BACKGROUND
nitrogen  represents one of the main minerals required throughout plant development.
in agronomic terms, this results in a worldwide ever-increasing use of fertilizers and its consequent environmental and socioeconomic costs  <cit> .
this n requirement is emphasized with regard to cereal crops  <cit> , for which maize  is a model species because of its economic importance and high metabolic capacity  <cit> .
in agricultural soils the main mineral n sources are nitrate  and ammonium .
in order to balance their n nutritional requirements with environmental availability, plants have to modulate the individual steps of n metabolism such as up-take, reduction of no3− to nh4+, nh4+ assimilation and n recycling.
the contribution of root and leaf systems depends on species, developmental stage and environmental conditions  <cit> , and it is also deeply influenced by c metabolism  <cit> .
all the nh4+ in the cell, derived from soil, from no3− reduction or from other metabolic processes, is channelled through the glutamine synthetase  reaction.
the gs catalyzes the fixation of nh4+ on glutamic acid  to form glutamine , and in the assimilation process it is generally coupled with plastidial glutamate synthase  that incorporates c skeletons.
gln and glu can be recruited as amino group donors as well as main n transport molecules  <cit> .
several evidence indicate that gs activity is deeply influenced by metabolic and environmental factors mainly linked to the balance between c and n metabolism  <cit> .
for instance, glu level seems to be fundamental in sensing plant nutritional status and in joining c and n metabolisms  <cit> .
moreover, the inter-conversion with other amino acids greatly influences n plant economy, especially regarding asparagine  and alanine   <cit> .
plant responses are deeply affected by the proportion of mineral n sources  <cit> .
while nh4+ as sole nutrient can induce toxicity symptoms, its co-provision with no3− generally promotes a synergistic effect leading to growth enhancement  <cit> .
it is noteworthy that nh4+ tolerance was related to high root n metabolism sustained by high gs activities  <cit> , which in maize appear to be associated with the capacity to cope with the c skeleton demands  <cit> .
the main gs are decameric enzymes  <cit>  classified on the basis of subcellular localization in cytosolic gs <dig> and plastidial gs <dig>  in plants, multigenic families encode several gs <dig> isoforms while the plastidial gs <dig> derives from one or few nuclear genes.
in general, gs <dig> is associated with the leaf nh4+ assimilation while gs <dig> is associated with plant n recycling.
but the relative activity of gs <dig> and gs <dig> is variable according to the species, organs, n sources, developmental stages and environmental conditions, suggesting a multifaceted participation of isozymes  <cit> .
moreover, recent studies conducted both in dicotyledonous  <cit>  and in monocotyledonous crops  <cit>  showed non-overlapping functions for the gs <dig> isoforms.
besides, distinct post-translational modifications were described for both isoenzymes  <cit> .
in maize, one gene for gs <dig>  and  <dig> genes codifying for different gs <dig> subunits were identified, named from gs1- <dig> to gs1- <dig> according to the reviewed uniprotkb/swiss-prot database  <cit>  .
gs <dig> and gs <dig> are differentially regulated in roots and leaves in response to growing conditions.
the cytosolic isoforms also have different kinetic properties, stabilities and tissue localizations  <cit> .
by means of quantitative trait loci analyses and characterization of maize mutants, hirel and co-workers indicated the key roles of gs1- <dig> and gs1- <dig> both in grain yield and germination  <cit> .
gs1- <dig> and gs1- <dig> represent the major leaf isoforms  <cit>  and in maize mutants the deficiency of these enzymes affects leaf gene transcripts, proteins and metabolite accumulations  <cit> .
moreover, the transcript localizations confirmed the involvement of gs1- <dig> in root metabolism and suggested that gs1- <dig> acts in n phloem translocation  <cit> .
it is worth noting that in mutants deficient for gs1- <dig> and gs1- <dig> the dry weight and total n content in the shoot vegetative parts were unaltered, providing evidence of how such parameters are prevalently determined by root metabolism  <cit> .
this observation, together with the finding that the n stored before silking supplies up to 70% of the grain n content  <cit> , draws attention to the need to study the root system during the early phases of maize development.
after a first localization of gs1- <dig> and gs1- <dig> in tip and/or cortex tissues  <cit> , root gs arrangement had scarcely been investigated, probably due to technical limitations.
n availability is associated with the accumulation of a specific root isoform  that is theoretically assigned to gs1- <dig>  but had not been precisely characterized.
similarly, the responses to different n sources were not fully elucidated and information about gs1- <dig> was still lacking.
by the means of two-dimensional western blotting  and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry  techniques, this work profiles the gs patterns in maize roots in response to no3−, nh4+ or both, during vegetative growth, describing for the first time the differential modulations of cytosolic and plastidial forms and the active involvement of gs1- <dig>  moreover, the determination of amino acid composition in roots, xylem sap and leaves provides new information about the roles of gln, glu, asn, and ala metabolisms in plant c/n balance.
RESULTS
effects of the nutritional treatments on leaf and root metabolic status
the aim of this work was to investigate the responses of the different root gs isoforms in maize plants exposed to different inorganic n sources, during early vegetative growth.
to better appreciate the effects at metabolic level, the changes in plant amino acid balance were also evaluated.
plantlets of the t <dig> inbred line were grown in a hydroponic system in the absence of n for 10 days to reach a developmental stage corresponding to the third-leaf expansion .
since in field conditions maize n fertilization consists of a single application at sowing  <cit> , the third-leaf stage corresponds to a vegetative phase in which plants are exposed to a high level of inorganic n and that is indicated as one of the more susceptible to nh4+ toxicity  <cit> .
moreover, it is important to note that the optimal dose of no3− fertilization also depends on maize varieties  <cit> .
in order to better appreciate the short-term responses and compare our proteomic results with previous works, the plants were exposed to a total n availability of 10 mm.
in details, plants were exposed for 30 h to four nutritional treatments: n absence , 10 mm no3− , 10 mm nh4+ , 5 mm no3− + 5 mm nh4+ .
the concentration of no3− and nh4+ in roots, xylem saps and leaves were measured , together with the content of the reducing sugars and sucrose in root and leaf systems .
moreover, the accumulation of the main n assimilative enzymes such as nitrate reductase  and gs in roots and leaves was estimated by one-dimensional western blotting  .
the analysis conducted against nr in roots detected the expected single band at 99 kda  while in leaf profiles two bands were visible .
the lower band at about 94 kda, corresponding to pyruvate phosphate dikinase  that is the most abundant enzyme in maize leaves , was considered as an unspecific signal.
on the base of molecular masses, the three bands in gs profiles  were assigned to gs <dig> , to gs <dig>  and to the root isoform gs1r , as described by sakakibara and co-workers  <cit> .
according to previous works  <cit> , higher levels of gs <dig> were detected in root profiles  while gs <dig> was predominant in leaves .figure  <dig>
nitrogen and carbon metabolites in maize in response to different inorganic n sources.
concentration of no3
−
, nh4
+
, reducing sugars  and sucrose  in roots , xylem sap  and leaves  in maize plants grown for 10 days without n sources and then exposed for the last 30 h to absence of n , to 10 mm no3
− , to 10 mm nh4
+  or to 5 mm no3
− + 5 mm nh4
+ .
graphs show average values ± se .
the upper letters indicate differences among the four treatments within each organ according to student’s t-test .
accumulation of n assimilatory enzymes in maize in response to inorganic n sources.
1d-wb performed against nr  and gs  in roots  and leaves  of maize plants grown without n sources for 10 days and then exposed for the last 30 h to absence of n , to 10 mm no3
− , to 10 mm nh4
+  or to 5 mm no3
− + 5 mm nh4
+ .
the analyses were conducted twice on two independent biological samples  for each condition.
b and d profiles were visualized by a global antibody for both gs <dig> and gs <dig> that recognized three different bands.
the molecular masses of electrophoretic standards are reported on the left .
it appeared evident that the exposure of the plants to no3− induced strong accumulation of the anion in all organs and especially in roots, reaching the value of  <dig>  ±  <dig>  μmol g−1fw in  plants .
this trend was similar to that observed in t <dig> by previous time-course experiments  <cit> .
the increment of no3− in xylem sap attested the concomitant induction of anion translocation.
in particular, in  and  xylem saps the values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μmol g−1fw, respectively , quite a lot higher than the  <dig>  mm value observed in maize crops  <cit> , suggested an extensive plant response to restoring tissue n levels.
the n management in plants exposed to nh4+ was different.
both  and  plants did not show any toxicity symptoms and the nh4+ root accumulation and translocation incremented proportionally to the medium concentrations .
on the contrary, the nh4+ levels in leaf systems were scarcely affected especially in comparison with roots in which nh4+ reached the highest value of  <dig>  ±  <dig>  μmol g−1fw in  condition .
in several plant species, a similar limitation of nh4+ translocation to the shoot, considered more sensitive to nh4+ than roots  <cit> , was associated to a high rate of metabolisation by gs in roots, which was proposed as one of the main traits of tolerance to high nh4+ inputs  <cit> .
finally, no synergistic effect on the ions accumulation emerged .
the availability of n led to an increase of both reducing sugars and sucrose levels in leaves, especially in response to nh4+ exposure .
however, only in the roots of  plants a peculiar doubling of reducing sugars  was observed .
considering that the sucrose slightly increased, it is possible that this increment originated from the delivery of photo-assimilates from the shoot.
interestingly, this trait was almost completely alleviated by the co-provision with no3− .
in a comparative study among maize genotypes, the physiological traits of nh4+ tolerance seemed to rely on the plant’s capability to shift the partitioning of carbohydrates towards the root system in order to sustain nh4+ assimilation  <cit> .
hence, the results confirmed that the t <dig> line shows some physiological traits typical of tolerant maize cultivars, especially those deriving from a high capability for nh4+ assimilation by the root system.
at the same time, the reduction of reducing sugars in  roots  indicated that the roots were also involved in no3− assimilation, as proposed by prinsi and co-workers  <cit> .
the 1d-wb supported the induction of the assimilatory pathways .
the nr accumulated proportionally to the no3− concentration in the external medium both in roots and leaves .
similarly, the gs <dig> levels in roots  responded to no3− availability.
this response was scarcely detected in leaves, where the enzyme level was quite constant .
this behaviour is similar to that observed in young vegetative maize plants grown at high and low n fertilization in the field  <cit> .
interestingly, in comparison to  plants, the exposure to nh4+ led to a reduction of nr level in roots and to a slight increment in leaves.
together with the increment of the no3− accumulated in the shoot , these results suggest that the nh4+ availability induced a shift in plant n economy, promoting the usage of no3− reserves by the shoot.
finally, the gs <dig> enzyme accumulated in response to the increasing cell nh4+ contents, by the specific induction of the gsr isoform .
in summary, the plant metabolic status results confirmed that the four experimental treatments induced the assimilation and translocation of the different n sources, highlighting the fundamental role of the root system in the plant physiological adaptation to both no3− and nh4+ nutrition.
characterization of the gs isoforms in maize roots
in order to distinguish at protein level the different gs isoforms accumulated in the maize root system, the wb with the gs global antibody  was applied on the 2d-electrophoretic map of root soluble proteins.
a preliminary investigation conducted in a wide ph range of 3– <dig> on 10% acrylamide sodium dodecyl sulphate-polyacrylamide gel electrophoresis  indicated that all recognizable isoforms were embraced in the acidic portion of the profile and in a molecular range of 35–50 kda .
to improve the analytical resolution, the next protein separations were performed in the narrower 4– <dig> ph range.
this approach allowed a good reproducibility among the four nutritional treatments , providing an overall proteomic map composed of about 1210 ±  <dig> spots .
considering the electrophoretic adjustments, this map compared well with the one proposed by prinsi and co-workers  <cit> .
the 2d-wb showed a unique pattern , recurrent in all experimental conditions, consisting of six stains that were numbered from  <dig> to  <dig> and assigned to six spots in the 2d-electrophoretic maps .
these  <dig> spots  were separately analysed by lc-nesi-ms/ms.
each spot was assigned to a specific gs isoform according to the discriminating peptides sequenced.
since for every reference spot  the assignment was independently confirmed among the four 2d profiles, the data were then pooled to get the overall spot characterization .
the spots  <dig>   <dig> and  <dig> were identified as different gs <dig> forms from the same gene, named gs2a, gs2b and gs2c .
this observation was in agreement with the fact that two distinct gs <dig> forms were highlighted in the maize leaf proteome  <cit> .
here, as well as in several other plant species, the gs <dig> protein forms exceed the number of encoding genes .
as a whole, this trait supports the presence of post-transcriptional/translational modifications .
the similar incidence of specific peptides for both the two isoforms revealed that spot  <dig> derived from an overlapping of gs1- <dig> and gs1- <dig>  explainable by the almost identical isoelectric point that made the two proteins inseparable by denaturing electrophoresis , as previously noted by martin and co-workers  <cit> .
conversely, spots  <dig> and  <dig> were distinctly identified as gs1- <dig> and gs1- <dig> isoforms, respectively .
this proteomic investigation revealed a pattern of gs accumulation very consistent with the expression profile proposed by li and co-workers  <cit> .
these authors, by means of a comprehensive analysis of the transcript levels of the six gs genes in maize, provided evidence that all isoforms were expressed in the roots, with the prevalence of gs1- <dig>  gs1- <dig> and gs1- <dig> mrnas.
at the same time, the fact that gs1- <dig> was characterized as a low abundance and vascular-specific isoform  <cit>  might explain why it was not detectable.figure  <dig>
localization of the gs isoforms in 2d profile of maize roots by western blotting.
representative 2d-electrophoretic map of soluble protein fraction from roots of maize plants.
proteins were analyzed by isoelectric focusing at ph 4– <dig> and 10% sds-page and visualized by ccbb staining .
for each nutritional treatments, the gel portion comprised in the broken-line was analyzed by wb against all gs .
the analyses were conducted twice on two independent biological samples  showing the same profile among all the experimental conditions.
the visualized spots are numbered  and traced on the 2d-electrophoretic map.
the lc-nesi-ms/ms characterizations of the six spots are reported in table  <dig>  the molecular masses of electrophoretic standards are reported on the left .
spot assignments to the specific isoform of glutamine synthetase  by lc-nesi-ms/ms
n
acr
protein an
pi/mw
pi/mw
u.p.
s/t
%cov.
avg n. p.
1
gs2a
2
gs2b
3
gs2c
4*
gs1-3/4
5
gs1-5
6
gs1-1
the different spots indicated in figure  <dig> were separately collected from the root 2d profile of each nutritional treatment.
values are the mean ± se of four independent biological samples  analysed in triplicate .
4*: the spot was assigned as a co-migration of the gs1- <dig> and gs1- <dig> isoforms.
n: spot number on figure  <dig>  acr: acronym reported in figure  <dig>  an: accession number.
p
i
/mw are expressed in kda and compare the theoretical values  with the experimental ones ; u.p.
: number of unique peptides identified, s: specific to the isoform, t: total.
%cov: amino acid coverage.
avg n. p.: number of distinct peptides.
detailed information on lc-nesi-ms/ms sequencing is reported in additional file  <dig>
considering the electrophoretic positions of the gs proteins in roots, it was possible to conclude that the middle band observed in the 1d-wb  included gs1- <dig> and gs1- <dig>  while the gsr form , accumulated in response to external n availability, was composed by gs1- <dig> with gs1- <dig>  this conclusion was previously suggested  <cit> , but to our knowledge, this proteomic approach allowed the first discrimination between the individual responses of gs1- <dig> and gs1- <dig>  the accumulation levels of the six gs proteins  in the 2d profiles showed different and specific changes in responses to inorganic n sources .
moreover, the 2d profiles were staining with the pro-q® diamond that is specific for phosphoproteins  <cit> , applying this approach to investigate the phosphorylation state of gs isoforms, to our knowledge for the first time.
the results showed that four of the six spots were phosphorylated and that the spot phosphorylation state was constant among all conditions .
the results, together with the evaluations of root gs activities , contributed to improve the characterization of the six proteins.figure  <dig>
accumulation of the gs isoforms in maize roots in response to n sources.
the graphs report the levels  of the different isoforms evaluated in the root 2d-electrophoretic profiles stained with ccbb  of maize plants grown without n sources for 10 days and then exposed for the last 30 h to absence of n , to 10 mm no3
− , to 10 mm nh4
+  or to 5 mm no3
− + 5 mm nh4
+ .
the graph number and isoform acronyms refer to figure  <dig> and table  <dig>  respectively.
a. spot  <dig> .
b. spot  <dig> .
c. spot  <dig> .
d. spot  <dig> .
e. spot  <dig> .
f. spot  <dig> .
graphs show average values ± se .
the upper letters indicate differences among the four treatments according to student’s t-test .
analysis of phosphorylation state of root gs isoforms.
the 2d-electrophoretic profiles in 4– <dig> pi range of soluble proteins from root of maize plants grown in the different experimental conditions were stained with sequential fluorescence staining procedures.
a. representative magnification of the profile containing the gs isoforms stained with sypro ruby®, showing all the proteins.
b. representative magnification of the same gel portion after the pro-q® diamond phosphoprotein gel staining that points out only the phosphorylated proteins.
the experiment was conducted for each nutritional condition showing a similar profile for all the samples.
the spots are numbered  according to figure  <dig> and table  <dig>  the molecular masses of electrophoretic standards are reported on the left .
effects of the different inorganic n sources on gs activity in maize root.
maize plants were grown without n sources for 10 days and then exposed for the last 30 h to absence of n , to 10 mm no3
− , to 10 mm nh4
+  or to 5 mm no3
− + 5 mm nh4
+ .
both the gs synthetase  and the transferase activities  were evaluated and expressed as specific activity.
the synthetase activity was measured in the enriched fraction of plastidial proteins  and in total protein fraction  representing the gs <dig> activity and the total activity, respectively.
the transferase activity was measured in total protein fraction prior to and after the exposure of the samples to 45°c for 10 min.
the % thermal inactivation  represents the portion of transferase activity lost after this thermic treatment.
graphs show average values ± se .
the upper letters indicate differences among the four treatments according to student’s t-test .
the responses to inorganic n sources of the gs <dig> proteins in maize roots
gs <dig> was a very faint protein in the maize roots, with a maximum value of only  <dig>  ±  <dig>  %vol for gsb in  plants .
similarly, the highest gs synthetase activity of the plastidial fraction was detected in  plants, where it represented about the  <dig> % of the total enzymatic activity .
this proportion appeared similar to that measured in rice  roots, in which the contribution of gs <dig> was less than 4% of the total gs activity  <cit> .
in detail, while in  and  plants the plastidial activity was almost undetectable, it increased proportionally to no3− availability in  and  roots, reaching the values of  <dig>  ±  <dig>  and  <dig>  ±  <dig>  μmol mg−1prot min− <dig>  respectively .
these results strongly support the idea that, in maize roots, gs <dig> is involved only in no3− assimilation and it is not influenced by nh4+ nutrition.
among the three identified gs <dig> forms, the gs2b is the only one of which the accumulation level was significantly increased in plants exposed to no3−.
this observation confirms our former proteomic analyses  <cit> .
the comparison of gs <dig> accumulation levels  and gs <dig> activity  suggests the hypothesis that gs2b represented the form really participating in catalytic activity.
interestingly, this spot represented also the only phosphorylated gs <dig> .
the reciprocal position of the gs <dig> spots was comparable with those observed in the leaf maize proteome.
indeed, in leaves two gs <dig> spots were identified of which the accumulation levels were differently related to the enzymatic activity.
in particular, the more acidic spot, matching with gs2b, was accumulated proportionally, while the basic one disappeared with the increment of the enzymatic activity  <cit> .
the gs2c electrophoretic position  as well as its trend of accumulation  showed a high degree of similarity with this last observation.
as a whole, the results suggest that gs2a and gs2c might be transitional forms of the enzyme, probably originated by ptms.
the chemical and/or immunological detection of sugar and nitric oxide moieties did not lead to any positive results , but it is not possible to exclude the occurrence of several other ptms.
the responses to inorganic n sources of the gs <dig> isoforms in maize roots
according to the proposed model, the presence of phosphorylation on gs <dig> is associated with active enzymatic forms, where it promotes protection from degradation and the interaction with activating 14-3- <dig> proteins  <cit> .
the evaluation that the spots  <dig>   <dig> and  <dig> were phosphorylated in all conditions tested  supports the hypothesis that they represented active subunits of the root gs <dig> enzyme.
firstly, it is possible to note that all three gs <dig> spots were detected in  plants, in which spot  <dig> was predominant .
their presence in roots of starved plants confirmed a gs <dig> involvement in the use and/or recycling of endogenous n reserves.
spot  <dig> was the least influenced by the plant nutritional status, as it only decreased by about 20% in response to nh4+ .
this trend was in agreement with previous transcriptional analyses highlighting that gs1- <dig> and gs1- <dig> mrnas slightly decreased in response to n as well as with the unchanged intensity of gs <dig> band in sds-page .
together with the fact that in maize gln1- <dig> and gln1- <dig> mutants the vegetative biomass is not affected  <cit> , these results reinforce the hypothesis that other root isoforms are able to sustain n assimilation during vegetative growth.
interestingly, spots  <dig> and  <dig> showed marked and different changes .
the gs1- <dig> isoform specifically increased in response to nh4+ nutrition, becoming the most abundant one in  roots with the highest measured value .
it is worth noting that the changes in gs1- <dig> accumulation reflected the nh4+ availability, reaching an increment of about +112% and +54% in  and  plants, respectively.
the responses of gs1- <dig> isoform attested an even higher increase of about +222% in  condition.
likewise, in  plants the gs synthetase activity significantly increased .
these results confirm that nh4+ induces gsr  and, for the first time, they allowed us to discern the differential contribution of its components.
moreover, gs1- <dig> showed a peculiar doubling  in  plants.
the fact that gs1- <dig> is metabolically active in  roots is reinforced by the estimation that its change is the only one to be associated with the increase in total gs synthetase activity in the  condition .
interestingly, these observations confirm at protein and enzymatic order the induction of gs1- <dig> transcript by no3− exposure recently observed in the t <dig> line  <cit> .
taken together, these results provide the first information about the functional role of the gs1- <dig> isoform, providing evidence of its involvement in root no3− metabolisation.
the gs synthetase activity in the total protein fraction, starting from  <dig>  ±  <dig>  μmol mg−1prot min− <dig> in starved plants , increased to a similar extent in n treated ones .
this feature suggests that gs <dig> followed a saturation kinetic, probably because the elevated concentrations of no3− and/or nh4+ overfilled the metabolic capability of the maize root organ.
however, the root metabolic capability seemed to be sustained by different gs <dig> isoforms, according to different mineral n sources.
this was particular evident in  roots where gs1- <dig>  gs1- <dig> and gs <dig> were accumulated to an intermediate level .
in order to get better information about this aspect, the percentage of thermal inactivation of total gs transferase activity, induced by 45°c for 10 min, was evaluated .
according to the physicochemical characterization, the isoleucine- <dig> in the gs1- <dig> sequence confers thermal stability, while the substitution with ala- <dig> clearly renders the gs1- <dig> more heat-labile than gs1- <dig>  <cit> .
considering that gs1- <dig> and gs1- <dig> shared this feature with gs1- <dig> and gs1- <dig> respectively, it is possible to assume that the thermal inactivation measured reflected the proportion of gs activity ascribable to the gs1- <dig> with gs1- <dig> subunits.
such evaluation allowed us to confirm that gs1- <dig> and gs1- <dig> were fundamental for the assembling of gs active enzyme in  and  roots, where the thermal treatment provoked a loss in activity of about 97% and 98%, respectively.
it is also worth noting how the contribution of gs1- <dig> and gs1- <dig> gained in importance in roots of plants exposed to nh4+, especially if in co-provision with no3− .
overall, this proteomic investigation confirmed the gs <dig> involvement in n recycling as well as in the root assimilation of no3− and nh4+, and at the same time, it allowed us to propose that specific and combined isoforms sustain these different metabolic tasks.
the inorganic n sources differently affected the amino acid accumulation and composition in root, leaf and xylem sap
in order to appreciate the extent by which the gs activation induced by the experimental treatments affected the plant n metabolism, the composition of amino acids in different tissues was measured.
figure  <dig> points out the changes related to the mostly more abundant amino acids, gathering the others in a single group, detected in roots , in xylem sap  and in leaves  of plants exposed to the four n conditions.
the comprehensive amino acid compositions of the three tissues are detailed in the additional file 1: tables a <dig>  a <dig> and a <dig>  respectively.figure  <dig>
levels of amino acids in maize plants in response to inorganic n sources.
concentration of the main amino acids in roots , xylem sap  and leaves  in maize plants grown for 10 days without n sources and then exposed for the last 30 h to absence of n , to 10 mm no3
− , to 10 mm nh4
+  or to 5 mm no3
− + 5 mm nh4
+ .
graphs show the amino acid concentrations  as average values ± se .
the letters above are assigned according to student’s t-test .
detailed quantification of individual amino acids in root, xylem and leaf is reported in additional file 1: table a <dig>  a <dig> and a <dig>  respectively.
the provision of n provoked a significant increase in amino acid level at whole plant scale, but the extent of these increments varied in relation to the inorganic n source and/or organ.
the total amino acids concentration in roots and leaves of  plants was almost double the amounts in  plants, confirming that nh4+ nutrition promoted a more intensive n assimilation than no3− .
this aspect was mirrored by the total protein amounts in the root systems .
because the total amino acid concentration reached similar values both in roots and leaves during the  and  treatments , the t <dig> line did not show a marked synergistic response to the co-provision of no3− with nh4+.
gln was the amino acid subjected to the greatest and most prevalent changes after the exposure of the plants to n as well as being the main compound for n translocation in the xylem sap, in which it reached the maximum value of  <dig>  ±  <dig>  mm in  plants .
moreover, all three organs showed high amounts of ala, which represented up to 40%, 44% and 55% of the total amino acids in roots, xylem sap and leaves, respectively .
considering that previous studies on maize plants grown in high n reported ala percentages in xylem and leaves ranging from 5% to 20-29%, respectively  <cit> , the higher percentage of ala may be a peculiarity of the t <dig> line.
taking into account the involvement of ala and aspartic acid  in the c <dig> photosynthesis, it is also possible that this variability derives from differences in the times of the day when leaves were sampled as well as from differences in leaf developmental stage and in plant n regime  <cit> .
looking at the root system, the gln level starting from  <dig>  ±  <dig>  mm in  condition increased to  <dig>  ±  <dig>  mm in  plants but it reached the higher values of  <dig>  ±  <dig>  mm and  <dig>  ±  <dig>  mm in  and  plants, respectively .
this trend was associated with comparable upsurges in the xylem sap .
since the root gs synthetase activity was very similar in all three n treatments , it is reasonable to suppose that the lowest level of gln in  roots did not result from an enzymatic control on gs but rather from a metabolic regulation on no3− reduction steps, limiting the free nh4+ in the cell.
the slower n assimilation in  roots was associated with an accumulation of glu and asp , supporting the idea that the gs/gogat and the tricarboxylic acid  cycles were reciprocally balanced, sufficiently to sustain the storage of intermediates.
in addition, the similar gln concentrations observed in roots and xylem saps of  and  plants  indicated that the maximum capacity of plants to synthesize and translocate gln was already reached during the exposure to the lowest availability of nh4+ .
this is also consistent with the saturation kinetic of the gs synthetase activity described above .
otherwise, asn significantly incremented only in roots exposed to nh4+, reaching the values of  <dig>  ±  <dig>  mm and  <dig>  ±  <dig>  mm in  and  conditions, respectively .
asn represents one of the main compounds for n storage and transport due to its high n/c ratio and stability.
it is synthesized by asn synthetase  by the amidation of asp using gln as amino donor, but several studies have indicated an nh4+-dependent synthetase activity in plants .
the asn changes are in agreement with the observation that in maize roots the as gene expression is influenced by c/n ratio since it is induced by carbohydrate limitation and by supplies of nh4+, gln, asn, asp but not of glu  <cit> .
moreover, in maize mutants deficient for gs1- <dig> and/or gs1- <dig> a higher leaf content of asn and ala compared with than of the wild-type was reported, suggesting compensatory involvement in nh4+ assimilation  <cit> .
in this work, the levels of asn in root tissues were quite proportional to the root nh4+ concentrations, but not to the gln ones, suggesting that the asn accumulation could be involved in a mechanism of cell protection from high nh4+, which appeared specifically induced by the cation and not by gln.
this induction was not associated with a comparable upsurge of asn translocation.
on the contrary, the ala concentration strongly increased in all three organs in response of n availability .
the highest increment of ala translocation was observed during the  treatment, showing a synergistic trait related to co-provision of no3− with nh4+.
recently it was proposed that ala and pyruvate translocations might have important roles for the maintenance of c/n balance throughout plants  <cit> .
considering that in leaves the asn re-assimilation necessarily releases free nh4+  <cit> , the sequestration of asn in roots and the preferential translocation of gln and ala might participate in the mechanism of nh4+ tolerance observed in t <dig>
the glu levels showed marked differences between root and leaf systems under the same nutritional treatment.
in roots, glu accumulation was increased in the presence of no3−, reaching the values of  <dig>  ±  <dig>  mm and  <dig>  ±  <dig>  mm in  and  plants, respectively.
however, the provision of nh4+  was associated with a very low level of  <dig>  ±  <dig>  mm, comparable with the  plants .
glu was scarcely translocated in the xylem sap, while in leaves the n availability sustained a generalized increment of glu that mirrored the total amino acid concentrations .
these observations suggest that the balancing of the gs/gogat cycle distinctly diverged between the two organs.
in particular, in leaf the high amount of gln received from the xylem sap appeared re-assimilated to restore glu.
on the contrary, in the root system the nh4+ exposure seemed to hamper the accumulation of glu, resulting in a large prevalence of gln.
several studies have given evidence of the existence of a glu homeostasis in plants, probably involved in plant c/n perception, which is perturbed by nadh  and 2og  availability  <cit> .
hence, it is possible to suppose that the peculiar shortage of glu in  roots was associated with a scarce provision of 2og by the tca cycle to the gs/gogat system.
the involvement of the tca cycle also seems to be supported by the concerted changes of asp .
considering that the  and  roots showed similar extent of n assimilation as well as that the  roots were characterized by the highest content of reducing sugars, it is unlikely that this imbalance derived from a lack of c skeletons.
instead, it is more conceivable that the lack of glu was related to an excess of reducing power.
in fact, the high n assimilation in roots exposed to nh4+ could be associated with a strong activation of anaplerotic reactions for c skeletons, leading to a production of nadh exceeding the metabolic requests.
considering that this excess could cause a feedback inhibition on the tca cycle  <cit>  it is possible that the outcome could be a very low availability of 2og in the cell.
this consideration is consistent with the evidence that in roots of arabidopsis thaliana the supply of nh4+ compared to no3− promotes a higher capacity of respiratory bypass pathways involved in the dissipation of excess of redox equivalents  <cit> .
in addition, the co-provision of no3− with nh4+ sustained a higher accumulation of glu in  roots .
this could be a synergistic effect by which, even if the n assimilation in  and  roots was similar, the consumption of reducing power by no3− reducing steps could be associated with a minor inhibition of the tca cycle.
it is interesting to note that, because the asn and ala are synthesized by the transfer of the amino group of gln on c skeletons  available out of the tca cycle  <cit> , their synthesis could contribute towards regenerating glu with a nadh production lower than the gs/gogat route.
overall, the analysis of the amino acid composition confirms the activation of gs observed by the proteomic and enzymatic approaches, highlighting the relevance of root responses in the n economy.
moreover, the results provide new information about the metabolic regulation of the gs/gogat cycle that seem to be deeply influenced by several aspects, such substrates and coenzymes, as well as by the biosynthetic pathways of other amino acids.
CONCLUSIONS
taken together, the results give novel insights about the multiplicity of factors involved in gs regulation.
firstly, the work provides new evidence that in maize different gs isoenzymes/isoforms have distinct metabolic functions, diverging between root and leaf system.
interestingly, the proteomic discrimination of the gs <dig> proteins revealed that in roots the cytosolic enzyme also contributes in no3− assimilation by gs1- <dig> activity, providing first indications about the role of this isoform.
at the same time, the changes in enzymatic properties as well as the presence of phosphorylation confirmed the involvement of ptms.
it is conceivable that these observations may be useful for future studies aimed to investigate the rearrangement of gs native enzyme and its interaction with regulatory proteins.
furthermore, the analyses of amino acid composition in roots, xylem sap and leaves provides novel information about the fact that in roots the gs/gogat cycle was not only regulated at molecular level but it was also deeply influenced by biochemical factors, like substrates and cell redox status.
finally, from a physiological point of view, it is interesting to note that the work gives new insights about the relevance of glu, asn and ala in plant c/n balance in response to nitrate and/or ammonia nutritions.

 nitrogen represents one of the main minerals required throughout plant development. in agronomic terms, this results in a worldwide ever-increasing use of fertilizers and its consequent environmental and socioeconomic costs. this n requirement is emphasized with regard to cereal crops, for which maize is a model species because of its economic importance and high metabolic capacity. in agricultural soils the main mineral n sources are nitrate and ammonium. in order to balance their n nutritional requirements with environmental availability, plants have to modulate the individual steps of n metabolism such as up-take, reduction of no3− to nh4+, nh4+ assimilation and n recycling. the contribution of root and leaf systems depends on species, developmental stage and environmental conditions, and it is also deeply influenced by c metabolism. all the nh4+ in the cell, derived from soil, from no3− reduction or from other metabolic processes, is channelled through the glutamine synthetase reaction. the gs catalyzes the fixation of nh4+ on glutamic acid to form glutamine, and in the assimilation process it is generally coupled with plastidial glutamate synthase that incorporates c skeletons. gln and glu can be recruited as amino group donors as well as main n transport molecules. several evidence indicate that gs activity is deeply influenced by metabolic and environmental factors mainly linked to the balance between c and n metabolism. for instance, glu level seems to be fundamental in sensing plant nutritional status and in joining c and n metabolisms. moreover, the inter-conversion with other amino acids greatly influences n plant economy, especially regarding asparagine and alanine. plant responses are deeply affected by the proportion of mineral n sources. while nh4+ as sole nutrient can induce toxicity symptoms, its co-provision with no3− generally promotes a synergistic effect leading to growth enhancement. it is noteworthy that nh4+ tolerance was related to high root n metabolism sustained by high gs activities, which in maize appear to be associated with the capacity to cope with the c skeleton demands. the main gs are decameric enzymes classified on the basis of subcellular localization in cytosolic gs and plastidial gs in plants, multigenic families encode several gs isoforms while the plastidial gs derives from one or few nuclear genes. in general, gs is associated with the leaf nh4+ assimilation while gs is associated with plant n recycling. but the relative activity of gs and gs is variable according to the species, organs, n sources, developmental stages and environmental conditions, suggesting a multifaceted participation of isozymes. moreover, recent studies conducted both in dicotyledonous and in monocotyledonous crops showed non-overlapping functions for the gs isoforms. besides, distinct post-translational modifications were described for both isoenzymes. in maize, one gene for gs and genes codifying for different gs subunits were identified, named from gs1- to gs1- according to the reviewed uniprotkb/swiss-prot database. gs and gs are differentially regulated in roots and leaves in response to growing conditions. the cytosolic isoforms also have different kinetic properties, stabilities and tissue localizations. by means of quantitative trait loci analyses and characterization of maize mutants, hirel and co-workers indicated the key roles of gs1- and gs1- both in grain yield and germination. gs1- and gs1- represent the major leaf isoforms and in maize mutants the deficiency of these enzymes affects leaf gene transcripts, proteins and metabolite accumulations. moreover, the transcript localizations confirmed the involvement of gs1- in root metabolism and suggested that gs1- acts in n phloem translocation. it is worth noting that in mutants deficient for gs1- and gs1- the dry weight and total n content in the shoot vegetative parts were unaltered, providing evidence of how such parameters are prevalently determined by root metabolism. this observation, together with the finding that the n stored before silking supplies up to 70% of the grain n content, draws attention to the need to study the root system during the early phases of maize development. after a first localization of gs1- and gs1- in tip and/or cortex tissues, root gs arrangement had scarcely been investigated, probably due to technical limitations. n availability is associated with the accumulation of a specific root isoform that is theoretically assigned to gs1- but had not been precisely characterized. similarly, the responses to different n sources were not fully elucidated and information about gs1- was still lacking. by the means of two-dimensional western blotting and liquid chromatography-nanoelectrospray ionization-tandem mass spectrometry techniques, this work profiles the gs patterns in maize roots in response to no3−, nh4+ or both, during vegetative growth, describing for the first time the differential modulations of cytosolic and plastidial forms and the active involvement of gs1- moreover, the determination of amino acid composition in roots, xylem sap and leaves provides new information about the roles of gln, glu, asn, and ala metabolisms in plant c/n balance.  effects of the nutritional treatments on leaf and root metabolic status the aim of this work was to investigate the responses of the different root gs isoforms in maize plants exposed to different inorganic n sources, during early vegetative growth. to better appreciate the effects at metabolic level, the changes in plant amino acid balance were also evaluated. plantlets of the t inbred line were grown in a hydroponic system in the absence of n for 10 days to reach a developmental stage corresponding to the third-leaf expansion. since in field conditions maize n fertilization consists of a single application at sowing, the third-leaf stage corresponds to a vegetative phase in which plants are exposed to a high level of inorganic n and that is indicated as one of the more susceptible to nh4+ toxicity. moreover, it is important to note that the optimal dose of no3− fertilization also depends on maize varieties. in order to better appreciate the short-term responses and compare our proteomic results with previous works, the plants were exposed to a total n availability of 10 mm. in details, plants were exposed for 30 h to four nutritional treatments: n absence, 10 mm no3−, 10 mm nh4+, 5 mm no3− + 5 mm nh4+. the concentration of no3− and nh4+ in roots, xylem saps and leaves were measured, together with the content of the reducing sugars and sucrose in root and leaf systems. moreover, the accumulation of the main n assimilative enzymes such as nitrate reductase and gs in roots and leaves was estimated by one-dimensional western blotting. the analysis conducted against nr in roots detected the expected single band at 99 kda while in leaf profiles two bands were visible. the lower band at about 94 kda, corresponding to pyruvate phosphate dikinase that is the most abundant enzyme in maize leaves, was considered as an unspecific signal. on the base of molecular masses, the three bands in gs profiles were assigned to gs, to gs and to the root isoform gs1r, as described by sakakibara and co-workers. according to previous works, higher levels of gs were detected in root profiles while gs was predominant in leaves.figure nitrogen and carbon metabolites in maize in response to different inorganic n sources. concentration of no3 −, nh4 +, reducing sugars and sucrose in roots, xylem sap and leaves in maize plants grown for 10 days without n sources and then exposed for the last 30 h to absence of n, to 10 mm no3 −, to 10 mm nh4 + or to 5 mm no3 − + 5 mm nh4 +. graphs show average values ± se. the upper letters indicate differences among the four treatments within each organ according to student’s t-test. accumulation of n assimilatory enzymes in maize in response to inorganic n sources. 1d-wb performed against nr and gs in roots and leaves of maize plants grown without n sources for 10 days and then exposed for the last 30 h to absence of n, to 10 mm no3 −, to 10 mm nh4 + or to 5 mm no3 − + 5 mm nh4 +. the analyses were conducted twice on two independent biological samples for each condition. b and d profiles were visualized by a global antibody for both gs and gs that recognized three different bands. the molecular masses of electrophoretic standards are reported on the left. it appeared evident that the exposure of the plants to no3− induced strong accumulation of the anion in all organs and especially in roots, reaching the value of  ±   μmol g−1fw in plants. this trend was similar to that observed in t by previous time-course experiments. the increment of no3− in xylem sap attested the concomitant induction of anion translocation. in particular, in and xylem saps the values of  ±  and  ±   μmol g−1fw, respectively, quite a lot higher than the  mm value observed in maize crops, suggested an extensive plant response to restoring tissue n levels. the n management in plants exposed to nh4+ was different. both and plants did not show any toxicity symptoms and the nh4+ root accumulation and translocation incremented proportionally to the medium concentrations. on the contrary, the nh4+ levels in leaf systems were scarcely affected especially in comparison with roots in which nh4+ reached the highest value of  ±   μmol g−1fw in condition. in several plant species, a similar limitation of nh4+ translocation to the shoot, considered more sensitive to nh4+ than roots, was associated to a high rate of metabolisation by gs in roots, which was proposed as one of the main traits of tolerance to high nh4+ inputs. finally, no synergistic effect on the ions accumulation emerged. the availability of n led to an increase of both reducing sugars and sucrose levels in leaves, especially in response to nh4+ exposure. however, only in the roots of plants a peculiar doubling of reducing sugars was observed. considering that the sucrose slightly increased, it is possible that this increment originated from the delivery of photo-assimilates from the shoot. interestingly, this trait was almost completely alleviated by the co-provision with no3−. in a comparative study among maize genotypes, the physiological traits of nh4+ tolerance seemed to rely on the plant’s capability to shift the partitioning of carbohydrates towards the root system in order to sustain nh4+ assimilation. hence, the results confirmed that the t line shows some physiological traits typical of tolerant maize cultivars, especially those deriving from a high capability for nh4+ assimilation by the root system. at the same time, the reduction of reducing sugars in roots indicated that the roots were also involved in no3− assimilation, as proposed by prinsi and co-workers. the 1d-wb supported the induction of the assimilatory pathways. the nr accumulated proportionally to the no3− concentration in the external medium both in roots and leaves. similarly, the gs levels in roots responded to no3− availability. this response was scarcely detected in leaves, where the enzyme level was quite constant. this behaviour is similar to that observed in young vegetative maize plants grown at high and low n fertilization in the field. interestingly, in comparison to plants, the exposure to nh4+ led to a reduction of nr level in roots and to a slight increment in leaves. together with the increment of the no3− accumulated in the shoot, these results suggest that the nh4+ availability induced a shift in plant n economy, promoting the usage of no3− reserves by the shoot. finally, the gs enzyme accumulated in response to the increasing cell nh4+ contents, by the specific induction of the gsr isoform. in summary, the plant metabolic status results confirmed that the four experimental treatments induced the assimilation and translocation of the different n sources, highlighting the fundamental role of the root system in the plant physiological adaptation to both no3− and nh4+ nutrition. characterization of the gs isoforms in maize roots in order to distinguish at protein level the different gs isoforms accumulated in the maize root system, the wb with the gs global antibody was applied on the 2d-electrophoretic map of root soluble proteins. a preliminary investigation conducted in a wide ph range of 3– on 10% acrylamide sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that all recognizable isoforms were embraced in the acidic portion of the profile and in a molecular range of 35–50 kda. to improve the analytical resolution, the next protein separations were performed in the narrower 4– ph range. this approach allowed a good reproducibility among the four nutritional treatments, providing an overall proteomic map composed of about 1210 ±  spots. considering the electrophoretic adjustments, this map compared well with the one proposed by prinsi and co-workers. the 2d-wb showed a unique pattern, recurrent in all experimental conditions, consisting of six stains that were numbered from to and assigned to six spots in the 2d-electrophoretic maps. these spots were separately analysed by lc-nesi-ms/ms. each spot was assigned to a specific gs isoform according to the discriminating peptides sequenced. since for every reference spot the assignment was independently confirmed among the four 2d profiles, the data were then pooled to get the overall spot characterization. the spots and were identified as different gs forms from the same gene, named gs2a, gs2b and gs2c. this observation was in agreement with the fact that two distinct gs forms were highlighted in the maize leaf proteome. here, as well as in several other plant species, the gs protein forms exceed the number of encoding genes. as a whole, this trait supports the presence of post-transcriptional/translational modifications. the similar incidence of specific peptides for both the two isoforms revealed that spot derived from an overlapping of gs1- and gs1- explainable by the almost identical isoelectric point that made the two proteins inseparable by denaturing electrophoresis, as previously noted by martin and co-workers. conversely, spots and were distinctly identified as gs1- and gs1- isoforms, respectively. this proteomic investigation revealed a pattern of gs accumulation very consistent with the expression profile proposed by li and co-workers. these authors, by means of a comprehensive analysis of the transcript levels of the six gs genes in maize, provided evidence that all isoforms were expressed in the roots, with the prevalence of gs1- gs1- and gs1- mrnas. at the same time, the fact that gs1- was characterized as a low abundance and vascular-specific isoform might explain why it was not detectable.figure localization of the gs isoforms in 2d profile of maize roots by western blotting. representative 2d-electrophoretic map of soluble protein fraction from roots of maize plants. proteins were analyzed by isoelectric focusing at ph 4– and 10% sds-page and visualized by ccbb staining. for each nutritional treatments, the gel portion comprised in the broken-line was analyzed by wb against all gs. the analyses were conducted twice on two independent biological samples showing the same profile among all the experimental conditions. the visualized spots are numbered and traced on the 2d-electrophoretic map. the lc-nesi-ms/ms characterizations of the six spots are reported in table  the molecular masses of electrophoretic standards are reported on the left. spot assignments to the specific isoform of glutamine synthetase by lc-nesi-ms/ms n acr protein an pi/mw pi/mw u.p. s/t %cov. avg n. p. 1 gs2a 2 gs2b 3 gs2c 4* gs1-3/4 5 gs1-5 6 gs1-1 the different spots indicated in figure  were separately collected from the root 2d profile of each nutritional treatment. values are the mean ± se of four independent biological samples analysed in triplicate. 4*: the spot was assigned as a co-migration of the gs1- and gs1- isoforms. n: spot number on figure  acr: acronym reported in figure  an: accession number. p i /mw are expressed in kda and compare the theoretical values with the experimental ones; u.p.: number of unique peptides identified, s: specific to the isoform, t: total. %cov: amino acid coverage. avg n. p.: number of distinct peptides. detailed information on lc-nesi-ms/ms sequencing is reported in additional file considering the electrophoretic positions of the gs proteins in roots, it was possible to conclude that the middle band observed in the 1d-wb included gs1- and gs1- while the gsr form, accumulated in response to external n availability, was composed by gs1- with gs1- this conclusion was previously suggested, but to our knowledge, this proteomic approach allowed the first discrimination between the individual responses of gs1- and gs1- the accumulation levels of the six gs proteins in the 2d profiles showed different and specific changes in responses to inorganic n sources. moreover, the 2d profiles were staining with the pro-q® diamond that is specific for phosphoproteins, applying this approach to investigate the phosphorylation state of gs isoforms, to our knowledge for the first time. the results showed that four of the six spots were phosphorylated and that the spot phosphorylation state was constant among all conditions. the results, together with the evaluations of root gs activities, contributed to improve the characterization of the six proteins.figure accumulation of the gs isoforms in maize roots in response to n sources. the graphs report the levels of the different isoforms evaluated in the root 2d-electrophoretic profiles stained with ccbb of maize plants grown without n sources for 10 days and then exposed for the last 30 h to absence of n, to 10 mm no3 −, to 10 mm nh4 + or to 5 mm no3 − + 5 mm nh4 +. the graph number and isoform acronyms refer to figure  and table  respectively. a. spot. b. spot. c. spot. d. spot. e. spot. f. spot. graphs show average values ± se. the upper letters indicate differences among the four treatments according to student’s t-test. analysis of phosphorylation state of root gs isoforms. the 2d-electrophoretic profiles in 4– pi range of soluble proteins from root of maize plants grown in the different experimental conditions were stained with sequential fluorescence staining procedures. a. representative magnification of the profile containing the gs isoforms stained with sypro ruby®, showing all the proteins. b. representative magnification of the same gel portion after the pro-q® diamond phosphoprotein gel staining that points out only the phosphorylated proteins. the experiment was conducted for each nutritional condition showing a similar profile for all the samples. the spots are numbered according to figure  and table  the molecular masses of electrophoretic standards are reported on the left. effects of the different inorganic n sources on gs activity in maize root. maize plants were grown without n sources for 10 days and then exposed for the last 30 h to absence of n, to 10 mm no3 −, to 10 mm nh4 + or to 5 mm no3 − + 5 mm nh4 +. both the gs synthetase and the transferase activities were evaluated and expressed as specific activity. the synthetase activity was measured in the enriched fraction of plastidial proteins and in total protein fraction representing the gs activity and the total activity, respectively. the transferase activity was measured in total protein fraction prior to and after the exposure of the samples to 45°c for 10 min. the % thermal inactivation represents the portion of transferase activity lost after this thermic treatment. graphs show average values ± se. the upper letters indicate differences among the four treatments according to student’s t-test. the responses to inorganic n sources of the gs proteins in maize roots gs was a very faint protein in the maize roots, with a maximum value of only  ±  %vol for gsb in plants. similarly, the highest gs synthetase activity of the plastidial fraction was detected in plants, where it represented about the % of the total enzymatic activity. this proportion appeared similar to that measured in rice roots, in which the contribution of gs was less than 4% of the total gs activity. in detail, while in and plants the plastidial activity was almost undetectable, it increased proportionally to no3− availability in and roots, reaching the values of  ±  and  ±   μmol mg−1prot min− respectively. these results strongly support the idea that, in maize roots, gs is involved only in no3− assimilation and it is not influenced by nh4+ nutrition. among the three identified gs forms, the gs2b is the only one of which the accumulation level was significantly increased in plants exposed to no3−. this observation confirms our former proteomic analyses. the comparison of gs accumulation levels and gs activity suggests the hypothesis that gs2b represented the form really participating in catalytic activity. interestingly, this spot represented also the only phosphorylated gs. the reciprocal position of the gs spots was comparable with those observed in the leaf maize proteome. indeed, in leaves two gs spots were identified of which the accumulation levels were differently related to the enzymatic activity. in particular, the more acidic spot, matching with gs2b, was accumulated proportionally, while the basic one disappeared with the increment of the enzymatic activity. the gs2c electrophoretic position as well as its trend of accumulation showed a high degree of similarity with this last observation. as a whole, the results suggest that gs2a and gs2c might be transitional forms of the enzyme, probably originated by ptms. the chemical and/or immunological detection of sugar and nitric oxide moieties did not lead to any positive results, but it is not possible to exclude the occurrence of several other ptms. the responses to inorganic n sources of the gs isoforms in maize roots according to the proposed model, the presence of phosphorylation on gs is associated with active enzymatic forms, where it promotes protection from degradation and the interaction with activating 14-3- proteins. the evaluation that the spots and were phosphorylated in all conditions tested supports the hypothesis that they represented active subunits of the root gs enzyme. firstly, it is possible to note that all three gs spots were detected in plants, in which spot was predominant. their presence in roots of starved plants confirmed a gs involvement in the use and/or recycling of endogenous n reserves. spot was the least influenced by the plant nutritional status, as it only decreased by about 20% in response to nh4+. this trend was in agreement with previous transcriptional analyses highlighting that gs1- and gs1- mrnas slightly decreased in response to n as well as with the unchanged intensity of gs band in sds-page. together with the fact that in maize gln1- and gln1- mutants the vegetative biomass is not affected, these results reinforce the hypothesis that other root isoforms are able to sustain n assimilation during vegetative growth. interestingly, spots and showed marked and different changes. the gs1- isoform specifically increased in response to nh4+ nutrition, becoming the most abundant one in roots with the highest measured value. it is worth noting that the changes in gs1- accumulation reflected the nh4+ availability, reaching an increment of about +112% and +54% in and plants, respectively. the responses of gs1- isoform attested an even higher increase of about +222% in condition. likewise, in plants the gs synthetase activity significantly increased. these results confirm that nh4+ induces gsr and, for the first time, they allowed us to discern the differential contribution of its components. moreover, gs1- showed a peculiar doubling in plants. the fact that gs1- is metabolically active in roots is reinforced by the estimation that its change is the only one to be associated with the increase in total gs synthetase activity in the condition. interestingly, these observations confirm at protein and enzymatic order the induction of gs1- transcript by no3− exposure recently observed in the t line. taken together, these results provide the first information about the functional role of the gs1- isoform, providing evidence of its involvement in root no3− metabolisation. the gs synthetase activity in the total protein fraction, starting from  ±   μmol mg−1prot min− in starved plants, increased to a similar extent in n treated ones. this feature suggests that gs followed a saturation kinetic, probably because the elevated concentrations of no3− and/or nh4+ overfilled the metabolic capability of the maize root organ. however, the root metabolic capability seemed to be sustained by different gs isoforms, according to different mineral n sources. this was particular evident in roots where gs1- gs1- and gs were accumulated to an intermediate level. in order to get better information about this aspect, the percentage of thermal inactivation of total gs transferase activity, induced by 45°c for 10 min, was evaluated. according to the physicochemical characterization, the isoleucine- in the gs1- sequence confers thermal stability, while the substitution with ala- clearly renders the gs1- more heat-labile than gs1-. considering that gs1- and gs1- shared this feature with gs1- and gs1- respectively, it is possible to assume that the thermal inactivation measured reflected the proportion of gs activity ascribable to the gs1- with gs1- subunits. such evaluation allowed us to confirm that gs1- and gs1- were fundamental for the assembling of gs active enzyme in and roots, where the thermal treatment provoked a loss in activity of about 97% and 98%, respectively. it is also worth noting how the contribution of gs1- and gs1- gained in importance in roots of plants exposed to nh4+, especially if in co-provision with no3−. overall, this proteomic investigation confirmed the gs involvement in n recycling as well as in the root assimilation of no3− and nh4+, and at the same time, it allowed us to propose that specific and combined isoforms sustain these different metabolic tasks. the inorganic n sources differently affected the amino acid accumulation and composition in root, leaf and xylem sap in order to appreciate the extent by which the gs activation induced by the experimental treatments affected the plant n metabolism, the composition of amino acids in different tissues was measured. figure  points out the changes related to the mostly more abundant amino acids, gathering the others in a single group, detected in roots, in xylem sap and in leaves of plants exposed to the four n conditions. the comprehensive amino acid compositions of the three tissues are detailed in the additional file 1: tables a a and a respectively.figure levels of amino acids in maize plants in response to inorganic n sources. concentration of the main amino acids in roots, xylem sap and leaves in maize plants grown for 10 days without n sources and then exposed for the last 30 h to absence of n, to 10 mm no3 −, to 10 mm nh4 + or to 5 mm no3 − + 5 mm nh4 +. graphs show the amino acid concentrations as average values ± se. the letters above are assigned according to student’s t-test. detailed quantification of individual amino acids in root, xylem and leaf is reported in additional file 1: table a a and a respectively. the provision of n provoked a significant increase in amino acid level at whole plant scale, but the extent of these increments varied in relation to the inorganic n source and/or organ. the total amino acids concentration in roots and leaves of plants was almost double the amounts in plants, confirming that nh4+ nutrition promoted a more intensive n assimilation than no3−. this aspect was mirrored by the total protein amounts in the root systems. because the total amino acid concentration reached similar values both in roots and leaves during the and treatments, the t line did not show a marked synergistic response to the co-provision of no3− with nh4+. gln was the amino acid subjected to the greatest and most prevalent changes after the exposure of the plants to n as well as being the main compound for n translocation in the xylem sap, in which it reached the maximum value of  ±   mm in plants. moreover, all three organs showed high amounts of ala, which represented up to 40%, 44% and 55% of the total amino acids in roots, xylem sap and leaves, respectively. considering that previous studies on maize plants grown in high n reported ala percentages in xylem and leaves ranging from 5% to 20-29%, respectively, the higher percentage of ala may be a peculiarity of the t line. taking into account the involvement of ala and aspartic acid in the c photosynthesis, it is also possible that this variability derives from differences in the times of the day when leaves were sampled as well as from differences in leaf developmental stage and in plant n regime. looking at the root system, the gln level starting from  ±   mm in condition increased to  ±   mm in plants but it reached the higher values of  ±   mm and  ±   mm in and plants, respectively. this trend was associated with comparable upsurges in the xylem sap. since the root gs synthetase activity was very similar in all three n treatments, it is reasonable to suppose that the lowest level of gln in roots did not result from an enzymatic control on gs but rather from a metabolic regulation on no3− reduction steps, limiting the free nh4+ in the cell. the slower n assimilation in roots was associated with an accumulation of glu and asp, supporting the idea that the gs/gogat and the tricarboxylic acid cycles were reciprocally balanced, sufficiently to sustain the storage of intermediates. in addition, the similar gln concentrations observed in roots and xylem saps of and plants indicated that the maximum capacity of plants to synthesize and translocate gln was already reached during the exposure to the lowest availability of nh4+. this is also consistent with the saturation kinetic of the gs synthetase activity described above. otherwise, asn significantly incremented only in roots exposed to nh4+, reaching the values of  ±   mm and  ±   mm in and conditions, respectively. asn represents one of the main compounds for n storage and transport due to its high n/c ratio and stability. it is synthesized by asn synthetase by the amidation of asp using gln as amino donor, but several studies have indicated an nh4+-dependent synthetase activity in plants. the asn changes are in agreement with the observation that in maize roots the as gene expression is influenced by c/n ratio since it is induced by carbohydrate limitation and by supplies of nh4+, gln, asn, asp but not of glu. moreover, in maize mutants deficient for gs1- and/or gs1- a higher leaf content of asn and ala compared with than of the wild-type was reported, suggesting compensatory involvement in nh4+ assimilation. in this work, the levels of asn in root tissues were quite proportional to the root nh4+ concentrations, but not to the gln ones, suggesting that the asn accumulation could be involved in a mechanism of cell protection from high nh4+, which appeared specifically induced by the cation and not by gln. this induction was not associated with a comparable upsurge of asn translocation. on the contrary, the ala concentration strongly increased in all three organs in response of n availability. the highest increment of ala translocation was observed during the treatment, showing a synergistic trait related to co-provision of no3− with nh4+. recently it was proposed that ala and pyruvate translocations might have important roles for the maintenance of c/n balance throughout plants. considering that in leaves the asn re-assimilation necessarily releases free nh4+, the sequestration of asn in roots and the preferential translocation of gln and ala might participate in the mechanism of nh4+ tolerance observed in t the glu levels showed marked differences between root and leaf systems under the same nutritional treatment. in roots, glu accumulation was increased in the presence of no3−, reaching the values of  ±   mm and  ±   mm in and plants, respectively. however, the provision of nh4+ was associated with a very low level of  ±   mm, comparable with the plants. glu was scarcely translocated in the xylem sap, while in leaves the n availability sustained a generalized increment of glu that mirrored the total amino acid concentrations. these observations suggest that the balancing of the gs/gogat cycle distinctly diverged between the two organs. in particular, in leaf the high amount of gln received from the xylem sap appeared re-assimilated to restore glu. on the contrary, in the root system the nh4+ exposure seemed to hamper the accumulation of glu, resulting in a large prevalence of gln. several studies have given evidence of the existence of a glu homeostasis in plants, probably involved in plant c/n perception, which is perturbed by nadh and 2og availability. hence, it is possible to suppose that the peculiar shortage of glu in roots was associated with a scarce provision of 2og by the tca cycle to the gs/gogat system. the involvement of the tca cycle also seems to be supported by the concerted changes of asp. considering that the and roots showed similar extent of n assimilation as well as that the roots were characterized by the highest content of reducing sugars, it is unlikely that this imbalance derived from a lack of c skeletons. instead, it is more conceivable that the lack of glu was related to an excess of reducing power. in fact, the high n assimilation in roots exposed to nh4+ could be associated with a strong activation of anaplerotic reactions for c skeletons, leading to a production of nadh exceeding the metabolic requests. considering that this excess could cause a feedback inhibition on the tca cycle it is possible that the outcome could be a very low availability of 2og in the cell. this consideration is consistent with the evidence that in roots of arabidopsis thaliana the supply of nh4+ compared to no3− promotes a higher capacity of respiratory bypass pathways involved in the dissipation of excess of redox equivalents. in addition, the co-provision of no3− with nh4+ sustained a higher accumulation of glu in roots. this could be a synergistic effect by which, even if the n assimilation in and roots was similar, the consumption of reducing power by no3− reducing steps could be associated with a minor inhibition of the tca cycle. it is interesting to note that, because the asn and ala are synthesized by the transfer of the amino group of gln on c skeletons available out of the tca cycle, their synthesis could contribute towards regenerating glu with a nadh production lower than the gs/gogat route. overall, the analysis of the amino acid composition confirms the activation of gs observed by the proteomic and enzymatic approaches, highlighting the relevance of root responses in the n economy. moreover, the results provide new information about the metabolic regulation of the gs/gogat cycle that seem to be deeply influenced by several aspects, such substrates and coenzymes, as well as by the biosynthetic pathways of other amino acids.  taken together, the results give novel insights about the multiplicity of factors involved in gs regulation. firstly, the work provides new evidence that in maize different gs isoenzymes/isoforms have distinct metabolic functions, diverging between root and leaf system. interestingly, the proteomic discrimination of the gs proteins revealed that in roots the cytosolic enzyme also contributes in no3− assimilation by gs1- activity, providing first indications about the role of this isoform. at the same time, the changes in enzymatic properties as well as the presence of phosphorylation confirmed the involvement of ptms. it is conceivable that these observations may be useful for future studies aimed to investigate the rearrangement of gs native enzyme and its interaction with regulatory proteins. furthermore, the analyses of amino acid composition in roots, xylem sap and leaves provides novel information about the fact that in roots the gs/gogat cycle was not only regulated at molecular level but it was also deeply influenced by biochemical factors, like substrates and cell redox status. finally, from a physiological point of view, it is interesting to note that the work gives new insights about the relevance of glu, asn and ala in plant c/n balance in response to nitrate and/or ammonia nutritions. 
in this work, proteomic and biochemical approaches have been used to study root gs enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both. the analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six gs proteins. the amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. moreover, the results give new insights about the influence of amino acid metabolism in plant c/n balance. in maize one gene for gs and five genes for gs subunits are known, but their roles in root metabolism are not yet well defined. glutamine showed the prevalent changes in all nitrogen nutritions. this work provides new information about the multifaceted regulation of the gs enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post-translational events and biochemical factors. for the first time, the proteomic approach allowed to discriminate the individual contribution of the gs isoforms, highlighting the participation of gs1- in nitrate metabolism. the plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. the main gs are classified as cytosolic gs and plastidial gs of which the functionality is variable according to the nitrogen sources, organs and developmental stages.
 glutamine synthetase catalyzes the first step of nitrogen assimilation in plant cell. the main gs are classified as cytosolic gs and plastidial gs of which the functionality is variable according to the nitrogen sources, organs and developmental stages. in maize one gene for gs and five genes for gs subunits are known, but their roles in root metabolism are not yet well defined. in this work, proteomic and biochemical approaches have been used to study root gs enzymes and nitrogen assimilation in maize plants re-supplied with nitrate, ammonium or both.  the plant metabolic status highlighted the relevance of root system in maize nitrogen assimilation during both nitrate and ammonium nutrition. the analysis of root proteomes allowed a study to be made of the accumulation and phosphorylation of six gs proteins. three forms of gs were identified, among which only the phosphorylated one showed an accumulation trend consistent with plastidial gs activity. nitrogen availabilities enabled increments in root total gs synthetase activity, associated with different gs isoforms according to the nitrogen sources. nitrate nutrition induced the specific accumulation of gs1- while ammonium led to up-accumulation of both gs1- and gs1- highlighting co-participation. moreover, the changes in thermal sensitivity of root gs transferase activity suggested differential rearrangements of the native enzyme. the amino acid accumulation and composition in roots, xylem sap and leaves deeply changed in response to mineral sources. glutamine showed the prevalent changes in all nitrogen nutritions. besides, the ammonium nutrition was associated with an accumulation of asparagine and reducing sugars and a drop in glutamic acid level, significantly alleviated by the co-provision with nitrate.  this work provides new information about the multifaceted regulation of the gs enzyme in maize roots, indicating the involvement of specific isoenzymes/isoforms, post-translational events and biochemical factors. for the first time, the proteomic approach allowed to discriminate the individual contribution of the gs isoforms, highlighting the participation of gs1- in nitrate metabolism. moreover, the results give new insights about the influence of amino acid metabolism in plant c/n balance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords amino acidsammoniumglutamine synthetasemaizenitraterootsissue-copyright-statement© the author 2015 