Increasing nutrient assimilation rates -
of Manitoba Agronomist Conf. Heard drybean nutrient uptake poster PDF. Source: Karamanos, R. Nutrient uptake and metabolism in crops. Nutrient accumulation and partitioning by grain corn in Manitoba. In: Great Plains Soil Fertility Conference Proceedings. Schlegel ed. March , Denver, Colorado.
Source: Hart, J. Horneck, D. Peek, and W. Young, III. Nitrogen and Sulfur Uptake for Cool Season Forage and Turf Grass Grown for Seed. Oregon State Crop and Soil Extension. No longer available online. Contact us if you need a copy of the document.
Source: Malhi. Seasonal Biomass Accumulation and Nutrient Uptake of Pea and Lentil on a Black Chernozem Soil in Saskatchewan.
Journal of Plant Nutrition. One putative sulfate transporter gene, an ortholog of AtSULTR3;5 Medtr6g , was highly induced by all CEP peptide domains. In our data, we found no PHT phosphate transporter genes significantly induced by the CEP1p application.
Finally, we wanted to identify signaling pathway genes that responded to CEP1 application, which might be interesting targets for breeding crops with enhanced sensitivity to such peptides. Four Myb transcription factors were also induced by all three peptides.
Small signaling peptides are known to perform a wide variety of roles in plant growth and development. However, studies exploiting synthetic SSPs to address agronomically important physiological traits such as root nutrient uptake are scarce.
Here, we devised a novel hydroponics-based nutrient uptake screen for high-throughput assessment of SSPs function in modifying root nutrient uptake in Medicago and Arabidopsis. We showed that exogenous application of synthetic SSPs can affect plant nutrient uptake rates, expressed per unit root length to avoid potential confounding effects related to changes in root system architecture.
Although treating M. truncatula plants with CEP1 peptides for short periods had no effect on total root length. As thousands of SSPs are produced by plants, this nutrient uptake phenotyping screen promises to be valuable for identifying and characterizing novel peptides involved in plant nutrition, which may find application as natural plant growth stimulants in agriculture.
Nitrate is a key macronutrient for plant growth and development and CEP1 peptides play a major role in ensuring plants have sufficient nitrogen for growth when N-availability in soil is heterogeneous or scarce Tabata et al.
Using the Arabidopsis cepr receptor mutants, Tabata et al. Multiple studies demonstrate the effect of externally applied synthetic CEP peptides on root architecture however, effects on nitrate uptake of direct CEP peptide application to plant roots has not been demonstrated before Imin et al.
These are physiologically relevant concentrations of nitrate typically found in agricultural soils, which are accessed by so-called high affinity nitrate transporters Lark et al.
In comparison to the study by Tabata et al. This might include additional CEP species that work at high concentrations of Nitrogen. Since gene overexpression studies fail to discriminate between D1 and D2 peptide domains within the polypeptide sequence encoded by the MtCEP1 gene, our study demonstrates that MtCEP1Domain1 alone is sufficient to induce uptake of nitrate from the surrounding media when N-availability is low.
Our nutrient uptake methodology can be scaled up or down depending on the seedling size. Using both Arabidopsis 10 plants per replicate and Medicago one plant per replicate we were able to detect measurable changes in uptake of nitrate, phosphate, and sulfate within 4—8 h Figures 3A,B and Supplementary Figure 2.
Additionally, both the Arabidopsis and Medicago CEP1 domains, AtCEP1 and MtCEP1D1, enhanced Medicago nitrate uptake rate indicating that the CEP signaling pathway and peptide function is conserved across species Delay et al.
However, given that effects on root system architecture, including important foraging traits such as initiation of lateral roots, are more negatively affected by AtCEP1 than by MtCEP1 peptide domain 1 or 2 Figures 2B,C , more work is needed to understand the differential effects of these peptides and to determine function of additional CEP peptide encoding genes.
Likewise, further investigation of CEP peptide dosage, length of exposure, and chemical structure is required before use in agriculture. Interestingly, we observed that application of CEP1 on both Medicago and Arabidopsis enhanced uptake not only of nitrate, but also phosphate and sulfate Figures 3A,B.
Given that MtCEP1 is uniquely responsive to nitrogen deficiency but not phosphate or sulfate deficiency Figure 2A , these results were unexpected. Tabata et al. However, recent work utilizing this uptake platform to screen for genetic diversity of nutrient uptake rates in maize germplasm found that the uptakes rates of various nutrients, as well as root respiration, are generally positively correlated Griffiths et al.
This presumably reflects the need to balance uptake of different nutrients with the demand for metabolism and growth, dictated by the overall stoichiometry of elements in the plant, with faster growth requiring increased uptake of all essential nutrients and greater energy consumption. Part of this energy consumption will drive energization of cellular membranes, which in turn drives transport of various nutrients into and around cells and tissues.
This may account for part of the apparent coordination in nutrient uptake observed in this and other studies Krouk and Kiba, No doubt, however, full coordination requires control at many levels, including the genetic level as exemplified by changes in gene expression, as observed here.
To begin to understand how CEP peptides alter root nutrient uptake and development, we conducted RNAseq on M. truncatula roots 3 h post treatment with the three different peptides Figure 4.
Our analysis revealed that the peptide MtCEP1D1 1, DEGs had the largest effect on the Medicago transcriptome, followed by MtCEP1D2 1, DEGs and AtCEP1 DEGs. Equally, upon comparison of nutrient uptake rates by MtCEP1D1;hyp4,11 and AtCEP1:hyp4,11 we observed that application of MtCEP1D1 on M.
GO enrichment analysis revealed that both MtCEP1 peptide domains decreased auxin related gene expression. This corroborates the finding that CEP1 application represses auxin biosynthesis and alters auxin transport in Medicago roots to affect gravitropic responses in roots Chapman et al.
Moreover, application of both MtCEP1 domain encoding peptides decreased energy metabolism-related processes and sugar metabolism required for plant growth and development, consistent with the associated decrease in total root length.
Enrichment of GO categories related to cell recognition MtCEP1D1 and phosphatase activity MtCEP1D2 are consistent with the role of MtCEP1 as a signaling peptide controlling various physiological responses.
A targeted search of transporters involved in N, P, and S uptake in Medicago yielded several nitrate transporters and one sulfate transporter that were upregulated by application of CEP peptides. Increased transporter density on the root exodermis is commonly believed to enhance uptake, but other mechanisms may exist such as allelic diversity, increased assimilation to decrease internal cellular concentrations, and increased counter-ion efflux Griffiths and York, The absence of a clear candidate phosphate transporter that is transcriptionally regulated by the CEP peptides points to alternative mechanisms of controlling phosphate uptake under these conditions.
One such possibility is the involvement of sulfate transporters in phosphate uptake, given the observation that SULTR3;5 was also shown to mediate accumulation of inorganic phosphate in rice Yamaji et al.
Finally, our data also revealed novel candidate genes that may be involved in CEP1 signal perception and relay. These included several Myb-domain containing transcription factors, WRKY, GRAS domain, and ERF AP2 ERF transcription factors.
Although a previous study overexpressing CEP1 in hairy roots of M. truncatula found the same family of TFs, the gene IDs were different Imin et al. We identified several LRR-RL kinases that were preferentially upregulated by MtCEP1D1 application Medtr5g, Medtr8g, and Medtr8g This suggests that MtCEP1D1 may initiate signaling in distinct downstream pathways.
Previously, synthetic peptides have been reported to affect developmental processes. Here we show that application of a peptide can affect transcription of transporter genes and enhance nutrient uptake processes.
Based on these results, SSPs show promise in horticulture, and agriculture more generally, through use in hydroponic and fertigation systems, as well as part of seed coat treatments, which would place them in close proximity to plant seedlings and roots upon germinations.
Implementation of nutrient uptake enhancing SSPs in agriculture could help drive greater nutrient capture whilst minimizing nutrient losses. Sonali Roy orcid. York orcid. The original contributions presented in the study are publicly available.
This data can be found here: National Center for Biotechnology Information NCBI BioProject database under accession number PRJNA SR, W-RS, and MU conceptualized the peptide assays and interpreted the results.
MG and LY conceptualized and implemented the nutrient uptake measurement platform. MG created the R scripts for data analyses. SR and MG designed the experiments.
SR, MG, IT-J, BS, EA, SZ, DJ, and NK performed the experiments and analyzed the data. SR, MG, W-RS, MU, and LY wrote this manuscript with input from all authors. This work was funded by the National Science Foundation award to W-RS and MU; USDA-NIFA award to LY; the Center for Bioenergy Innovation, a United States Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science; and the Lloyd Summer Noble Summer Scholar grant to SR and MG.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. We would like to thank David Huhman for help with ion content measurement, Lynne Jacobs for help growing plants, and Kim Spiering for technical support.
Supplementary Figure 1 Dot plots showing nutrient uptake in 48 different plant roots treated with different peptides over 8 h as measured by the uptake platform. Nutrient depletion plots show raw data not normalized for root length.
Uptake was measured for A nitrate, B phosphate, C sulfate, and D potassium. Each peptide and control had six replicates each. Supplementary Figure 2 A Rhizovision output showing representative root scan used for measuring total root length.
B RVExplorer output image to show an example segmentation of the root scan in A. C Box plot showing difference in root length post treatment with peptides. Supplementary Figure 3 CEP1 has no significant effect on uptake of additional nutrients tested.
Specific nutrient uptake rates of ammonium, nitrite, potassium, magnesium, sodium, and chloride as indicated in M. truncatula in the presence of the synthetic AtCEP1 peptide and MtCEP1D1 at a concentration of 1 μM. Supplementary Figure 4 Principal component analysis graph showing distribution of biological replicates in peptide treated and control samples.
Plot was generated using DESeq2 using a read count filter of 30 and a Median Ratio Normalization. Green dots represent the Control CEP1C , blue dots represent CEP1D1, purple dots represent CEP1D2, and red dots represent AtCEP1 treatment.
Supplementary Table 1 Transcript per million TPM counts, DESeq2 results and differentially expressed genes under MtCEP1D1, MtCEP1D2, and AtCEP1 application. Supplementary Table 2 Gene Ontology analysis of DEGs under application of synthesized CEP1 peptide domains and unique genes in top GO terms.
Supplementary Table 5 Table showing RNAseq read statistics for peptide treatments presented in this manuscript. Bao, Z. Identification of Novel Growth Regulators in Plant Populations Expressing Random Peptides. Plant Physiol. doi: PubMed Abstract CrossRef Full Text Google Scholar. Breakspear, A.
Plant Cell 26, — CrossRef Full Text Google Scholar. Chapman, K. CEP receptor signalling controls root system architecture in Arabidopsis and Medicago. New Phytol. Dai, X. LegumeIP V3: from models to crops—an integrative gene discovery platform for translational genomics in legumes.
Nucleic Acids Res. Delay, C. CEP genes regulate root and shoot development in response to environmental cues and are specific to seed plants. Google Scholar. de Bang, T. Small peptide signaling pathways modulating macronutrient utilization in plants. Plant Biol. Genome-Wide Identification of Medicago Peptides Involved in Macronutrient Responses and Nodulation.
Fageria, N. The Use of Nutrients in Crop Plants. Boca Raton: CRC Press, doi: Ghorbani, S. Expanding the repertoire of secretory peptides controlling root development with comparative genome analysis and functional assays.
Griffiths, M. A multiple ion-uptake phenotyping platform reveals shared mechanisms affecting nutrient uptake by roots. Targeting Root Ion Uptake Kinetics to Increase Plant Productivity and Nutrient Use Efficiency.
Hastwell, A. Author Correction: CLE peptide-encoding gene families in Medicago truncatula and Lotus japonicus, compared with those of soybean, common bean and Arabidopsis. Hawkesford, M.
The Molecular and Physiological Basis of Nutrient Use Efficiency in Crops. Hoboken: Wiley-Blackwell. Imin, N. The peptide-encoding CEP1 gene modulates lateral root and nodule numbers in Medicago truncatula. Krouk, G. Nitrogen and Phosphorus interactions in plants: from agronomic to physiological and molecular insights.
Laffont, C. The senescence-induced aspartic protease CND41 encoded by the nuclear genome has been localized specifically to the chloroplast and an in vitro analysis confirmed that CND41 showed a Rubisco proteolytic activity at physiological pH Kato et al.
However, data suggest that CND41 could not act on Rubisco unless its structure was denatured. Therefore, active Rubisco in the chloroplast would be resistant to CNDcatalysed degradation until leaf senescence was under way. CND41 homologues identified in Arabidopsis accumulate with ageing.
Their role in the regulation of Rubisco turnover and senescence in Arabidopsis remains to be studied Kato et al. It seems likely that degradation of chloroplast protein starts within the plastid but it is far less clear whether chloroplastic proteases drive protein degradation to the end.
The central vacuole that remains intact and for which compartmentation is maintained during senescence might be the end point of chloroplast protein degradation. The increase of vacuolar protease mRNA levels in senescing leaves is in good agreement with this scenario Martinez et al.
Several possible routes for internalization of chloroplast components by the central vacuole are proposed, such as autophagosome and senescence-associated vesicle SAV trafficking. Autophagy is a well-known process in yeast and animals but it has only been recently established in plants.
Macro-autophagy involves formation of autophagosomes, which are double membrane-bound structures enclosing macromolecules and organelle residues. Transcriptome analysis has shown that most of the autophagy genes are upregulated during senescence and in response to nitrogen limitation Thompson and Vierstra, ; Wingler et al.
Chiba et al. When leaves of transgenic Arabidopsis plants expressing stromal-targeted fluorescent proteins were incubated with concanamycin A, spherical bodies exhibiting protein-specific fluorescence were observed within the vacuolar lumen.
These bodies — corresponding to RCBs — were, however, not observed in the concanamycin A-treated leaves of the atg5 T-DNA insertion mutant impaired for autophagy.
In addition, stromal-targeted DsRed proteins and GFP-ATG8 fusion proteins were observed together in autophagic bodies within the vacuole. The authors concluded that Rubisco and stromal proteins can be mobilized to the vacuole through an ATG gene-dependent autophagic process without prior chloroplast destruction Ishida and Yoshimoto, The role of autophagy in recycling cell proteins is now accepted, although the premature leaf senescence and accelerated chloroplast degradation observed in autophagy knockout lines is less well understood.
As shown by yeast and animal studies, autophagy might have a dual role preventing or triggering cell death depending on its fine-tuning. By removing cell waste, autophagy could be essential for cell longevity.
In contrast, excessive autophagy activity could lead to cell death. SAVs differ from autophagosomes in that they occur only in chloroplast-containing cells whereas autophagosomes have been observed in leaf and root cells Otegui et al.
As with RCBs, evidence has shown that SAVs contain chloroplast proteins such as Rubisco and GS2 Martinez et al. Because of their high protease activity, SAVs are a likely site for chloroplast protein degradation. The senescence-associated cysteine-protease encoded by the SAG12 senescence-associated gene gene has been detected in SAVs Otegui et al.
SAG12 may then participate in the intense proteolytic activity contained in this type of vesicle. Because SAG12 is the only SAG whose expression is uniquely controlled by natural senescence, a specific role of SAV in the natural senescence process has been proposed.
Regulation of proteolysis during senescence is then likely to integrate the regulation of chloroplastic and vacuolar proteases and the regulation of various trafficking pathways. Desclos et al. The protease activation state might thus be tightly controlled during leaf development in relation with N remobilization Etienne et al.
Depending on the species, nitrogen uptake could be negatively regulated or even in some cases totally inhibited during seed production. This change is correlated with a strong repression of gene expression, the BnNRT2 mRNA being undetectable at the flowering stage Beuve et al.
This residual influx is correlated with a decreased yet significant expression of both NRT2·1 and NRT1·1 genes Fig.
Dechorgnat et al. However, due to this decrease in N uptake activities, another nitrogen source such as remobilization is necessary to cope with the strong N demand from seed filling. A Root nitrate influx in plants at the vegetative Veg. and reproductive Repro. B Expression of NRT left and NRT right genes, at the vegetative Veg.
Expression of nitrate transporter genes was measured using quantitative PCR and expressed as a percentage of the tubulin 4 gene, used as a control.
There is evidence that plants share common N remobilization mechanisms whether they are monocotyledonous, dicotyledonous, C 3 or C 4 photosynthesis types.
Grain N accumulation usually appears to be regulated by the N source. In wheat, the kinetics of Rubisco content and grain N accumulation suggest that during grain filling N translocation from the vegetative organs is mainly limited by the availability of the substrate in the source organs Bertheloot et al.
To investigate whether NRE is controlled by source availability or by the transfer processes located in the source leaves and the phloem pathway efficiency, functional genomic and mutant approaches have been used.
Genes encoding enzymes involved in nitrogen metabolism and specifically induced during N remobilization have been identified Masclaux et al. These enzymes are a major focus of plant physiologists, with special focus on cytosolic glutamine synthetase GS1 , glutamate dehydrogenase GDH and AS reviewed by Masclaux-Daubresse et al.
Chloroplast breakdown during senescence involves de facto NiR, GS2 and GOGAT proteolysis. In senescing leaves, nitrogen recycling and re-assimilation needs then to be catalysed by enzymes other than chloroplastic ones.
The metabolic model proposes that glutamine is mainly synthesized in senescing leaves by newly expressed GS1 isoforms Fig. Using the amino acid pool released via the proteolysis of chloroplast proteins, a series of transamination reactions would lead to an increase in the glutamate pool that could serve immediately as a substrate for GDH, deaminating activity thus providing 2-oxoglutarate and ammonia.
Ammonia released this way is in turn re-assimilated by GS1 to produce glutamine for export. The importance of GS1 in nitrogen management, growth rate, yield and grain filling has been emphasized by functional genomics and quantitative trait loci QTL approaches mainly performed on rice and maize Hirel et al.
Correlation between GS activity and the amount of N remobilized from shoot to the grain was demonstrated in wheat using cultivars exhibiting contrasted NUE Kichey et al. However, the role of the GS1 enzyme is complex — numerous isoforms encoded by a multigenic family exist.
In rice, three genes have been identified, while maize and Arabidopsis contain five GLN1 genes coding for GS1 Bernard and Habash, These genes are not regulated in a similar manner and GS1 isoforms are located in different plant tissues and do not have the same kinetic properties Ishiyama et al.
It is thus clear that not all GS1 isoforms participate equally in N remobilization. In Arabidopsis , transcriptomic data show that all GLN1 genes except GLN1·5 are induced by leaf senescence Guo et al.
GLN1·1 was induced more than five-fold. Functional genomics are in progress to determine the extent of the participation of each of the four senescence-induced GLN1 genes in N remobilization. Our recent results on gln1·2 mutants show that GLN1·2 is expressed in companion cells and that the mutant plants accumulate amides in their old leaves.
However, 15 N labelling experiments did not show significant differences between mutant and wild-type for N-remobilization J. Lothier et al. Among the five GS1-encoding genes Gln to Gln in maize, only Gln is upregulated during senescence Martin et al.
Gln and Gln knockout mutants have been isolated Martin et al. The gln , gln and gln gln double mutants showed a sharp reduction of kernel yield whereas nitrogen concentration in the kernels was increased. The gln and gln gln mutants accumulated large amount of amino acids and ammonia in the source leaf located below the ear and dedicated to grain feeding.
Amino acid accumulation in the blade was mainly due to an increase in glutamate and asparagine levels as a consequence of a dysfunction in N export that reduced the total amino acid concentration and especially glutamine amounts in the phloem sap of mutants. Interestingly, the Gln locus co-localized with a maize QTL for thousand-kernel weight, and the Gln locus co-localized with two QTLs for thousand-kernel weight and yield.
More recently, 15 N tracing experiments showed that the Gln and Gln loci co-localized with a QTL for N remobilization Hirel et al.
The role of Gln remains to be determined. In rice, mutants lacking OsGS1·1 were severely impaired in growth rate and grain filling. Total free amino acid concentration was reduced in leaf blades of this mutant due to low glutamine levels. The OsGS1·1 gene product, which is located in companion cells and parenchyma cells of leaf tissues, is likely to be responsible for the generation of glutamine for remobilization via the phloem Obara et al.
Efforts to study nitrogen management during leaf senescence have mainly focused on GS1 and to a lesser extent on GDH. However, much data support the idea that GS1 and GDH are not the only limiting factors in N remobilization for a review see Masclaux et al.
Transcriptomic studies of leaf senescence have shown that several aminotransferase and AS genes are also induced during senescence. In sunflower, expression of two AS genes HAS1 and HAS1·1 detected only during leaf senescence, when asparagine amounts increased, suggest a role of these enzymes in N remobilization Herrera-Rodriguez et al.
In Arabidopsis , among the three asparagine synthetase genes ASN1 , ASN2 and ASN3 , only one is over-expressed during leaf senescence Lam et al.
A study of the asn1 mutant in our laboratory revealed early senescing phenotypes Fig. Although Lam et al. The role of ASN1 in nitrogen remobilization from leaf to leaf and from rosette to seeds was investigated using 15 N tracing as described by Diaz et al.
Results presented in Fig. Such a finding is in good agreement with the relationship between severity of leaf-senescence and NRE at the vegetative stage described by Diaz et al.
By contrast, we found that 15 N-remobilization from rosette to seeds was slightly impaired in the asn1 mutant Fig. This preliminary result shows that the role of asparagine synthetase is certainly complex and further investigations taking into account the contribution of the other asparagine synthetases, ASN2 and ASN3, are needed to understand the role of AS in nitrogen recycling and mobilization at the whole-plant level.
Asparagine synthetase AS1 might play a role in nitrogen recycling and mobilization. A Phenotypes of asn1 mutant Gabi B05 and Col0 wild-type grown in greenhouse with 10 m m nitrate. C Nitrogen remobilization from the vegetative tissues to the seeds was monitored according to Diaz et al.
At the end of the plant cycle, the dry weight of seeds and dry remains were recorded and used to calculate harvest index HI, seed d. wt as a percentage of the whole-plant d. Such preliminary finding needs confirmation using a second mutant allele.
Prior to phloem loading the central vacuole of mesophyll cells might be a site for transient storage of amino acids released from protein degradation. In tobacco, the total amount of amino acids exported from leaf blades increased five-fold during leaf ageing Masclaux-Daubresse et al.
Asparagine is the major translocated amino acid in pea. In cereals, tomato and tobacco, glutamine is the preferentially exported N-compound. In Arabidopsis , phloem sap mainly contains asparagine, glutamate and glutamine J. Lothier, INRA, Versailles, France, unpubl.
During senescence, both asparagine and glutamine concentrations increase in the phloem sap and both amino acids are likely to play a key role in rendering nitrogen available for remobilization from the senescing leaf.
The Arabidopsis genome encodes 67 putative amino acid transporters belonging to 11 gene families reviewed by Rentsch et al. The nature of the amino acid transporter involved in phloem loading during senescence is, however, poorly understood van der Graaff et al. N-storage and remobilization potential are important for both annual and perennial plants.
For annual plants, as mentioned above, nitrogen remobilization is important for seed production and seed nitrogen content. Nitrogen content in the seeds further determines germination efficiency and survival of young seedlings.
Nitrogen remobilization is also important for perennial plant survival. Trees, which grow in low nitrogen environments most of the time, have two phases of nitrogen remobilization. Nitrogen is remobilized from the senescing leaves in autumn to be stored in trunks during winter.
N is remobilized a second time from trunks to developing organs in spring before root N uptake becomes the main process to meet tree N needs.
As trees age, the internal cycling of N becomes more and more important in the whole-tree N-budget. Both nitrogen withdrawal from senescing leaves and root N uptake contribute to the build-up of N storage pools and to the efficient nitrogen management that are essential for plant survival over years Millard et al.
Forage grasses are subject to frequent defoliation by herbivores or mechanical harvesting. Recovery of grasses after defoliation is related to the availability of carbon and nitrogen reserves in the remaining tissues Volenec et al.
Decreasing mineral N supply before defoliation was shown to decrease the availability of N reserves in leaves and as a result the absolute amount of N subsequently remobilized to roots.
Interestingly, it was shown that N remobilization and senescence can be induced prematurely by environmental factors, such as pathogen attack or heavy metals.
Evidence corroborates that the N remobilization process is enhanced by biotic and abiotic stresses through the induction of the GLN1 , GDH and ASN genes Pérez-Garcia et al.
Chaffei et al. The induction of AS in roots might facilitate amino acid recycling and storage of asparagine in this organ. The co-ordinated leaf N remobilization and root N storage is certainly essential for plant recovery.
Pageau et al. N mobilization promoted by infection could be considered on the one hand as part of a slash-and-burn strategy that should deprive the pathogen of nutrients by exporting nutrients away from the developing infection site, and on the other hand as a strategy to save nutrients in healthy organs involved in recovery.
N uptake by the roots and further N assimilation are integrated in the plant to match the nutrient demand of the whole organism. The first mechanism operates at the transcriptional level and includes the induction by the substrates and the repression exerted by endogenous N assimilates.
This results in an upregulation when N is low and a downregulation when N is high. Accordingly, several NRT2 and AMT1 transporters as well as Nia and Nii genes were found to be transcriptionally repressed by N metabolites such as amino acids like glutamine Tsay et al.
On the other hand, in response to N deprivation, expression of many ammonium and high-affinity nitrate transporters is induced or repressed reviewed by Tsay et al. In response to N deprivation, expression of GLN and GDH genes is also up- or downregulated.
In tobacco, it was shown that ammonium and amino acids regulate GLN and GDH transcript levels Masclaux-Daubresse et al. Glutamate feeding over 5 h increased GLN1 mRNA while both glutamate and proline feeding decreased GLN2 mRNA.
Ammonium, proline, glutamine and glutamate increased GDH transcripts. Effects of N-metabolites on GLN and GDH transcript levels proved to be sensitive to calcium blockers and the Ka protein-kinase inhibitor.
Evidence showed that ammonium itself regulates GLN genes at the transcriptional level. In soybean, co-operation between three distinct promoter regions is necessary for ammonium-stimulated expression of the GS15 gene.
The interaction among these regions may be facilitated by an HMG A high-mobility group A -like protein that binds to the proximal and distal promoter regions of the soybean GS15 gene Reisdorf-Cren et al.
Global transcriptome studies after nitrate induction Scheible et al. Using NR mutants, it was shown that much of this regulation is exerted by nitrate itself Wang et al. The stimulation of N uptake and N assimilation by photosynthesis for a review see Lillo, ensures that N uptake is correlated with C status.
For example, nitrate uptake and reduction are co-ordinately regulated by a circadian control. This control has often been attributed to the regulatory action on gene expression of sugars produced by photosynthesis and transported downward to the roots.
This has been shown for the ammonium and nitrate transporters, NR and NiR. The regulation of nitrate uptake and transporters seems to be independent of the known sugar regulation pathways, such as hexokinase signalling Lillo, Wirth et al.
In contrast, the diurnal regulation of Nia transcripts is governed not only by sugars but also by light regulation via phytochrome Lillo, In addition, it was observed that Nia expression is controlled by signals from photosynthetic electron flow, which adds a new facet to the intracellular cross-talk between chloroplasts and the nucleus Lillo, HY5 and its homologue HYH, two transcription factors from the bZIP family, are essential for phytochrome-dependent light-activated expression of NR Lillo, ChIPchip analyses showed a binding site for HY5 in the Nia2 promoter Lillo, The NRT1·1 promoter also has three binding sites for HY5, although HY5 seems to have a negative effect on transcription in this case Lillo, Castaings et al.
Arabidopsis nlp7 mutants are defective in the nitrate induction of Nia genes and NRT2·1 and NRT2·2. Interestingly, mutants in the CIPK8 gene, which encode a protein kinase Hu et al.
It is tempting to speculate that CIPK8 might be involved in the same regulation pathway as NLP7. NLP7 belongs to a gene family with nine different members, but the functions of the other NLP proteins are still unknown.
In Arabidopsis , compelling evidence shows that SnRK1s Snf1-related protein kinases are central integrators of transcription networks in plant stress and energy signalling that are inactivated by sugars Baena-Gonzalez et al.
SnRK1 proteins were shown to specifically regulate the ASN1 gene, encoding the dark-induced asparagine synthetase also known as DIN6. The protein kinase inhibitor Ka abolishes such induction and the two ubiquitously expressed members of the SnRK1 group, Kin10 and Kin11, specifically activate a DIN6promoter::LUC fusion.
Mutation of the G-box CACGTG, G1 proximal to the TATA box abolished most of the activation of DIN6promoter::LUC by KIN10, hypoxia, darkness and DCMU.
Rapid post-translational regulation such as protein modification is the second mechanism that controls nitrogen uptake and assimilation. Post-transcriptional regulation of nitrate transporters by phosphorylation has recently been described for the nitrate transporter NRT1·1.
When phosphorylated, AtNRT1·1 functions as a high-affinity transporter whereas it is active in the low affinity range when dephosphorylated for a review see Tsay et al. Tsay and co-workers obtained significant insight into the role of the CIPK23 kinase in the specific phosphorylation of the AtNRT1·1 protein in response to nitrate levels, demonstrating AtNRT1·1-mediated sensing in the primary nitrate response Ho et al.
The best studied post-translational regulation in N metabolism is the regulation of NR in higher plants. NR is inactivated by a two-step process that involves the phosphorylation of ser , as shown in spinach, followed by the binding of an inhibitory protein kinase.
In addition, both CDPK calcium-dependent protein kinases and SnRK1 protein kinases are able to phosphorylate NR at least in vitro reviewed by Lillo, When a modified form of NR, no longer susceptible to post-translational dark inactivation, was over-expressed, the resulting protein did not decline in the second part of the photoperiod.
The inactive phosphorylated form is re-activated by dephosphorylation, probably by PP2A. Moreover, evidence showed that there is a correlation between the phosphorylation state or the activation state of NR and the rate at which NR protein decreases. Plastidic glutamine synthetase from Medicago truncatula is also regulated through phosphorylation and interactions.
The GS2 phosphorylation site ser 97 , critical for the interaction with and subsequent proteolysis, was identified by directed mutagenesis. Cytosolic glutamine synthetases from M.
truncatula are also regulated by phosphorylation but by calcium-independent kinases Lima et al. Phosphorylation occurs at more than one residue and increases affinity for the substrate glutamate.
In addition to phosphorylation, several chloroplastic enzymes of nitrogen assimilation such as NIR, GS2 and Fd-GOGAT are also redox-regulated through the thioredoxin system for reviews see Lemaire et al.
With the aim of improving NUE, many critical candidate genes have been manipulated, over-expressing them or using knockout mutations, in order to test their effects on biomass and plant nitrogen status.
Several good reviews have been written on this subject that provide more detail than mentioned in this section Andrews et al.
Until now, probably because of strong post-transcriptional controls see above , manipulating nitrate uptake through the over-expression of HATS-like NRT2·1 led to increased nitrate influx under some conditions but did not change the phenotypic NUE or nitrate utilization Fraisier et al.
NR has long been considered to be the rate-limiting step in nitrate assimilation. Nicotiana tabaccum plants constitutively expressing NR from N. plumbaginifolia showed delay in NR-activity loss under drought, which allowed them to present a more rapid recovery after short-term water deficit Ferrario-Mery et al.
Then, under field conditions of fluctuating water availability, constitutive NR expression may confer some physiological advantage.
Over-expressing NR or NiR in Arabidopsis , potato or tobacco reduced nitrate levels in plant tissues but did not increase biomass yield, tuber numbers or seed yields. Over-expression of Nia or Nii genes in plants increased mRNA levels and often affected N uptake without modifying yield or plant growth regardless of the nitrogen source available.
This is believed to be due in part to the complex post-transcriptional regulation of NR reviewed by Pathak et al. Over-expression of cytosolic glutamine synthetase GS2 genes was performed in N. tabaccum and Oryza sativa using the Rubisco small subunit promoter and the CaMV 35S promoter, respectively Hoshida et al.
tabaccum , over-expression enhanced growth rate and in O. sativa it increased photorespiration and drought tolerance. Attempts to over-express GS1 genes are more numerous and have used different promoter combinations, including CaMV 35S, RolD and small Rubisco subunit rbcS.
Effects on plant biomass and grain yield were also more successful. For example, over-expression of the Phaseolus vulgaris GS1 gene under the control of the rbcS promoter in wheat resulted in significantly higher root and grain yield with higher N content in grain in some lines Habash et al.
Over-expression of the Pisum sativum GS1 gene under the control of the CaMV 35S promoter in N. tabaccum resulted in biomass and leaf protein increases Oliveira et al.
In summary, several studies have demonstrated a direct correlation between an enhanced GS activity in transgenic plants and biomass or yield Good et al. Although over-expression of GOGAT genes has been rare, Yamaya et al. In conclusion, studies show that over-expression of GS or GOGAT genes can improve biomass and grain yields depending on which gene allele and which promoters are used.
This indicates that further characterization is required to demonstrate the beneficial effects of such strategies for crops and in field conditions. Attempts to over-express AS were carried out in tobacco and Arabidopsis for a review see Good et al.
Interestingly, over-expression of ASN1 in Arabidopsis enhanced soluble seed protein content and total protein and increased fitness of plants grown under nitrogen-limiting conditions Lam et al.
Alanine is a major amino acid for nitrogen storage under anaerobic stress such as flooding. Over-expression of barley alanine amino transferase under the control of root promoters in canola and rice had interesting effects, considerably increasing plant biomass, seed yield, NUE and shoot nitrogen concentration when plants were grown at low nitrate supply Good et al.
These results are of particular interest, showing that it is possible to improve NUE by manipulating downstream steps in N-remobilization. In addition to manipulating enzymes involved in nitrogen assimilation of amino acid metabolism, the generation of plants modified for the expression of transcription factors has also been attempted.
For example, ectopic expression of the maize Dof1 transcription factor, which regulates the expression of genes involved in organic acid metabolism, led in Arabidopsis to the accumulation of amino acids and to an increase of growth under N-limiting conditions.
These effects suggest that NUE could also be improved by manipulating carbon metabolism pathways. PII-like, NLP7 and TOR target of rapamycin proteins, which are potentially linked to C and N sensing in plants, are other candidates for further engineering as shown by the increased plant growth, yield and stress resistance acquired by TOR-overexpressing plants Ferrario-Mery et al.
Hibberd et al. The Rubisco protein is known to be used as a storage protein in C 3 herbaceous plants and trees Millard et al. In elevated atmospheric CO 2 , Rubisco carboxylase activity is increased and Rubisco protein content is decreased.
Incrdasing global climate change is a potential threat to nutrient utilization in agroecosystems. However, the combined effects of elevated nutfient 2 Healthy sodium levels and canopy warming Increasing nutrient assimilation rates plant nutrient concentrations and translocations are not well understood. Compared to ambient conditions, soil nutrient status was generally unchanged under elevated [CO 2 ] and canopy warming. In contrast, elevated [CO 2 ] decreased K concentrations by Canopy warming increased shoot N, P and K concentrations by 8. Site Menu expand. Invisalign for straighter teeth of the nutrient uptake curves nurient this page show nutrient Youth athletes on the vertical axis and nufrient stage on the horizontal axis. In general, the curves show that the highest rate of uptake steepest part of curve occurs mid-season. Estimated amount of nutrients removed by harvest are also listed for most crops. Source: Malhi, S. Johnston, J.
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