon temperature and mechanical stress  <cit> .
phosphate  is an essential macronutrient for plants.
in addition to its structural role in nucleic acids and cell membranes, pi has important functions in lipid and energy metabolism.
limited bioavailability of pi often restricts growth in natural and agricultural ecosystems.
due to intense use of p fertilizers in agriculture, pi resources have become limited.
therefore, understanding how plants adapt to low pi availability is of critical importance to generate pi-efficient plants.
as a coping mechanism to limited pi supply, plants undergo dramatic changes in metabolism, physiology, hormonal balance and gene expression  <cit> .
similarly, iron  is essential for the survival and proliferation of virtually all organisms.
iron is required for many vital enzymatic reactions such as dna replication, lipid metabolism, and nitrogen fixation.
in plants, fe deficiency is a common nutritional disorder worldwide, causing low yield and quality of crop plants.
to acquire sufficient fe while avoiding toxicity, plants tightly control uptake, utilization, and storage in response to environmental availability  <cit> .
at present, most studies on plant rps focus on the developmental effects of rp mutants.
we recently showed that fe deficiency alters the expression of several rps, presumably resulting in biased translation  <cit> .
in the present study, changes in global expression pattern of rp genes in arabidopsis roots upon pi and fe deficiency were compared at the transcriptomic and proteomic level in order to gain insights into the regulation of the response to pi or fe deficiency.
it is shown that the expression of rps followed stress-specific pattern, revealing a novel layer of regulation that may be critical for the fitness of plants under nutrient shortage.
RESULTS
comprehensive expression analysis of ribosomal protein genes in arabidopsis roots
the rna-seq technology has proven to provide precise gene expression information  <cit>  and accurate discrimination of differentially expressed genes under various conditions.
using this technology, we previously analyzed global gene expression changes upon pi-deficiency and fe-deficiency in arabidopsis roots  <cit> .
a total of  <dig>  genes were detected in roots from pi-deficient and pi-sufficient plants by alignment to the tair <dig> annotation of the arabidopsis genome .
a set of  <dig>  genes  was differentially expressed under pi deficiency .
using the same criteria, a total of  <dig>  genes  were found to be differentially expressed between fe-sufficient and fe-deficient conditions among the  <dig>  detected genes.
thus, a comparable number of genes were affected in both growth types.
a subset of  <dig> genes was differentially expressed under both conditions.
ribosomal proteins play critical roles in ribosome biogenesis, translation and post-translational modifications of proteins.
a complete sequence data set for the  <dig> known rp families was compiled from the tair <dig> release of the arabidopsis genome   <cit> , this data set  comprises all arabidopsis paralogous genes associated with any specific rp family and other ribosome-related genes including eif <dig> , rack <dig>   <cit>  and fer <dig>   <cit> , which was shown to be tightly associated with ribosomes  <cit> .
we defined the expression level of all rp genes in this data set according to an earlier study  <cit> .
if the number of unique reads was zero in all three biological repeats under normal  condition, the gene was defined as not expressed; low abundance of a transcript was defined by a number of unique reads < <dig> in either of the three biological repeats; medium abundance was defined for genes with  <dig> to  <dig> reads, and genes with more than  <dig> reads in either of the three repeats were defined as highly expressed.
on the basis of these criteria, in arabidopsis roots subjected to pi deficiency seven rp genes were not detected, transcripts of  <dig> rp genes were lowly abundant,  <dig> rp genes showed medium transcript levels, and  <dig> rp genes were highly expressed .
the average read number of rp genes was  <dig> ; while on average  <dig> reads were associated with non-rp transcripts, indicating that rp paralogs were highly expressed in arabidopsis roots.
previous data suggested that rp genes are retained in the arabidopsis genome at higher levels than other types of gene  <cit> , suggesting a high biological importance of this plant-specific diversity of rp genes.
in growing yeast cells,  <dig> ribosomes were produced per minute, and transcription of ribosomal rna and rp genes account for 60% of total transcription  <cit> .
the synthesis of rps, ribosome assembly factors, and extra-ribosomal components involved in translation must simultaneously satisfy the plant’s demand for metabolism and growth, comprising an important component of the total cellular translational activity.
in many cases, different rp paralogs from the same rp families displayed differential expression.
for example, among three paralogs of the rpl <dig> family, rpl18b and rpl18c were highly expressed with more than  <dig>  reads, while the third member of this family, rpl18a, was not expressed.
by analyzing public transcriptomic data   <cit> , we found that expression of rpl18a is particularly low among all tissues and treatments, whereas rpl18b and rpl18c are highly expressed in some tissues.
this suggests that rpl18a may be not functional or of minor importance, although retained in the genome during evolution.
pronounced differences were also observed for the expression of genes encoding the proteins of the large subunits l <dig>  l <dig>  l27a, l <dig>  l37a, and l <dig>  in a previous report, rps15aa, rps15ad and rps15af, but not rps15ac transcripts were detected in all mature tissues by semi-quantitative rt-pcr  <cit> .
taken together, the differential expression of genes encoding rps within specific ribosomal gene families indicates extensive ribosome heterogeneity at the intercellular level  <cit> .
within the  <dig> rp gene families with high read numbers  in arabidopsis roots were six small  and seven large  ribosomal subunit proteins .
the highest expression level was observed for s3a, which was detected with an average read number of  <dig>  for each paralog.
high steady state levels of some rp gene transcripts may be indicative of fundamental roles of the encoded proteins in plant growth and development.
at the protein level, for  <dig> rp genes and  <dig> ribosome-related genes a total of  <dig> cognate proteins, accounting for nearly 50% of the detected genes, were identified in a proteomic approach .
this accounts for 75% of the large subunit proteins,  <dig> % of the small subunit proteins, and 100% of the ribosome-related proteins.
these results suggest that some rp families are necessary for translation in arabidopsis roots, but not all of the paralogous proteins within one rp family take part in this process.
our data further demonstrate that rps were more likely to be detected among the highly expressed genes than in genes with transcripts of medium or low abundance .
notably,  <dig> highly expressed rp genes had no cognate peptides, supporting the observation that the gene expression level is not always correlated with protein abundance  <cit> .
many rp paralogs might have been detected, but without unique matches due to their high sequence identity.
for example, for three paralogous proteins of the rpl <dig> family, rpl6a , rpl6b  and rpl6c ,  <dig> matching peptides have been detected, but only for rpl6a seven unique peptides were found, defining rpl6b and rpl6c as not present at the protein level although their corresponding transcript are high.
gene ontology  enrichment analysis revealed that the products of most of the rp genes were localized in the cytosolic small and large ribosomal subunits, especially proteins encoded by medium and highly expressed genes .
in addition, some biological processes such as ’cellular response to uv-b’ , ’response to cold’ , and ’response to salt stress’  were overrepresented among the highly expressed genes , indicating that rps may also participate in the response to abiotic stresses.
in addition, the go categories ’seed germination’ , ’embryo development ending in seed dormancy’ , and ’leaf morphogenesis’  were overrepresented in this group of rp genes .
in previous studies, mutations in some rp genes  caused an embryo-lethal phenotype,  <cit> ; thus, shortage in specific rps could lead to severe growth defects.
mutations in the rp genes s <dig> and s <dig> were reported to cause abnormal leaf development  <cit> .
changes in leaf morphology in rp gene mutants might be caused by a diminished capacity to maintain protein synthesis at a level sufficient for proliferative cell divisions in the vasculature and palisade mesophyll, and for cell division progression at the leaf margin  <cit> .
expression of rp genes in pi-deficient arabidopsis roots
out of the  <dig> rp genes and six ribosomal-related genes annotated in tair <dig>  only three were differentially expressed between pi-sufficient and pi-deficient arabidopsis roots , all of which were up-regulated.
among the three genes, induction of at1g <dig>  was more pronounced when compared with the other two genes, at3g <dig>  and at3g <dig> .
in rice flowers, rpl35a was highly expressed under high temperature after the flowering stage  <cit> , suggesting that this rp family is not only involved in pi deficiency but may also play a role in heat stress response.
in a previous report, transcript levels of rps <dig>  rpl <dig> and rpl13a from lupine cotyledons were found to be increased during cytokinin treatment relative to a 0 h water control  <cit> .
expression of rps <dig> was decreased after 5 h of incubation with aba, indicating that rps <dig> may have different functions in abiotic stresses.
the rapid accumulation of genome-wide gene expression data allows the creation of co-expression networks by examining the pair-wise relationship of genes over a large number of experimental conditions.
in the co-expression network, a node is represented by a gene, and an edge is drawn between gene a and b if the correlation coefficient between these two genes is above a defined threshold  <cit> .
genes with similar expression pattern may be involved in the same biological process and potential functions of the unknown genes in a co-expression network can be predicted.
<cit> .
in an attempt to identify pi-responsive non-rp genes that are co-regulated with rp genes, we analyzed the correlation of the expression of the  <dig>  differentially expressed genes including three rp genes.
the results show that only one rp genes  interacted with the 2-oxoglutarate  and fe -dependent oxygenase superfamily protein at3g <dig> with a pearson correlation coefficient  cutoff of  <dig> .
with this cf threshold decreased to  <dig> , all three differentially expressed rp genes and  <dig> other differentially expressed genes including some transcription factors and protein kinases could form a larger co-expression network ; and these genes were involved in diverse biological processes .
at the protein level,  <dig> rps accumulated differentially upon pi deficiency .
among them, only rpl12b  was up regulated, all other proteins showed decreased abundance.
interestingly, among the three paralogous proteins in the rpl <dig> family, only rpl12b was induced, while the abundance of the other two rpl <dig> members was unchanged under pi-deficient conditions.
this suggests that rpl12b may play an important role in the pi-stress response.
to get a better understanding of the function of differentially expressed rps, a protein-protein interaction network was constructed using the string database   <cit>  the network is presented in the ’evidence’ view, whereby lines connecting proteins represent the type of evidence  used in predicting the association.
all  <dig> differentially expressed rp proteins were clustered in a large protein-protein interaction module .
alternative splicing  is a mechanism that increases transcriptome and proteome complexity and controls developmental programs and responses to the environment in higher eukaryotes  <cit> .
previously, genome-wide differential as features induced by pi deficiency have been analyzed in arabidopsis roots  <cit> .
further analysis revealed that a subset of  <dig> rp genes produced transcripts with retained introns; all of these intron retention events were induced by pi shortage .
three out of the  <dig> encoded rps were decreased in abundance under pi deficiency.
these results suggest that post-transcriptional regulatory processes such as alternative splicing may have affected the efficiency of translation  <cit> .
expression of ribosomal protein genes in fe-deficiency arabidopsis roots
using the criteria listed above, a total of  <dig> rp genes were differentially expressed in fe-deficient arabidopsis roots.
in contrast to the up-regulated pattern of differentially expressed rp genes in roots of pi-deficient plants, expression of all the  <dig> rp genes was decreased upon fe deficiency.
the rp gene with lowest relative expression level was the ribosome-associated protein gene ferritin <dig> , which was found in small polysome fractions isolated form the leaves of arabidopsis plants in the dark  <cit> .
fer <dig> was reported to be associated with the chloroplast and suggested to mediate coordination between the translation activity of cytosolic ribosomes and the function of chloroplasts in response to iron-based signals  <cit> .
thus, decreased expression levels of fer <dig> under fe deficiency may be associated with the regulation of cellular fe homeostasis.
gene ontology analysis revealed that the  <dig> differentially expressed rp genes were chiefly involved in translation and ribosome biogenesis.
in addition, the biological processes ’response to salt stress’, ’cellular response to uv-b’, and ’leaf morphogenesis’ were also enriched .
the  <dig> differentially expressed genes were derived from  <dig> small and  <dig> large ribosomal subunit rps , in which the transcript abundance of all paralogous genes from five rp families , was decreased.
in the rps15a family, three of the six paralogous genes  were down-regulated under fe deficiency.
these three genes showed increased transcript abundance following treatment with bap, rps15af was also induced by iaa treatment.
a similar trend of expression in paralogous genes with rps15a family was observed under heat and cold stress  <cit> .
it might thus be assumed that rps15a is an important regulator involved in multiple abiotic and nutrient stresses.
co-expression analysis of the  <dig> differentially expressed rp genes under fe deficiency yielded a network consisting of  <dig> nodes and  <dig>  edges .
this network could be further divided into one larger and one smaller clusters, the larger of which contained much more nodes  and edges, and every node  was correlated with other rp genes .
similarly, with the aim of identifying other fe-responsive non-rp genes that interact with rp genes, we put the  <dig>  differentially expressed genes including  <dig> rp genes together to do the co-expression analysis.
results showed that a total of  <dig> rp genes and  <dig> fe-responsive non-rp genes comprise one bigger and two smaller clusters with pearson correlation coefficient cutoff of  <dig>  .
proteomic profile analysis showed that only two rps were differentially expressed between fe-deficient and fe-sufficient arabidopsis roots  <cit> .
the two proteins were from the rpl <dig> family and showed decreased abundance, although the transcriptional expression of these two genes remained unchanged .
analysis of as revealed three rp genes with differential alternative donor/acceptor splices sites and six rp genes with differential intron retention in roots of fe-deficient plants .
none of these rp genes were transcriptionally or translationally fe-responsive.
comparative expression pattern of rp genes under pi and fe deficiency
comparison of the differentially expressed rp genes and rps between pi- and fe- deficient arabidopsis roots showed no overlap between the two growth types, suggesting that distinct rps are involved in the responses of plants to pi- and fe-deficient.
interestingly, most rp genes encoding proteins of the large subunit were affected by pi deficiency, while the majority of differentially rp genes encoding rps under fe deficiency were from both the small and the large subunit .
furthermore, the expression pattern of rps upon pi deficiency was more complex than that observed under fe-deficient conditions.
nevertheless, one of the common observations for these two treatments is that there was a large discrepancy between the rp genes and pr proteins affected by pi deficiency  as well as under fe deficiency conditions .
our previous studies  <cit>  have found that the concordance between differences in the abundance of mrna and its encoded protein is not always high due to several factors: firstly, proteins with low abundance might not be reliably detected and thus did not match the criteria that define them as differentially accumulated whereas the transcript levels of the corresponding genes are significantly changed.
secondly, several proteins with high abundance accumulated differentially upon fe/pi deficiency, but they have important housekeeping functions and the transcript levels were not changed.
finally, a change in transcript abundance may or may not be translated into changes in protein level; posttranscriptional regulatory processes such as alternative splicing, rna processing, and other processes may affect the efficiency of translation.
a generally low congruency of proteomic and transcriptional profiles has been reported previously  <cit> .
CONCLUSIONS
in summary, our results suggest that the composition of ribosomes is changed in a stress-specific manner as part of an adaptation to pi and fe deficiency , probably biasing protein translation .
our findings further suggest that rps are not only involved in protein translation but also in the response of plants to nutrient deficiency, possibly altering the composition of ribosomes in roots of pi- or fe-deficient plant.
the functional divergence of rp paralogs could be attributable to genetically- or epigenetically-driven neofunctionalization and/or subfunctionalization, leading to highly specialized paralogs associated with pi- and fe-deficient processes.
these findings may have important implications for understanding the molecular functions of rp genes in response to changing environmental conditions.

 ribosomes are large ribonucleoprotein complexes which synthesize the vast majority of cellular peptides. the structure of ribosomes is essentially the same in prokaryotes and eukaryotes in that they are composed of ribosomal rnas and ribosomal proteins, organized in a large and a small subunit. however, ribosomes of eukaryotes exhibit greater structural diversity, reflecting the higher complexity of the molecular mechanism underlying translation in the latter group. in arabidopsis thaliana, cytoplasmic ribosomes are composed of four distinct rrnas and distinct ribosomal proteins. the rps are encoded by a total of genes; thus, several paralogous genes encode the same rp. these paralogous genes could be redundant or, as indicated for certain rp genes, may be involved in specific plant processes or developmental stages. for example, among the four paralogous genes encoding rpl one gene was highly expressed in cotyledons, whereas the other three genes were preferentially expressed in roots, developing anthers and pollen. in addition, it has been reported that a mutation in rpl23aa lead to impaired growth and developmental abnormalities, suggesting that rpl23aa plays an essential role in fitness traits. by contrast, knockdown of the closely related gene family member rpl23ab had little effect on the phenotype. similarly, genes encoding rps15a were differentially expressed in arabidopsis, with one paralogous gene being completely transcriptionally quiescent, while the other three were highly expressed in mitotically active regions. ribosomal proteins are essential for protein synthesis and, consequently, play an important role in metabolism, cell division, and growth. in addition to their housekeeping functions, the phenotypes resulting from mutations in several different rp genes provide strong evidence that rps participate as regulatory components in developmental processes. generally, rp mutants share developmental abnormalities such as reduced shoot growth, reduced cell proliferation and increased nuclear ploidy in leaf cells. for example, a semi-dominant mutation in rpl27ac affected multiple aspects of plant shoot development, including leaf patterning, inflorescence and floral meristem function, as well as seed set. silencing of rps disturbed the ratio between the small and large subunits of mitoribosomes, causing an excess of the latter. introducing rps antisense and rpl23aa rnai constructs resulted in an altered number of cotyledons. also, a dominant missense mutation in rpl10a suppressed stem-elongation in comparison with the wild type. less severe phenotypes were reported for mutations in other rp genes. pointed first leaf encodes the small subunit rps. pfl mutants showed changes in the shape of early vegetative leaves from the spatulate wild-type shape to a pointed, narrow shape. mutations in the pfl gene caused a delay in the transition to flowering and the production of more vegetative leaves than in the wild type. in addition to effects on plant development and growth, rps also took part in the response to stress. under uv-b stress, rpl genes were differentially regulated in a dosage- and time-dependent manner; while rpl10c was induced and rpl10b was down-regulated at high uv-b intensity, rpl10a was not responsive to uv-b. another study showed that specific rps changed in abundance in response to sucrose feeding, implying that different rps are incorporated into ribosomes depending on the growth condition. transcripts of rps15af increased following treatment with cytokinin 6-benzylaminopurine and auxin indole acetic acid, while abscisic acid treatment decreased transcript abundance. in addition, transcripts of rps15aa, rps15ad and rps15af showed increased abundance upon temperature and mechanical stress. phosphate is an essential macronutrient for plants. in addition to its structural role in nucleic acids and cell membranes, pi has important functions in lipid and energy metabolism. limited bioavailability of pi often restricts growth in natural and agricultural ecosystems. due to intense use of p fertilizers in agriculture, pi resources have become limited. therefore, understanding how plants adapt to low pi availability is of critical importance to generate pi-efficient plants. as a coping mechanism to limited pi supply, plants undergo dramatic changes in metabolism, physiology, hormonal balance and gene expression. similarly, iron is essential for the survival and proliferation of virtually all organisms. iron is required for many vital enzymatic reactions such as dna replication, lipid metabolism, and nitrogen fixation. in plants, fe deficiency is a common nutritional disorder worldwide, causing low yield and quality of crop plants. to acquire sufficient fe while avoiding toxicity, plants tightly control uptake, utilization, and storage in response to environmental availability. at present, most studies on plant rps focus on the developmental effects of rp mutants. we recently showed that fe deficiency alters the expression of several rps, presumably resulting in biased translation. in the present study, changes in global expression pattern of rp genes in arabidopsis roots upon pi and fe deficiency were compared at the transcriptomic and proteomic level in order to gain insights into the regulation of the response to pi or fe deficiency. it is shown that the expression of rps followed stress-specific pattern, revealing a novel layer of regulation that may be critical for the fitness of plants under nutrient shortage.  comprehensive expression analysis of ribosomal protein genes in arabidopsis roots the rna-seq technology has proven to provide precise gene expression information and accurate discrimination of differentially expressed genes under various conditions. using this technology, we previously analyzed global gene expression changes upon pi-deficiency and fe-deficiency in arabidopsis roots. a total of genes were detected in roots from pi-deficient and pi-sufficient plants by alignment to the tair annotation of the arabidopsis genome. a set of genes was differentially expressed under pi deficiency. using the same criteria, a total of genes were found to be differentially expressed between fe-sufficient and fe-deficient conditions among the detected genes. thus, a comparable number of genes were affected in both growth types. a subset of genes was differentially expressed under both conditions. ribosomal proteins play critical roles in ribosome biogenesis, translation and post-translational modifications of proteins. a complete sequence data set for the known rp families was compiled from the tair release of the arabidopsis genome, this data set comprises all arabidopsis paralogous genes associated with any specific rp family and other ribosome-related genes including eif, rack and fer, which was shown to be tightly associated with ribosomes. we defined the expression level of all rp genes in this data set according to an earlier study. if the number of unique reads was zero in all three biological repeats under normal condition, the gene was defined as not expressed; low abundance of a transcript was defined by a number of unique reads < in either of the three biological repeats; medium abundance was defined for genes with to reads, and genes with more than reads in either of the three repeats were defined as highly expressed. on the basis of these criteria, in arabidopsis roots subjected to pi deficiency seven rp genes were not detected, transcripts of rp genes were lowly abundant, rp genes showed medium transcript levels, and rp genes were highly expressed. the average read number of rp genes was; while on average reads were associated with non-rp transcripts, indicating that rp paralogs were highly expressed in arabidopsis roots. previous data suggested that rp genes are retained in the arabidopsis genome at higher levels than other types of gene, suggesting a high biological importance of this plant-specific diversity of rp genes. in growing yeast cells, ribosomes were produced per minute, and transcription of ribosomal rna and rp genes account for 60% of total transcription. the synthesis of rps, ribosome assembly factors, and extra-ribosomal components involved in translation must simultaneously satisfy the plant’s demand for metabolism and growth, comprising an important component of the total cellular translational activity. in many cases, different rp paralogs from the same rp families displayed differential expression. for example, among three paralogs of the rpl family, rpl18b and rpl18c were highly expressed with more than reads, while the third member of this family, rpl18a, was not expressed. by analyzing public transcriptomic data, we found that expression of rpl18a is particularly low among all tissues and treatments, whereas rpl18b and rpl18c are highly expressed in some tissues. this suggests that rpl18a may be not functional or of minor importance, although retained in the genome during evolution. pronounced differences were also observed for the expression of genes encoding the proteins of the large subunits l l l27a, l l37a, and l in a previous report, rps15aa, rps15ad and rps15af, but not rps15ac transcripts were detected in all mature tissues by semi-quantitative rt-pcr. taken together, the differential expression of genes encoding rps within specific ribosomal gene families indicates extensive ribosome heterogeneity at the intercellular level. within the rp gene families with high read numbers in arabidopsis roots were six small and seven large ribosomal subunit proteins. the highest expression level was observed for s3a, which was detected with an average read number of for each paralog. high steady state levels of some rp gene transcripts may be indicative of fundamental roles of the encoded proteins in plant growth and development. at the protein level, for rp genes and ribosome-related genes a total of cognate proteins, accounting for nearly 50% of the detected genes, were identified in a proteomic approach. this accounts for 75% of the large subunit proteins, % of the small subunit proteins, and 100% of the ribosome-related proteins. these results suggest that some rp families are necessary for translation in arabidopsis roots, but not all of the paralogous proteins within one rp family take part in this process. our data further demonstrate that rps were more likely to be detected among the highly expressed genes than in genes with transcripts of medium or low abundance. notably, highly expressed rp genes had no cognate peptides, supporting the observation that the gene expression level is not always correlated with protein abundance. many rp paralogs might have been detected, but without unique matches due to their high sequence identity. for example, for three paralogous proteins of the rpl family, rpl6a, rpl6b and rpl6c, matching peptides have been detected, but only for rpl6a seven unique peptides were found, defining rpl6b and rpl6c as not present at the protein level although their corresponding transcript are high. gene ontology enrichment analysis revealed that the products of most of the rp genes were localized in the cytosolic small and large ribosomal subunits, especially proteins encoded by medium and highly expressed genes. in addition, some biological processes such as ’cellular response to uv-b’, ’response to cold’, and ’response to salt stress’ were overrepresented among the highly expressed genes, indicating that rps may also participate in the response to abiotic stresses. in addition, the go categories ’seed germination’, ’embryo development ending in seed dormancy’, and ’leaf morphogenesis’ were overrepresented in this group of rp genes. in previous studies, mutations in some rp genes caused an embryo-lethal phenotype,; thus, shortage in specific rps could lead to severe growth defects. mutations in the rp genes s and s were reported to cause abnormal leaf development. changes in leaf morphology in rp gene mutants might be caused by a diminished capacity to maintain protein synthesis at a level sufficient for proliferative cell divisions in the vasculature and palisade mesophyll, and for cell division progression at the leaf margin. expression of rp genes in pi-deficient arabidopsis roots out of the rp genes and six ribosomal-related genes annotated in tair only three were differentially expressed between pi-sufficient and pi-deficient arabidopsis roots, all of which were up-regulated. among the three genes, induction of at1g was more pronounced when compared with the other two genes, at3g and at3g. in rice flowers, rpl35a was highly expressed under high temperature after the flowering stage, suggesting that this rp family is not only involved in pi deficiency but may also play a role in heat stress response. in a previous report, transcript levels of rps rpl and rpl13a from lupine cotyledons were found to be increased during cytokinin treatment relative to a 0 h water control. expression of rps was decreased after 5 h of incubation with aba, indicating that rps may have different functions in abiotic stresses. the rapid accumulation of genome-wide gene expression data allows the creation of co-expression networks by examining the pair-wise relationship of genes over a large number of experimental conditions. in the co-expression network, a node is represented by a gene, and an edge is drawn between gene a and b if the correlation coefficient between these two genes is above a defined threshold. genes with similar expression pattern may be involved in the same biological process and potential functions of the unknown genes in a co-expression network can be predicted.. in an attempt to identify pi-responsive non-rp genes that are co-regulated with rp genes, we analyzed the correlation of the expression of the differentially expressed genes including three rp genes. the results show that only one rp genes interacted with the 2-oxoglutarate and fe -dependent oxygenase superfamily protein at3g with a pearson correlation coefficient cutoff of. with this cf threshold decreased to, all three differentially expressed rp genes and other differentially expressed genes including some transcription factors and protein kinases could form a larger co-expression network; and these genes were involved in diverse biological processes. at the protein level, rps accumulated differentially upon pi deficiency. among them, only rpl12b was up regulated, all other proteins showed decreased abundance. interestingly, among the three paralogous proteins in the rpl family, only rpl12b was induced, while the abundance of the other two rpl members was unchanged under pi-deficient conditions. this suggests that rpl12b may play an important role in the pi-stress response. to get a better understanding of the function of differentially expressed rps, a protein-protein interaction network was constructed using the string database the network is presented in the ’evidence’ view, whereby lines connecting proteins represent the type of evidence used in predicting the association. all differentially expressed rp proteins were clustered in a large protein-protein interaction module. alternative splicing is a mechanism that increases transcriptome and proteome complexity and controls developmental programs and responses to the environment in higher eukaryotes. previously, genome-wide differential as features induced by pi deficiency have been analyzed in arabidopsis roots. further analysis revealed that a subset of rp genes produced transcripts with retained introns; all of these intron retention events were induced by pi shortage. three out of the encoded rps were decreased in abundance under pi deficiency. these results suggest that post-transcriptional regulatory processes such as alternative splicing may have affected the efficiency of translation. expression of ribosomal protein genes in fe-deficiency arabidopsis roots using the criteria listed above, a total of rp genes were differentially expressed in fe-deficient arabidopsis roots. in contrast to the up-regulated pattern of differentially expressed rp genes in roots of pi-deficient plants, expression of all the rp genes was decreased upon fe deficiency. the rp gene with lowest relative expression level was the ribosome-associated protein gene ferritin, which was found in small polysome fractions isolated form the leaves of arabidopsis plants in the dark. fer was reported to be associated with the chloroplast and suggested to mediate coordination between the translation activity of cytosolic ribosomes and the function of chloroplasts in response to iron-based signals. thus, decreased expression levels of fer under fe deficiency may be associated with the regulation of cellular fe homeostasis. gene ontology analysis revealed that the differentially expressed rp genes were chiefly involved in translation and ribosome biogenesis. in addition, the biological processes ’response to salt stress’, ’cellular response to uv-b’, and ’leaf morphogenesis’ were also enriched. the differentially expressed genes were derived from small and large ribosomal subunit rps, in which the transcript abundance of all paralogous genes from five rp families, was decreased. in the rps15a family, three of the six paralogous genes were down-regulated under fe deficiency. these three genes showed increased transcript abundance following treatment with bap, rps15af was also induced by iaa treatment. a similar trend of expression in paralogous genes with rps15a family was observed under heat and cold stress. it might thus be assumed that rps15a is an important regulator involved in multiple abiotic and nutrient stresses. co-expression analysis of the differentially expressed rp genes under fe deficiency yielded a network consisting of nodes and edges. this network could be further divided into one larger and one smaller clusters, the larger of which contained much more nodes and edges, and every node was correlated with other rp genes. similarly, with the aim of identifying other fe-responsive non-rp genes that interact with rp genes, we put the differentially expressed genes including rp genes together to do the co-expression analysis. results showed that a total of rp genes and fe-responsive non-rp genes comprise one bigger and two smaller clusters with pearson correlation coefficient cutoff of. proteomic profile analysis showed that only two rps were differentially expressed between fe-deficient and fe-sufficient arabidopsis roots. the two proteins were from the rpl family and showed decreased abundance, although the transcriptional expression of these two genes remained unchanged. analysis of as revealed three rp genes with differential alternative donor/acceptor splices sites and six rp genes with differential intron retention in roots of fe-deficient plants. none of these rp genes were transcriptionally or translationally fe-responsive. comparative expression pattern of rp genes under pi and fe deficiency comparison of the differentially expressed rp genes and rps between pi- and fe- deficient arabidopsis roots showed no overlap between the two growth types, suggesting that distinct rps are involved in the responses of plants to pi- and fe-deficient. interestingly, most rp genes encoding proteins of the large subunit were affected by pi deficiency, while the majority of differentially rp genes encoding rps under fe deficiency were from both the small and the large subunit. furthermore, the expression pattern of rps upon pi deficiency was more complex than that observed under fe-deficient conditions. nevertheless, one of the common observations for these two treatments is that there was a large discrepancy between the rp genes and pr proteins affected by pi deficiency as well as under fe deficiency conditions. our previous studies have found that the concordance between differences in the abundance of mrna and its encoded protein is not always high due to several factors: firstly, proteins with low abundance might not be reliably detected and thus did not match the criteria that define them as differentially accumulated whereas the transcript levels of the corresponding genes are significantly changed. secondly, several proteins with high abundance accumulated differentially upon fe/pi deficiency, but they have important housekeeping functions and the transcript levels were not changed. finally, a change in transcript abundance may or may not be translated into changes in protein level; posttranscriptional regulatory processes such as alternative splicing, rna processing, and other processes may affect the efficiency of translation. a generally low congruency of proteomic and transcriptional profiles has been reported previously.  in summary, our results suggest that the composition of ribosomes is changed in a stress-specific manner as part of an adaptation to pi and fe deficiency, probably biasing protein translation. our findings further suggest that rps are not only involved in protein translation but also in the response of plants to nutrient deficiency, possibly altering the composition of ribosomes in roots of pi- or fe-deficient plant. the functional divergence of rp paralogs could be attributable to genetically- or epigenetically-driven neofunctionalization and/or subfunctionalization, leading to highly specialized paralogs associated with pi- and fe-deficient processes. these findings may have important implications for understanding the molecular functions of rp genes in response to changing environmental conditions. 
transcripts from and rp genes showed low and medium abundance, respectively; rp genes were highly expressed in arabidopsis roots. these findings may aid in uncovering an unexplored mechanism by which plants adapt to changing environmental conditions. our study shows that the expression of paralogous genes encoding rps was regulated in a stress-specific manner in arabidopsis roots, presumably resulting in an altered composition of ribosomes and biased translation. little information is available on how rp genes respond to a shortage of essential mineral nutrients such as phosphate or iron. in the present study, the expression of rp genes and the differential accumulation of rps upon pi or fe deficiency in arabidopsis roots were comprehensively analyzed. among the rp genes retrieved from the tair release of the arabidopsis genome, seven rp genes were not detected in arabidopsis roots by rna sequencing under control conditions. as anticipated, gene ontology analysis indicated that most rp genes were related to translation and ribosome assembly, but some of the highly expressed rp genes were also involved in the responses to cold, uv-b, and salt stress. only three rp genes derived from three ‘sets’ of paralogous genes were differentially expressed between pi-sufficient and pi-deficient roots, all of which were induced by pi starvation. in fe-deficient plants, rp genes from ’sets’ of paralagous rp genes were significantly down-regulated in response to fe deficiency. the biological processes ’translation’, ’ribosome biogenesis, and ’response to salt, cold, and uv-b stresses ’ were enriched in this subset of rp genes. neither the differentially expressed rp genes nor the differentially expressed rps showed any overlap between the two growth types. in the present study three and differentially expressed rp genes were identified under pi and fe deficiency, respectively. at the protein level, and two rps accumulated differentially under pi- and fe-deficient conditions, respectively. at protein level, and two rp proteins were differentially accumulated under pi- and fe-deficient conditions. arabidopsis thaliana ribosomes are composed of distinct ribosomal proteins, each of which is encoded by two to seven highly similar paralogous genes. comparison of pi-responsive genes with fe-responsive genes revealed an overlap of genes that were differentially expressed under both conditions in arabidopsis roots.
 ribosomes are essential ribonucleoprotein complexes that are engaged in translation and thus indispensable for growth. arabidopsis thaliana ribosomes are composed of distinct ribosomal proteins, each of which is encoded by two to seven highly similar paralogous genes. little information is available on how rp genes respond to a shortage of essential mineral nutrients such as phosphate or iron. in the present study, the expression of rp genes and the differential accumulation of rps upon pi or fe deficiency in arabidopsis roots were comprehensively analyzed.  comparison of pi-responsive genes with fe-responsive genes revealed an overlap of genes that were differentially expressed under both conditions in arabidopsis roots. gene ontology analysis revealed that these genes were mainly associated with abiotic stress responses. among the rp genes retrieved from the tair release of the arabidopsis genome, seven rp genes were not detected in arabidopsis roots by rna sequencing under control conditions. transcripts from and rp genes showed low and medium abundance, respectively; rp genes were highly expressed in arabidopsis roots. as anticipated, gene ontology analysis indicated that most rp genes were related to translation and ribosome assembly, but some of the highly expressed rp genes were also involved in the responses to cold, uv-b, and salt stress. only three rp genes derived from three ‘sets’ of paralogous genes were differentially expressed between pi-sufficient and pi-deficient roots, all of which were induced by pi starvation. in fe-deficient plants, rp genes from ’sets’ of paralagous rp genes were significantly down-regulated in response to fe deficiency. the biological processes ’translation’, ’ribosome biogenesis, and ’response to salt, cold, and uv-b stresses ’ were enriched in this subset of rp genes. at the protein level, and two rps accumulated differentially under pi- and fe-deficient conditions, respectively. neither the differentially expressed rp genes nor the differentially expressed rps showed any overlap between the two growth types.  in the present study three and differentially expressed rp genes were identified under pi and fe deficiency, respectively. at protein level, and two rp proteins were differentially accumulated under pi- and fe-deficient conditions. our study shows that the expression of paralogous genes encoding rps was regulated in a stress-specific manner in arabidopsis roots, presumably resulting in an altered composition of ribosomes and biased translation. these findings may aid in uncovering an unexplored mechanism by which plants adapt to changing environmental conditions. iron deficiencyphosphate deficiencyproteomicsribosomal proteinsrna-seqtranslation 