In a comprehensive pot experiment with two controls, without and with basal NPK application, Oprica et al. It is likely that the availability of micronutrients in the foliar fertiliser formulation may also have stimulated the uptake efficiency of the soil-applied NPK and, to our knowledge, is currently a subject of investigation at the AfricaRice Center.

Besides the well-documented role of N-fixation by symbiotic e. Rhizobia and free-living e. Azotobacter and Azospirillum spp. diazotrophs, soil microbes contribute to the nutrition of plants through various other processes.

Bacillus subtilis can acidify the root environment, potentially helping to increase the solubility of fixed nutrients Zhang et al. Pseudomonads streptomycetes and Bacilli serve as bio-fertilisers, producing phytohormones, siderophores and other growth-inducing compounds Bulgarelli et al.

Yet, other soil microbes function as biological control agents that negate the effects of pathogenic organisms, improving plant fitness, including fitness for nutrient assimilation and resistance to diseases, drought and metal toxicity Koele et al. For example, Prasanna et al.

Therefore, maintaining a diverse population of rhizosphere microorganisms by adequate management may be beneficial in the long run. The strong and multiple interactions imply, however, that the beneficial processes could be highly specific regarding plant species, soil, micro-organism and nutrients.

In this regard, a role for bacteria, mainly of the Bacillus , Pseudomonas and Penicillium genera, as well as arbuscular mycorrhizal fungi AMF in nutrient acquisition is further demonstrated in their ability to solubilise P mainly from tricalcium phosphate TCP.

Phosphate solubilising microbes PSMs perform their role by exuding organic acids such as citrate, acetate, succinate and gluconate, as well as by the enzymatic activities of phosphatases and phytases Richardson and Simpson ; Bulgarelli et al.

Such formulations could contribute in the recycling of P fixed in soil from fertiliser treatments, thus reducing the entry of new P into the fertiliser system. Moreover, as P has been shown to increase the proliferation of root hairs, the effect on root density in turn could contribute in the better mining and uptake of other nutrients.

In this regard, AMF, as part of the root system, are more extensive in nature and could explore spaces not reached by roots to exploit P for plant use. There has been a call to more accurately identify true PSMs based on their ability to solubilise P from several, instead of single TCP , P-metal complexes Bashan et al.

Indeed, identifying true PSMs with the most promising agronomic potentials is vital, considering the increasing depletion of quality global phosphate reserves and competition for rock phosphate by non-agro industries, both leading to a skyrocketing of the prize of P fertilisers Bashan et al.

In contrast to rhizosphere microbes, the potential involvement of phyllosphere shoot surface-dwelling or endophytic bacteria in plant nutrient acquisition is not well resolved. Nonetheless, certain phyllospheric microbes could play a role in crop nutrition.

Likewise, endophytes could contribute to plant nutrition of other minerals. For example Bacillus sp. B55 enhances S content and growth in tobacco seedlings under S-deficient conditions Meldau et al.

Notably, not only does this bacterium reduce organic S, but it also exudes the volatile, plant-assimilable S compound, dimethyl disulfide DMDS.

Therefore, for practical application, considering that DMDS is an organic, and thus biodegradable, compound, the question arises as to whether compound such as DMDS, or the bacteria producing them, could form a component of S nutrition in fertiliser formulations for S-deficient soils.

Opportunities, therefore, exist to systematically deploy specific plant-beneficial microbes as part of an integrated crop fertilisation management strategy. Indeed, different microbial inoculants are currently being commercially formulated for use in plant growth. Presently, an inventory of these formulations is being made and is the subject of an upcoming paper.

Nevertheless, the beneficial impact of currently available bioinoculants seems to vary greatly due to the complexity of the interactions, as well as potential issues with the stability of the inoculants over time, and under different climatic conditions. Moreover, many of the commercially available products may lack rigorous scientific evidence explaining their impact, warranting continued systematic research to clarify these controversies.

Nanotechnology is an emerging field with a strong promise to affect the current status of fertilisers. As such, this topic has been explored in some detail in this review. Nanomaterials having sizes in the 1—nm range are highly reactive due to their small size and large surface area, compared to bulk materials.

Thus, it is anticipated that before long, the fertiliser industry will fully join in the nanotechnology revolution. Indeed, available evidence indicates that the chemical and physical attributes of nanomaterials can be exploited to achieve useful benefits in crop fertilisation DeRosa et al.

Recently, patents and products containing nanomaterials for crop nutrition and protection are increasing e. Gogos et al. Different kinds of nanomaterials, including those manufactured from elements not traditionally classified as nutrients e.

Often, the positive effects of nanoparticles NPs on crop growth occur to a greater extent than with the equivalent dose of the same mineral nutrient presented in ionic salt form Alidoust and Isoda ; Pradhan et al.

The enhanced beneficial effects of NPs are due likely to the fact that unlike ionic fertilisers where a significant portion of the nutrients could be lost due to the formation of phosphate and carbonate precipitates or other soil factors, exposure to NPs is potentially controlled by the sustained but low release of the functional ions from the particles which serve as reservoirs of ions Dimkpa et al.

Moreover, ions from the immediately soluble salts are readily available to the roots and could rapidly reach undesirable doses, subject to interactions with soil factors. In addition to solubilisation in the soil Antisari et al.

For example, Cu presented as CuO NPs was taken up by maize and wheat in the particulate form Wang et al. Similarly, the presence of Fe and Mn NPs also has been observed in plants exposed to particulate Fe oxide and Mn Ghafariyan et al.

Notably, the same crop could differentially absorb different nutrient elements provided to it in particulate form through the root, as observed in wheat for CuO vs.

ZnO NPs, where Cu existed in wheat shoot mainly as CuO particles and a lower amount of dissolved forms, and Zn as Zn-phosphate Dimkpa et al. Given the possibility that particulate forms of mineral nutrients could be mobilised and remobilised via the xylem and phloem respectively Wang et al.

A recent study Liu and Lal demonstrated that synthetic nanohydroxyapatite [Ca 5 PO 4 3 OH] as a source of P supply modestly increased soybean growth, biomass and yield relative to regular triple super phosphate [Ca H 2 PO 4 2 ] application.

Beyond single nutrients, composite NPs of different but compatible nutrients also can be delivered into plant tissues via soil or foliar application, where they slowly dissolve to release ions for plant assimilation, triggered by specific environmental signals.

These applications would require an understanding of soil physicochemical properties as they relate to NP availability see for e.

Servin et al. Despite its immense benefits, nanotechnology also comes with risks because, similar to all chemical processes, it could have undesirable effects on non-target organisms, including plants and plant-associated soil microbes, depending on NP, dose applied and the biological species Dimkpa et al.

Accordingly, any large-scale adoption of nanotechnology for agricultural purposes must be supported by rigorous research to provide a better understanding of its agro-ecological ramifications, including the plant-specificity of the activity of the different nanomaterials, as well as any potential, dose-dependent, biotoxicity.

Fortunately, such research endeavours are ongoing in many centres globally, funded by government agencies such as USDA, EPA and their EU and Asian equivalents, as indicated by funding agency disclosures in the published literature. The VFRC is presently starting to engage along these lines with agricultural nanoscientists see for e.

No doubt, fertilisers have made, and will continue to make, notable contributions towards global provision of sufficient and nutritious food.

However, due to under-use, fertilisers have not met the needs increasing crop yield and quality and securing livelihoods for which they are made, for many members of the global farming community in poor countries of Africa and some other regions.

Increasing fertiliser use efficiency is the end result of the interplay among agro-technological adjustment of the use of current fertilisers, ecological literacy, the socio-economic realities of farmers and an improved scientific knowledge base.

In the latter case, the continuous, even if fragmented wealth of knowledge being gained from i edaphic and soil ecological processes such as interactions among nutrients, ii interaction between plants and microorganisms, and between nutrients and soil water—that determine nutrient solubility and availability— iii alternative nutrient uptake forms e.

Macronutrients may be sufficiently present in poor soils but not exploitable by crops because of limited root capacity, which could be increased through foliar application of micronutrients Thus, the array of packaging and delivery mechanisms could exploit synergistic processes while by-passing antagonism.

While some new strategies may entail adjustments of farm practices, fertiliser products could easily be integrated in current practices, while new approaches might even reduce input costs and increase farm produce and income.

Fertigation, the delivery of nutrients through irrigation, is one such strategy that can be integrated into fertiliser regimes, tuned to appropriate application rates and crop demand, to potentially improving nutrient uptake efficiency Yasuor et al.

Foliar fertilisers of leaf-requiring nutrients could be provided along with current applications of other agro-chemicals. Fertilisers packaged in tablets, much like dishwasher tablets, would contain the right composition of micronutrients in right quantities for one application per knapsack for a specific crop and area, easing application practices.

Nutrient-coating of seeds may not significantly alter existing farm practices. Recapturing of nutrients either directly lost from the field or after consumption by humans and animals has to become a much more integral part of fertiliser production.

Recycling nutrient fertilisers should not only be encouraged because of the finite nature of mined nutrients but as an essential strategy for reducing the amount of new inert nutrients converted into reactive nutrients and released into the environment.

Pursuing these different avenues to prompt the uptake of nutrients by crop plants and to recycle nutrients implies that increasing global food production may require the use of less, rather than more, mineral nutrients, globally Withers et al.

However, mineral fertilisers use should still be increased in continents e. Africa that currently underutilise them. Clearly, transforming from bulk to targeted fertilisers calls for a transition by the fertiliser and related industry. Valuable lessons could be learned from developments in pesticides over the past decades that moved from toxic, persistent chemicals towards targeted, systemic bio-pesticides based on understanding of the relevant biological processes.

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Agron Sustain Dev — Download references. Funding for this work is provided by the United States Agency for International Development USAID.

Thanks to Susan Yiapan for help with manuscript editing. Virtual Fertilizer Research Center, Washington, DC, NW, , USA. International Fertilizer Development Center, Muscle Shoals, AL, USA. Wageningen University and Research Center, Wageningen, The Netherlands. You can also search for this author in PubMed Google Scholar.

Correspondence to Prem S. Open Access This article is distributed under the terms of the Creative Commons Attribution 4. Reprints and permissions. Bindraban, P. et al. Revisiting fertilisers and fertilisation strategies for improved nutrient uptake by plants. Perhaps the best example where competition for nutrients between immune cells can play a role in shaping immune responses comes from studying DC—T cell interactions.

There is evidence that an antigen-presenting DC can become starved of nutrients, such as glucose, due to competitive nutrient uptake by neighbouring cells, in particular activating CD8 T cells Interestingly, glucose deprivation of DC can result in increased DC proinflammatory outputs, including the expression of interleukin and costimulatory molecules, which leads to enhanced CD8 T cell responses It is well established that T lymphocytes greatly increase nutrient uptake in response to antigen stimulation through up-regulating the expression of nutrient transporters.

This is critically important in the generation of effector cells; indeed T cells lacking certain glucose or amino acid transporters fail to differentiate into effector cells. During activation, CD8 T cells cluster around antigen-presenting DCs within the lymph node 62 , 63 , These clustering T cells can potentially deplete the nutrients from the microenvironment surrounding the DCs Fig.

In support of this, co-cultures of clustering CD8 T cells can inactivate the nutrient-sensitive mammalian Target of Rapamycin Complex 1 mTORC1 signalling pathways in the interacting DCs 25 Fig.

In fact, antigen-presenting DCs can be found at the centre of cell clusters consisting of numerous different types of activated immune cells with elevated nutrient uptake rates in addition to CD8 T cells, including NK cells, CD4 T cells and pDC 65 , 66 , 67 , Therefore, it is tempting to speculate that starvation of DCs, and the resultant increase in DC outputs, is a physiological mechanism for the regulation of DC-induced T cells responses, a scenario where nutrients are acting as an immunological signal Fig.

This is an interesting concept that remains to be formally tested. Competition for nutrients between immune cells. Antigen-presenting dendritic cells DC can be found at the centre of cell clusters consisting of numerous different types of activated immune cells, including CD8 T cells, CD4 T cells, NK cells and plasmacytoid dendritic cells pDC , with elevated nutrient uptake rates that will compete for nutrients blue dots.

Depending on the number of clustering cells surrounding an antigen-presenting DC, nutrients may be available left panel or depleted right panel in the immediate surrounding microenvironment due to competitive uptake. Nutrient starvation will have consequences for the DC including the inactivation of mTORC1 signalling, which has been linked to increased proinflammatory DC functions.

Competition for nutrients between T cells has also been proposed as a mechanism for the selection of T cells that recognise antigen with high affinity Compared with those from low-affinity TCR, high-affinity TCR-antigen interactions induce a more robust and sustained metabolic response, with increased expression of glucose transporters and glycolytic genes Therefore, it is suggested that high-affinity T cell clones could outcompete their low-affinity counterparts for nutrients leading to nutrient starvation and apoptosis of these low-affinity T cell clones It is easy to imagine other situations where neighbouring immune cells would compete for nutrients in similar ways.

For example, during B cell germinal centre responses, a solitary follicular helper T cell is surrounded by a large number of activating B cells with elevated nutrients demands. However, the inability to visualise nutrient abundance at the single-cell level represents a technical barrier that currently limits further exploration of nutrients as important immunological signals.

Nutrient-restrictive microenvironments will directly impinge upon metabolic pathways in immune cells, but will also impact upon nutrient-sensitive signalling pathways important in immune regulation. Glucose and glutamine can impact multiple signalling pathways that are integral to the control of immune responses Fig.

AMP-activated protein kinase AMPK is an indirect glucose sensor that becomes activated when ATP, or glycolytic intermediate fructose-1,6-bisphosphate, levels are decreased due to glucose restriction In effector T cells, AMPK can be activated within an hour of being placed in low concentrations of glucose 72 , Glutamine is also important for ATP production in effector T cells and AMPK can be activated by glutamine restriction in these cells AMPK negatively regulates the mTORC1, an important metabolic regulator with widespread roles in controlling immune cell functions 72 , 73 , 74 Fig.

Roles for mTORC1 include shaping T cell differentiation, controlling NK cells differentiation and effector function, and regulating the function of antigen-presenting DCs 74 Fig.

Competition for nutrients and the impact on signal transduction. Decreased levels of various nutrients within immune microenvironments could occur due to competitive uptake by surrounding cells. Alternatively, the expression of enzymes that consume nutrients, such as arginase, inducible nitric oxide synthase iNOS and Indoleamine-pyrrole 2,3-dioxygenase IDO , can lead to reduced levels of arginine Arg and tryptophan Trp.

Limiting levels of nutrients will affect various signalling pathways. Mammalian target of rapamycin complex 1 mTORC1 signalling is sensitive to levels of arginine, leucine Leu and glutamine Gln. Glucose deprivation will also activate AMP-activated protein kinase AMPK due to reduced levels of ATP or fructose-1,6-bisphosphate FBP leading to the inhibition of mTORC1 activity.

The metabolite phosphoenolpyruvate PEP , generated when glucose is metabolised by glycolysis, can affect the duration of NFAT signalling. Gln and glucose are required for the production of uridine diphosphate N -acetylglucosamine GlcNAc that is important in sustaining the expression of the transcription factor cMyc.

Decreased levels of amino acids in general will lead to the activation of general control nonderepressible 2 GCN2. The product of IDO-mediated Trp metabolism, kynurenine Kyn , can promote signalling through the aryl hydrocarbon receptor AhR.

NFAT nuclear factor of activated T cells. Immunological consequences of changes in nutrient signalling. Activation of AMP-activated protein kinase AMPK or inhibition of mammalian Target of Rapamycin Complex 1 mTORC1 signalling promotes the differentiation of regulatory T T Reg cells over effector T cell subsets T E , inhibits natural killer NK cell functions, and increases the proinflammatory outputs of dendritic cells DC.

Loss of cMyc expression inhibits the functions of T E subsets and NK cells. Activation of general control nonderepressible 2 GCN2 signalling promotes T Reg differentiation, inhibits Th17 differentiation, inhibits CD8 T cell function, and enhances the function of DC. Kynurenine Kyn -mediated aryl hydrocarbon receptor AhR signalling promotes the differentiation of T Reg.

Interestingly, fructose-1,6-bisphosphate is not the only glycolytic intermediate that impacts important immune signalling pathways. In addition to fueling glycolysis and OXPHOS, glucose and glutamine are also used for generation of uridine diphosphate N -acetylglucosamine UDP-GlcNAc ; this is the substrate for O -GlcNAcylation, which is the reversible addition of N -acetylglucosamine GlcNAc to proteins on serine or threonine residues by O-linked N -acetylglucosaminyltransferase OGT.

O -GlcNAcylation is dependent on the supply of both glucose and glutamine in T cells, suggesting that OGT and O -GlcNAcylation are important nutrient-sensing mechanisms in these cells 9. Indeed, OGT is reported to be essential for normal T cell development, activation and clonal expansion 9 , Mechanistically, a number of signalling molecules that are important for T cell function are found to be O -GlcNAcylated, including c-Myc, NFAT and nuclear factor-κB 9 , 75 , This protein modification has not yet been studied in depth in other immune cell subsets.

Apart from glutamine, other amino acids also control numerous signalling pathways that are important for immune function. For example, the activity of mTORC1 is acutely sensitive to the levels of a number of amino acids including leucine, arginine and glutamine In addition, the transcription factor c-Myc is also regulated by amino acid availability.

cMyc protein has a very short half-life in lymphocytes and sustained expression of cMyc is only possible in cells that have high rates of amino acid uptake and protein synthesis 6 , 9 , 19 , cMyc plays a crucial role during the activation and differentiation of T cell subsets and also of other lymphocytes including B cells and NK cells Fig.

GCN2 activity has been linked to the functions of various immune cells. In DCs, GCN2 activation results in enhanced antigen presentation to CD8 cells Conversely, GCN2 activity in gut antigen-presenting cells restrains excessive Th17 responses, with mice deficient of GCN2 developing stronger Th17 responses and more severe colitis in an induced colitis model IDO suppresses T cell responses, at least in part, by depleting tryptophan levels, leading to the activation of GCN2 within the T cell Fig.

Activation of GCN2 in CD8 T cells results in proliferative arrest and anergy, while activation of GCN2 in CD4 T cells can lead to the generation of regulatory T cells Fig. In vitro or ex vivo metabolic analyses have helped bring forth advances in our understanding of the metabolic phenotypes adopted by immune cells.

While these studies have been extremely informative, the reported metabolic phenotypes may not be recapitulated in vivo. The metabolic phenotypes of immune cells are dependent on the supply of the relevant fuels such as glucose and glutamine, which are certainly less abundant in vivo than in culture conditions used in the laboratory.

The consequence of a limiting supply of these fuels in vivo, within discrete immune microenvironments will be the restriction of metabolic pathways and the alteration of nutrient-sensitive signalling pathways that affect immune cell fate and function.

However, our understanding of when and where nutrients are available in vivo is severely hampered by the lack of research tools to measure nutrient distribution at the single-cell level. Therefore, elucidating how nutrient supply affects the metabolism, signalling and thus function of immune cells in diverse and complex immune microenvironments remains a significant challenge for the immunometabolism field.

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It focuses on producing crops with high levels of micronutrients in addition to agronomic traits such as high yield and disease resistance 67 , Biofortification differs from food fortification in that the former involves the addition of nutrients to food crops prior to harvesting whilst the latter adds nutrients to foods during post-harvest processing 3 , Food fortification repeatedly adds nutrients to foods whilst biofortification of varieties of food crops occurs once 74 , Biofortification has been projected to be the most sustainable solution to malnutrition and hidden hunger At present, Harvest Plus, the Biocassava project, and the National Agricultural Research Organization NARO are the major projects initiated for nutritional security via the development of biofortified varieties The initiation of The Harvest Plus Program in , aimed to improve the quality nutritional value of food crops through biofortification The Harvest Plus Program targeted Asia and African countries to ensure the availability and accessibility of high-quality biofortified varieties of staples and the bioavailability of nutrients after consumption 11 , Interventions such as food supplementation and industrial food fortification usually benefit the people of developed and industrialized countries with little to no impact in most developing countries.

On the other hand, biofortification targets the developing and rural world and extends greatly to the developed world as well 5 , 6. To fully implement biofortification, there is a need to assess the bioavailability of the nutrients, set targeted nutrient levels, assess the nutritional requirement of the targeted population, and enhance the absorption and retention levels of nutrients when subjected to processing and storage conditions 15 , In , a total of 33 million people across Africa, Asia, Latin America, and the Caribbean consumed biofortified crops Common examples of biofortification include OFSP, golden rice, yellow and orange maize biofortified with vitamin A, Zn and Fe biofortified-rice and wheat, and beans 15 , Biofortification programs implemented in most countries have yielded positive results.

In Nigeria, a 6-month study involving two groups of pre-school children aged 3—5 years was conducted. One group was fed with foods prepared using biofortified yellow cassava while the other group was fed with white cassava.

The finding showed that the status of vitamin A determined using serum retinol and hemoglobin concentrations of the group that consumed the biofortified cassava significantly improved relative to the group fed with white cassava While in Rwanda, hemoglobin, serum ferritin, and body Fe levels increased among reproductive women after consuming beans biofortified with Fe 85 Table 1.

Biofortification has certain advantages over other interventions to alleviate malnutrition and hidden hunger. It addresses the nutritional needs of both urban and rural populations and could be implemented at low costs after the initial developmental stages It is the most sustainable method among other interventions 6.

Biofortification of nutrients into food crops has less impacts on their organoleptic properties 16 , The main disadvantage associated with biofortification is its inability to rapidly improve the nutritional status of populations who are highly deficient in nutrients 6.

Global production, consumption, and sales of PBFs have significantly increased Additionally, vegetables can contribute to combating undernutrition, poverty, and hunger, since they can be locally cultivated and consumed Many consumers opt for exclusive PBFs due to the established relationships with health improvement, reduction in environmental impacts, and promoting food security 95 , PBFs have also been shown to provide nutritional benefits, specifically increased fiber, vitamin K and C, folate, magnesium, beta-carotene, and potassium consumption Additionally, Ca, I 2 , and Se present in vegetable-rich diet, are beneficial for optimal bone strength, blood pressure, hormone production, heart, and mental health However, it is important that consumers of exclusive plant-based diets select and combine PBFs to help prevent the risk of micronutrient deficiencies 95 ; particularly vitamin B12 needed for neurological and cognitive health , which is mainly animal-derived nutrient, unless supplemented or provided in Bfortified products PBFs also contain high levels of anti-nutritional factors such as phytates and tannins known to reduce the bioavailability of minerals by preventing their absorption in the intestine Additionally, processes like polishing, milling, and pearling of cereals can reduce their nutritional value Biofortification of PBFs presents a way to reach populations where supplementation and conventional fortification activities may be challenging and may serve as an essential step in preventing nutrient deficiencies, especially among consumers of exclusive PBFs.

Biofortification of PBFs involves increasing the levels of nutrients and their bioavailability. This is dependent on enhancing the bioaccessibility of the nutrients in the soil, uptake and transportation of the nutrients through the plant tissues, and their accumulation in non-toxic quantities in edible parts of the plants 22 , Biofortification of PBFs addresses two main challenges; the inability of the plants to synthesize certain nutrients and the uneven distribution of nutrients in different parts of the plant 22 , For example, the grains of rice are the consumed portion of the rice plant, however, pro-vitamin A synthesis and accumulation occurs in the leaves hence limited in quantities and bioaccessibility in the edible portion.

Therefore, biofortifying the rice plant with pro-vitamin A may enhance its accumulation and bioaccessibility in the grains The Harvest Plus Program has designed suitable steps involved in biofortification of PBFs.

These steps Figure 3 can be grouped into four categories; breeding, nutrition and food technology, impact and socioeconomics, and consumer response 11 , 15 , Figure 3. Harvest plus impact pathway—Plant-based biofortification steps [Modified from ].

For example, Se play important roles and is required in extremely minute quantities in the human body with recommended level of Se intake for adults being 0. Excessive intake of Se causes toxicity which is characterized by adverse health effects such as muscle soreness, intestinal complications, cardiovascular diseases and can extend to extreme cases of mortalities The consumption of biofortified foods should help consumers meet their nutritional needs without any risk of toxicity.

Both bioavailability fraction of nutrient that is stored or available for physiological functions and bioaccessibility fraction of the total nutrient that is potentially available for absorption 80 are important in PB biofortified foods.

In a study reported by significant improvement in contents, bioaccessibility and bioavailability of Fe- and Zn-biofortified cowpea cultivars were shown. The values obtained for Fe bioaccessibility [ In another study, increasing doses of selenate during wheat biofortification enhanced both Se content 0.

However, increasing the selenate doses did not necessarily improve the bioavailability of Se in both apical applications. For the basal application, it increased from Although biofortification might improve micronutrients bioaccessibility in food crops; dietary, physiological, human, and genetic factors can affect the bioavailability of these micronutrients in the human body when consumed — Due to the presence of antinutrients, biofortified crops showed limited improvement in the bioavailability of certain nutrients , It also binds with proteins to form complexes which decrease their solubility, limiting nutrient digestion, release and absorption 25 , To overcome this problem, phytase may be added to degrade phytic acid, reducing its ability to form complexes and, enhance micronutrient absorption Tannins, a polyphenol is mostly found in legumes, berry fruits and cocoa beans thereby causing reduction in Fe bioavailability by forming tannin-Fe complexes 25 , , Lectins are common in legumes, cereals and fruits and can damage the cells of the gut epithelium limiting its efficiency in nutrient absorption , Saponins are commonly found in crops such as legumes, tea leaves and oats, and have the potential to form complexes with sterols which affect the absorption of fat-soluble vitamins such as vitamin A and vitamin E Oxalic acids form strong bonds with Ca, Mg, K, and Na forming soluble or insoluble oxalate salts, which prevent the absorption of these nutrients for metabolic activities , Furthermore, the effects of these anti-nutrients are at sub-lethal levels.

Pre-processing and processing conditions such as soaking, germination, cooking, extrusion, milling, and chemical treatments have been used to reduce the antinutrient contents in foods , , Although these antinutrients negatively affect the absorption of essential nutrients, they have health-promoting properties which could be beneficial to the body Phytate has been shown to have hypoglycemic, anti-inflammatory and anti-carcinogenic properties 25 , Polyphenols aid in removing free-radicals and limiting low density lipoproteins , Lectins promote mitotic cell divisions and destruct cancer-affected cells Saponins have great antimicrobial, cancer-prevention and cholesterol-reducing properties which reduce the risk of cancer and heart diseases , The food environment from which biofortified foods are consumed can influence bioavailability of micronutrients.

When micronutrients are entrapped in macronutrients matrix, their bioavailability may depend on the breakdown of the macronutrient The presence of fats in the food environment promotes the absorption of fats soluble vitamins such as vitamin D, subsequently enhancing Ca absorption Dietary fibers with increased solubility tend to bind with minerals and reduce their bioavailability Age is one of the human factors that influence bioavailability of micronutrients; increasing age decreases the bioavailability of micronutrients Micronutrient absorption may increase at certain periodic stages such as pregnancy, lactation and breastfeeding.

Bioavailability of calcium increases at these stages in women to meet the nutritional requirements of the infant Also, the health condition of consumers may have limiting effects on bioavailability of micronutrients.

Conditions such as diabetes, obesity, celiac disease, hypochlorhydria, chronic pancreatitis, and parasite infections limit the absorption of micronutrients , People from different ethnic groups may have variations in genes involved in micronutrient absorption, affecting their bioavailabilities Three biofortification strategies including agronomic intervention, conventional plant breeding and genetic engineering have been described for PBFs 1.

These strategies have been applied to cereals, legumes, oilseeds, vegetables and fruits, with cereals having the largest number of biofortified varieties. Due to limited genetic variability in oilseeds, the transgenic approach is well-suited 14 , Agronomic biofortification involves the application of mineral fertilizers to soil or crops to increase the concentration and bioaccessibility of specific nutrients in the crops Initially, agronomic practices were done to improve the health of crops and increase yield.

However, the importance of nutrition has been highlighted over the years; hence agronomic practices have been expanded to improve the nutritional qualities of crops 5 , 65 , Changes in climate conditions and rapid depletion of soil nutrients is an indication of the need to improve and expand agronomic practices to include improving the nutritional qualities of crops Agronomic biofortification focuses on improving solubilization and mobilization of minerals 55 , The effectiveness of agronomic interventions depends on the soil composition, the solubility and mobility of minerals, the ability of crops to absorb minerals, and the accumulation of bioavailable minerals in non-toxic levels in the edible parts of the crops 25 , 55 , Agronomic biofortification mainly covers minerals and not vitamins because vitamins are synthesized in the crops.

Hence, agronomic biofortification cannot be used as a single strategy in eliminating micronutrient deficiencies and should complement other strategies for effective biofortification 1 , 5 , The use of fertilizers for agronomic biofortification must be performed carefully as an improper application of fertilizer can have unanticipated, and sometimes severe, consequences on the environment and crops.

In contrast, a balanced fertilization strategy is both economically more beneficial and environmentally more sustainable.

Additionally, soil microorganisms play a crucial role in the soil ecosystem and are highly sensitive to fertilization. A deficient fertilization regime results in nutrient deficiency and subsequent modifications of the microbial community of the soil.

Unbalanced fertilizations can have detrimental effects on soil biological health over the long-term 55 , Mineral fertilizers are mostly applied to the soil or directly sprayed on the leaf of crops. The former is more common and applicable when nutrients are required in higher amounts.

Foliar application is more economical and applicable when nutrient deficiency symptoms in crops are visible 25 , , when mineral elements are not translocated and accumulated in adequate amounts in the edible parts of the crop 1 , Foliar application tends to be more effective than soil applications because unlike soil application, it increases micronutrient contents rather than just promoting yield 25 , , Foliar application is dependent on several factors including the type of fertilizer, characteristics of crops, time of application, and environmental conditions , Agronomic biofortification of crops with minerals such as Fe and Zn require certain adjustments.

Due to their low mobility, adding metal chelators to the fertilizer is essential 5. Foliar application of FeSO 4 has proven effective for Fe biofortification For I 2 , potassium iodate has been effective as seen in countries like China 5 , , Inorganic fertilizers such as ZnSO 4 , ZnO, and Zn-oxy-sulfate are suitable for Zn agronomic biofortification.

Just like Fe, foliar application of Zn chelators such as ZnEDTA is highly effective — Se is agronomically fortified as selenate which is converted into organic selenomethionine in the crop. Both foliar and soil applications are suitable for Se biofortification, but dependent on soil type and timing of the application However, foliar applications are costly and could easily be rinsed off by raining water , The characteristics of the leaf play an important role in absorbing nutrients during foliar applications.

Nutrients from foliar application penetrate the cuticle to leaf cells and are transported to other parts through the plasmodesmata. The age, structure and permeability of the leaf affect nutrients absorption Foliar application is mostly effective during the flowering and early milk phases than booting and elongation phases of the developmental stages of crops.

The flowering and early milk stages are among the earliest phases where absorption of nutrients for fruit formation begins, hence, foliar application of nutrients at this stage would contribute greatly to increasing the micronutrient contents of the fruits , This was experienced during Zn agronomic biofortification of wheat using foliar application, which was attributed to enhanced phloem mobility and active photo-assimilate allocation to reproductive silk organs that enhanced remobilization of nutrients , , Also, environmental conditions such as time of the day, humidity, temperature, and wind speed affect the efficiency of foliar applications Warm and moist conditions in the early morning and late evening promote permeability of nutrients whilst low relative humidity and high temperature evaporate water from sprayed solution, leading to concentration of minerals on surfaces which reduces mineral permeability Other strategies that are used for agronomic biofortification include coating and priming of seed with mineral fertilizers.

These strategies aid in promoting crop yield and development but have minimal effects on the nutritional qualities of crops 55 , Agronomic biofortification has been used effectively in several countries to combat micronutrient deficiencies and promote agricultural productivity.

The effect of agronomic biofortification of selected underutilized vegetables in Ghana has been assessed Increasing application rate of K fertilizer increased fruits and vegetables weight. Also, the application rate of K fertilizer and the type of K fertilizer synergistically affect K concentration in the fruit.

In another study that assessed the influence of irrigation and fertilizer application on β-carotene yield and productivity of OFSP in South Africa , the total storage root yield increased by 2—3 folds and β-carotene content increased from Agronomic biofortification is simple and yields results rapidly in the short term 5 , However, mineral fertilizers used in agronomic biofortification is costly which increases the prices of biofortified crops, making them inaccessible to poorer populations Also, agronomic biofortification is highly dependent on farmers.

Application of mineral fertilizers is a regular activity hence may be omitted by farmers if they do not gain profits from the process 25 , 80 , Application of mineral fertilizers repeatedly may also cause accumulation, leading to toxicity 1 , In addition, increasing demand for mined minerals such as Se may cause exhaustion and negative impact on the environment 80 , Plant breeding involves producing genetically different or new varieties of crops with improvements in essential micronutrients 55 , , Biofortification through plant breeding aims at improving the concentration and bioaccessibility of minerals in crops by utilizing the genetic differences between crops of similar species 19 , Plant breeding initially focused on promoting yield and improving agronomic traits of crops however, recent plant breeding techniques have been geared toward promoting both the nutritional quality and agronomic traits 54 , Plant breeding techniques should focus on introducing genotypes that would enhance the uptake, transport and redistribution of minerals to improve the efficiency of biofortification In order to achieve this goal, there is a need to enhance mineral mobility in the phloem vessels responsible for redistributing and remobilizing these minerals The translocation and redistribution of Zn from the shoot to fruits or edible portions of crops has been a challenge due to the low mobility of Zn in phloem vessels, leading to lower Zn concentrations in the edible portions as compared to the leaves or the root system 60 , Plants have been bred using three main techniques—conventional, molecular and mutation breeding 25 , Conventional breeding is the most common and accepted form of plant breeding for biofortification 14 , Conventional breeding enhances improvement in the nutritional qualities of crops without compromising other agronomic traits 54 , 55 , Biofortification through conventional breeding involves crossing crops with genotypic characteristics of high nutrient density and other agronomic traits to produce new varieties with desirable nutrient and agronomic traits It requires identifying the biodiverse varieties of crops, assessing traits and amounts of target nutrients in these varieties, and determining the effects of growing conditions on the stability of these traits Currently, about varieties of biofortified cops have been released in over 30 countries via conventional breeding A typical crop biofortified through conventional breeding is OFSP which has been biofortified with pro-vitamin A and with increased yield traits 19 , , Quality Protein Maize QPM is also a product of conventional breeding 25 , Mutation breeding differs from conventional breeding such that, differences in genetic traits among crops are created by introducing mutations through chemical treatments or physical methods such as irradiation 25 , Mutation breeding has been recently adapted to biofortify resistant chickpea mutants like Pusa Ajay , Pusa Atul , Pusa Girnar , and Pusa, developed at I.

Crop improvements via mutation in Pusa include: thin testa, attractive bold seeds, better cooking quality and high yield performance Unlike conventional breeding, differences in genetic traits among crops are created by introducing mutations through chemical treatments or physical methods such as irradiation 25 , Biofortification through molecular breeding involves identification of the position of a gene responsible for improving the nutritional quality and closely linked markers to that specific gene.

With the aid of the marker, the desirable traits can then be bred into the crop using conventional breeding 1 , Molecular breeding can be used to determine if a desirable trait is present or absent in a specific crop during developmental stages.

Hence, it is more rapid as compared to other forms of plant breeding 25 , These varieties have been released in countries such as Zambia, Nigeria and India. Also, it has been reported that several rice varieties have been bred to produce a variety with high Fe and Zn contents and improved agronomic traits Plant breeding is sustainable and less costly as compared to other biofortification strategies 1 , 14 , and financial investments occur only at the research and development stages.

Also, unlike agronomic biofortification, plant breeding has little to no impacts on the environment Consumers generally accept crops that are biofortified through conventional plant breeding and easy to obtain regulatory approval as compared to genetically modified GM foods However, conventional breeding is labor intensive and takes longer time to develop varieties with both desirable traits such as nutrient densities and agronomic traits 19 , Also, there may be limited genetic variations among crops, making it impossible to biofortify these crops via plant breeding 14 , 79 , , and may not be successful for all nutrients.

For instance, breeding varieties of rice with improved vitamin A content initially proved to be challenging, but recent advances in omics technologies have provided the opportunity to practically biofortify varieties of rice with pro-vitamin A Also, crops such as banana that are propagated by vegetative means are not suitable for conventional breeding Plant breeding relies heavily on genetic variations among crops and when variation is limited, it hinders the opportunity of biofortification through plant breeding Unlike plant breeding, genetic engineering is not limited to crops of related species.

Genetic engineering has demonstrated to be a viable solution to this problem 14 , 65 and has been shown to effectively biofortify crops such as banana and rice, which cannot be subjected to conventional plant breeding 79 , , Genetic engineering provides the platform for introducing nutrient or agronomic traits new to specific crop varieties by applying plant breeding and biotechnology principles , and when employed in biofortification, it identifies and characterizes suitable genes which could be introduced into crops to translate into desirable nutritional qualities It utilizes genes from vast array of species, including bacteria, fungi and other organisms.

Certain microorganisms enhance the uptake of nutrients by plants. Genes from these microorganisms can be genetically engineered into crops to enhance nutrient absorption, transportation, and concentration 25 , Fluorescent pseudomonas is a bacterium that enhances plant Fe uptake.

Plants growth-promoting rhizobacteria and mycorrhizal fungi enhance the absorption of minerals from the soils and promote plants growth. Genes from bacteria and Aspergillus species have been used to adjust the lysine and phytate contents of crops such as rice and wheat, respectively 25 , Genome editing, also known as gene editing, corrects, introduces or deletes almost any DNA sequence in many different types of cells and organisms Gene editing provides an opportunity to develop GMOs without the use of transgenes; in addressing regulatory challenges associated with transgenic crops , These off-targets can be overcome by the dimeric nuclease method, which is highly precise and specific According to , low levels of knowledge about gene editing occur because information generated in scientific studies has not been communicated effectively to consumers Biofortification through transgenic approach has been greatly explored in most developed countries.

The most notable example is golden rice which was developed by biofortifying rice with pro-vitamin A 1. This was done by expressing genes encoding phytoene synthase and carotene desaturase which are responsible for β-carotene pathway In golden rice, the expression of these genes caused an increase in pro-vitamin A levels by 1.

The overexpression of Arabidopsis thaliana vacuolar Fe transporter VIT1 in cassava caused fold increase in Fe contents in the storage roots The overexpression of Zn transporters and expression of the gene responsible for phytase activity in barley enhances the levels and bioavailability of Zn 55 , In order to improve the efficiency of the transgenic approach as a biofortification strategy, omics technology has been introduced 20 , Omics technology explains the interrelationship between genes, proteins, transcripts, metabolites, and nutrients 20 — Specific genes control the uptake, transport, concentration, and bioavailability of nutrients by crops.

Hence, genomics omics technology of genes is important since it presents the opportunity to study these specific genes and design suitable ways of improving and inducing them into crops , Transcriptomics omics technology of transcripts aids in conducting full-spectrum analysis to identify a specific expressed gene 20 , , Proteomics omics technology of proteins helps to understand the role of proteins in nutrient synthesis, uptake and transport pathways 21 , Metabolomics omics technology of metabolites aids in assessing metabolic pathways that control the biosynthesis of natural metabolites , , while ionomics considers how minerals present in crops undergo changes in response to genetic and environmental factors These omics technologies have been used in studies involving biofortification of lysine, Ca, Zn, Fe, and vitamin C in PBFs such as maize, finger millet, wheat and tomatoes, respectively PBFs such as cauliflower, cassava, and banana have been biofortified by both transgenic and breeding approaches while barley, soybean, lettuce, canola, carrot, and mustard have been biofortified with transgenic and agronomic approaches The transgenic approach has been shown to be sustainable and rapid when introducing desired traits into crops 25 , Table 2 summarizes a selection of biofortified crops developed by transgenesis.

Biofortification through the transgenic approach has its limitations. The transgenic approach requires huge investments in financial, time and human resources at the research and developmental stages 1 , 5 , Transgenic crops are not generally accepted due to concerns over GMOs 5 , Also, there are several regulations governing the production of transgenic crops Interactions among genes introduced into crops during genetic engineering may reduce the efficacy of the biofortification process 14 , Agronomic biofortification, plant breeding and genetic engineering, including omics technology, are suitable strategies that could be used for plant-based biofortification to help reduce the occurrence of malnutrition and hidden hunger.

The successful implementation of biofortification programs depends on the acceptance of biofortified crops by farmers and consumers The acceptance of GM crops is different for customers and farmers. In general, consumers have expressed a lower level of acceptance for GM crops and foods, because they are skeptical about the risks and benefits associated with these products , Many factors influence consumer attitudes, including information, trust, beliefs, perceptions of benefits and risks.

Several concerns have been raised about the human health implications from gene flow and transfer, environmental impacts from possible development of resistant weeds and crops, impacts on conventional methods, artificial-like methods, toxicity, and allergenicity of GM crops , It is because of this that genetically engineered plants and their products are often rejected based on unverified grounds.

The overall inclination toward avoidance have been directed toward GM crops, even though several scientific reports have shown that GM crops are safe to consume , Therefore, it may be necessary to create adequate informational programs which would highlight the importance of biofortified-genetically engineered plants and quell misconceptions about GM crops In many parts of the world where biotech seeds are available, farmers are highly embracing and accepting biotech seeds because of the benefits they receive from GM crops Globally, GM crops are governed by different regulations These regulations and legislations have huge effects on the commercialization and adoption of GM crops Strict labeling rules of GM crops have been set by over 40 countries The European Union introduced a very strict authorization system for GM crops and foods over a decade ago, as a precaution.

All food derived from GM plants were required to be labeled based upon the process, even if no traces of the genetic modification could be detected in the purified end-product. Accordingly, recent trends in many European countries have created an environment that makes the cultivation of GM crops and foods extremely difficult.

The US legislation is more lenient in that novel GM foods do not have to be labeled if they do not differ in composition from established non-GM foods. As with Europe, China has been requiring the labeling of foods derived from GM crops for more than a decade , Also, most agri-business companies patent newly developed GM crops, monopolizing the commercialization of the GM crops.

Several debates have been raised about the motive for developing GM crops—for privatization and profit-making or for the purpose of promoting food security Therefore, regulations and legislations governing GM crops should be adjusted, especially in developing countries, to be less rigorous, and cost and time-effective to promote the adoption of GM crops , In many countries, researchers and seed companies are predicting that new breeding techniques such as intragenesis and cisgenesis, which transfer only genetic information from the same species without transferring foreign genes, but could provide smoother routes to market for plants with improved traits, thereby avoiding the roadblocks presented by transgenic GMOs , These newly developed breeding techniques do not fit within the traditional definition of GMOs, and a debate is taking place in many areas regarding how they should be regulated.

In view of this regulatory uncertainty, it is possible for GM products to be distributed differently in the market and for customer acceptance to differ globally What does the future hold for GM crops?

Will there be a universal labeling system for GM crops? Should there be a change in regulations governing Intellectual property rights of GM crops? The adaptation of GM crops in the production of biodegradable polymers has been discussed.

Does this indicate that GM crops have strong positive environmental impacts in the future , ? The possibility of using transgenic crops as means of providing vaccines and medications can be exploited in the future Can GM crops be adapted to have other desirable traits aside agronomic and nutritional traits?

These questions could be the focal points of future research in promoting the development and commercialization of GM crops. Malnutrition and hidden hunger are both present in developed and developing countries and have devastating effects globally.

The recent implications of the global pandemic have shown that food systems need to be adapted to advance global changes that can limit deficiencies in our food supply.

Furthermore, climate change projections predict higher inequality and poverty for developing countries and hence, the need to augment the nutritional content in PBFs.

Biofortification is the most sustainable and cost-effective method for alleviating malnutrition. Biofortification of PBFs has been used to produce crops with adequate nutrient density and bioavailability and help to combat hidden hunger.

Through plant breeding, transgenics, and mineral fertilizer applications, micronutrient malnutrition can potentially be tackled. For future actions, an integrated approach is required, where politicians, farmers, food product developers, genetic engineers, dietitians, and educators need to be included in the developing efforts.

One of the biggest challenges of biofortification aside from the methods to strengthen the nutritional value of crops is the public acceptance. Especially for the transgenic techniques more education and marketing should be invested for the success of biofortified products in the market as only few cultivars are finally released for costumers.

Globally, the specificity of biofortification techniques should tackle regional nutritional challenges and should be chosen based on the likelihood of acceptance of cultural difference in consumers. Overall, biofortification represents a promising group of techniques that can improve the global nutritional wellbeing and lead us closer to minimize hunger and malnutrition.

AA conceptualized the manuscript, crafted the outline, led the manuscript writing, and formatted the final version of the manuscript. KO and SA wrote the initial draft.

SA prepared the illustrations. AA and ME reviewed and edited the manuscript. All authors approved the submitted version of the manuscript. 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.

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J Nutr. Ser is transported to the peroxisome and converted in hidroxypyruvate and glycerate by the action of serine:glyoxilate aminotransferase SGAT and hydroxypyruvate reductase HPR , respectively. In the chloroplast, glycerate results in glyceratephosphate by the glycerate kinase GK , which is finally converted to RuBP in the Calvin cycle Wingler et al.

Therefore, photorespiration arises as a relevant physiological process that might optimize NUE by specifically providing intermediates and reducing molecules to concomitantly assure energy dissipation and photoprotection and enhancing N assimilation Wingler et al.

Both anions share very similar physical properties in solution, playing important roles in charge balance and cell osmoregulation, showing strong dynamic interactions in land plants Wege et al.

As an anion, this implies a high cost of metabolic energy Brumós et al. When supplied to levels in excess to satisfy micronutrient requirements but insufficient to cause toxicity i. NUE and plant growth in different species of agronomic interest Rosales et al.

Seeds of tobacco Nicotiana tabacum L. At 15 days after sowing DAS , seedlings were transferred to 7. During the experiments, plants were watered with a basal nutrient solution containing: 4 mM KNO 3 , 1 mM Mg NO 3 2 , 1 mM Ca NO 3 2 , 0. To counterbalance all cations supplemented with the three CL treatments and to fulfil plant nutritional requirements, phosphate and sulphate salts were added to 0CL, 2CL and 6CL treatments as follows: 4.

All nutrient solutions were adjusted to pH 5. Pots were watered every two days, allowing adequate drainage to keep the soil close to field capacity 3. When the flowering stage started 60 DAS , half of the plants used in the assay were randomly harvested and weighted before noon between This material was further divided into two parts: one part was frozen with liquid nitrogen and conserved at °C in an ultra-low temperature freezer for further measurements of enzymatic activities; the other part was dried in a forced-air oven at 75°C for hours, grounded to powder in a mortar and preserved dry for further recording of plant organ dry weight DW and for determination of different nutrient contents.

The other half of the plant samples were used for xylem sap extraction and ion content determination. Xylem sap was collected from tobacco plants of 60 DAS grown in the 0CL containing 75 uM of Cl — , 2CL and 6CL treatments.

Pots were transferred to a airtight container, and submerged in the appropriate nutrient solution almost up to substrate level. Each plant was detached using a razor blade, and the root sap exudate was collected 1h later.

Shoots were harvested and weighted to obtain fresh weight, and samples were dried in a force-oven at 75°C for hours to obtain dry weight. Xylem sap samples were dilluted in a rate of For the determination of anion content, colorimetric assays were conducted using a microplate spectrophotometer reader Omega SPECTROstar, BMG LABTECH GmbH, Germany.

Total N accumulation TNA was calculated as the result of TNC multiplied by total DW as described in Rubio-Wilhelmi et al. Frozen leaf tissues were ground in a chilled mortar with 1 mL of extract buffer pH 7.

Homogenate was centrifuged for 20 min at 30, g. The upper fraction was used to quantify nitrate reductase NR; EC 1. For glutamine synthetase GS; EC 6. Homogenate was centrifuged at 30, g for 20 min. Supernatant was used for colorimetric and kinetic assays GS and AAT, respectively , based on methods described by Wallsgrove et al.

All these activities are shown in Figure S1. Protein assay was done according to the colorimetric method Bradford using bovine serum albumin BSA as standard. The mixture was stirred for 30 minutes at room temperature and centrifuged at 8, g for 10 minutes. Samples were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography as described by Megías et al.

Total RNA was extracted from ground leaf samples using TRIsure reagent procedure BIOLINE, London, UK. One µg of total RNA was used as a template for first strand cDNA synthesis in a 10 μl reaction using the iScript cDNA synthesis kit BIO-RAD, Hercules, CA, USA.

Diluted cDNA was used as a template for gene expression level quantification by quantitative real-time reverse transcription Q-RT -PCR. PCR was carried out with previously designed gene-specific primers Tables S1 , S2.

Data were analysed using the Bio-Rad CFX Manager software. Six biological replicates and two technical repeats were used per treatment in every run.

All mRNA levels were calculated from threshold cycle values and normalized with respect to the transcript level of the housekeeping genes NtL25 Nitab4. Reactions were performed on two independent RNA batches and results were comparable in the different assays. After fixation, fragments were dehydrated through graded alcohols and embedded in Epon resin Epoxy Embedding Medium, Sigma.

Sections were cut with an ultramicrotome Reichert-Jung Ultracut with a diamond knife and mounted on nickel grids. The Shapiro—Wilk W test was used to verify the normality of the datasets; and the Levene test to determine the homogeneity of variance. Each variable assayed was analysed using one-way Analysis of variance ANOVA or multivariate ANOVA MANOVA including treatments as grouping factors.

Whether homogeneity of variance or normality was not reached after data transformation, non-parametric Kruskal-Wallis ANOVA was followed by post hoc Mann-Whitney U test. These nutritional replacements did not induce nutritional deficiencies since no symptoms of wilting, bronzing or chlorosis were observed Table S3 ; Figure S4.

However, no significant differences were found in leaf biomass between 2CL and 6CL treatments. The results showed that the increased total biomass was mainly due to higher growth of root and leaf organs i.

e, stem and flowers values Figure 1. No differences in the root:shoot ratio were found between treatments Figure 1D. A Total dry weight DW ; B total leaf DW; C total root DW; and D root:shoot ratio are shown.

DW, dry weight. Results showed no significant differences in the total N content per biomass unit TNC between the three nutritional treatments 0CL, 2CL and 6CL. Statistics in B was calculated through ANOVA. The dotted line represents the normalized activity in 0CL plants.

Proteins and their elemental units, amino acids, are the major components and sources of organic N in plants. In addition, no significant differences in the content of alanine, cysteine, histidine, leucine, lysine, phenylalanine, threonine, tyrosine and valine were found Figure 5C.

A Total proteins content; B Total free amino acids content; C Amino acids profile. Given the tetraploid nature of N. tabacum genome, we conceived three different SHMT candidate genes according to the homology in their A. Furthermore, TEM micrographs showed a higher association of organelles involved in photorespiration i.

C Transmission electron micrographs showing the ultrastucture of leaf mesophys cells. Organelles that contribute to photorespiration were labelled as M, mitochondrion; P, peroxisome; C, chloroplast;. Both anions share similar physical properties in solution and show strong dynamic interactions in land plants, playing important roles in charge balance and cell osmoregulation Wege et al.

Plants utilize metabolic energy to take up nutrients from the soil. To do so, plant cells include semi-permeable plasmatic membranes with transmembrane protein channels for the intake of nutrients.

In agriculture, N fertilization represents a major bottleneck for crop yield due to its well-known crucial role in plant growth and metabolic processes. The high energy cost for the synthesis of N fertilizers, as well as its intrinsic mobility in the complex atmosphere-plant-soil system, have highlighted the environmental drawbacks of the unsustainable N use in agriculture Rothstein, ; Keeney and Hatfield, ; Garnett et al.

Therefore, NUE parameters specifically NUE and NU T E are considered important crop traits and a potential tool to reduce the abusive use of N fertilizers or to improve plant growth when low N is available Baligar et al.

From all N forms, organic N represents the main component of TNC in plants, being a crucial element to estimate NUE. This finding could be interpreted as the pool of soluble amino acids is optimized with N assimilation and C fixation by the RuBisCO activity to increase the synthesis of proteins.

These results are in line with those reported by Findenegg et al. Thereby, the analysis of the free amino acids profile can confer new perspectives to elucidate possible metabolic adjustments which remained unknown Florencio-Ortiz et al.

From the amino acids studied, arginine, aspartic acid, glutamic acid, glutamine, Gly, isoleucine and proline were decreased in 6CL plants, which is consistent with the decline of total amino acids content.

The significant reduction of aspartic acid to synthesize asparagine might directly affect other aspartate-derived amino acids such as methionine, threonine, isoleucine, and lysine Azevedo et al.

The conversion of glutamine to glutamic acid and vice versa represents the core of the N assimilation pathway, as the α-amino group of glutamic acid is transferred to other amino acids by the action of multispecific aminotransferases Forde and Lea, ; Bender, Additionally, photorespiration provides intermediates and reduced equivalents for the Krebs cycle and it is a sink for ROS elimination and energy dissipation to photoinhibition avoidance Wingler et al.

Then, the depletion of the photorespiration pathway could strongly condition plant survival, being suggested as a target for molecular genetic engineering in agriculture to improve crop yield Betti et al.

Consistently, Busch et al. Thus, photorespiration favors the metabolism of N, C and sulfur, generating Ser i. The physical association between chloroplasts, peroxisomes, and mitochondria is important in the regulation of the photorespiratory process Rivero et al.

The co-ordination of C and N assimilation has been studied extensively over many years because it is a key determinant of plant productivity. Previous appreciations by Franco-Navarro et al. Nonetheless, the low g s rates were counteracted by the higher mesophyll diffusion conductance to CO 2 g m , avoiding a negative effect on photosynthetic performance, due to a higher surface of chloroplasts exposed to the intercellular airspace of mesophyll cells.

The complex interaction of this process with chloroplast, peroxisomes, mitochondria, and cytosol compartments led us to believe that macronutrient Cl — could acquire other specific roles in higher plants. The leaf anatomical and cellular parameters Franco-Navarro et al.

The blue arrows represent the up-regulatory enzymatic responses observed. The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. PP-T, RA, ML, JDF-N, and FD-G performed the experiments, participated in the conception of experiments and research plans, analyzed and plotted the data; PP-T participated in the writing of the article; JC-F participated in the conception of research plans, co-funded to finance the project, supervised the experiments, and participated in the writing of the article; and MR supervised and participated in the performance of the experiments, conceived research plans, co-funded the project and wrote the article.

All authors contributed to the article and approved the submitted version. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research URICI.

Help, expertise and technical assistance of B. Beas, F. Moreno-Racero, D. Romero-Jiménez, E. Sánchez-Rodríguez, M. Rubio-Wilhelmi and J. Ruiz are gratefully acknowledged.

The authors thank at the University of Seville, research, technology and innovation centre CITIUS for the service for the use of electron microscope and also like to thank Dr. Purificación Calvo Dept. Microbiology, US for her unvaluable help in preparating the samples for microscopy.

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. Abdo, A. Mitigating nitrate accumulation in potato tubers under optimum nitrogen fertilization with K-humate and calcium chloride.

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Talanta , 95— Miranda-Apodaca, J. N metabolism performance in Chenopodium quinoa subjected to drought or salt stress conditions. Misra, N. When grown on the medium containing different concentrations of nitrate, the NLP7 -overexpressing plants showed obvious growth advantages compared with wild type WT and nlp plants.

The nlp plants showed constitutive N-deficient phenotypes on both nitrate-rich and -poor media Fig. Statistical analyses indicated that overexpression of NLP7 could increase biomass of the shoot under both nitrate-rich and -deficient conditions, and NLP7 knockout impaired plant growth even under nitrate-rich conditions Fig.

We also found that the NLP7 -overexpressing plants had higher chlorophyll contents than the mutant and WT plants under different nitrate conditions Fig.

These phenotypes were further proved by vertical growth assay as showed in Supplementary Fig. Considering the influences of plant density on nitrate concentration, we grew 28 plants 7 plants in a row per plate for further analysis.

Results showed that the NLP7 -overexpressing plants grew much better than the WT and nlp plants on medium containing 0. Compared with WT and nlp plants, the NLP7 -overexpressing plants had significantly increased fresh weight FW with the raising nitrate concentrations of the medium Fig.

Moreover, we conducted functional complementation analysis by expressing a functional pNLP7:NLP7-GFP fusion construct in the nlp plants. The results showed that nlp plants showed N-deficient phenotypes with lower FW and longer primary roots PRs on N-rich medium.

However, these phenotypes could be restored by expressing pNLP7:NLP7-GFP construct Supplementary Fig. Taken together, these results demonstrate that the growth of nlp is relatively insensitive to N level while the NLP7 -overxpresssing plants improved growth and response to N availability.

a The phenotypes of the day-old WT, nlp and NLP7 -overexpressing plants grown on medium plates containing different concentrations of nitrate with a density of 80 plants per plate.

Diameter of the plate is b Shoot fresh weight of the day-old plants grown under different nitrate conditions. c—d The chlorophyll a c and b d contents of the day-old plants. e The phenotypes of the day-old plants grown with a density of 28 plants per plate under different nitrate conditions.

f Fresh weight of the day-old plants under different nitrate conditions. Additionally, we also found that the nlp plants displayed severe N-starved phenotypes with yellow leaves, while the NLP7 -overexpressing plants still remained green after 3 days N starvation in liquid culture Supplementary Fig.

Moreover, NLP7 transgenic plants grew much bigger in N-limiting soil with significantly higher rosette surface area and rosette biomass than the WT and nlp plants under short-day conditions.

The mutant and WT plants showed much more severe N-deficient phenotypes with discolored rosette leaves compared with the NLP7 -overexpressing plants Supplementary Fig.

Overexpression of NLP7 not only increased the shoot biomass, but also enhanced root growth with higher root biomass and longer roots, while nlp mutant had much lower shoot and root biomass and delayed flowering compared with the WT Supplementary Fig. These results demonstrate that overexpression of NLP7 in Arabidopsis enhanced the plant growth and N use as well as tolerance to N-deficiency.

To verify the root architecture under different nitrate conditions, we checked the root system of the plants vertically grown on media containing different concentrations of nitrate.

The results showed in Fig. The nlp plants showed reduced shoot FW compared with WT plants under different nitrate conditions, with more significantly reduced under higher nitrate conditions Fig.

However, there was no significant difference of root FW between the nlp and WT plants. Consequently, the shoot to FW weight ratio, an important parameter for nutrient starvation 31 , was significantly higher for NLP7 -overexpressing plants and much lower for the nlp plants under different nitrate conditions, especially under N-rich conditions Fig.

More detailed analysis of the root showed that the NLP7 -overexpressing plants significantly increased PR length and lateral root LR density under both nitrate-rich and -poor conditions. These results were confirmed by the data of time-course analysis of root development as shown in Supplementary Fig.

S6 , where NLP7 -overexpressing plants had better-developed root system with increased LR number and slightly longer PRs compared with WT under both low and high nitrate conditions. The phenotypes of nlp plants agree with the previous results that NLP7 knockout confers constitutive N-starved phenotypes under different N conditions These data indicate that NLP7 may be involved in root development and overexpression of NLP7 promotes root development.

a The phenotypes of the day-old plants on vertical plates containing different concentrations of nitrate. To gain molecular insights for the improved growth of the NLP7 transgenic plants under different N conditions, we measured several metabolite markers for N assimilation.

Contents of glutamine Gln and glutamate Glu , markers for N utilization, were measured respectively. However, under this condition, Gln content was significantly higher in the NLP7 -overexpressing plants, whereas was significantly lower in the nlp mutant Fig.

These data implies that more Glu is converted to Gln in the NLP7 -overexpressing plants than in the mutant plants under nitrate-rich conditions. In fact, we found the GS enzyme activities were much higher in the NLP7 -overexpressing plants while significantly lower in the mutant Fig.

Interestingly, we found that nitrate accumulated considerably in the nlp mutant under different nitrate conditions, especially under high nitrate conditions.

Conversely, nitrate content in the NLP7 -overexpressing plants decreased significantly Fig. These results led us to test the enzymatic activities of NR.

Figure 3f shows that NR activities increased markedly in the NLP7 -overexpressing plants in contrast to the dramatic decrease in the nlp mutant, suggesting higher nitrate assimilation efficiency in the NLP7 -overexpressing plants. c Enzyme activities of glutamine synthetase in the plants under different nitrate conditions.

d,e Content of total protein d and nitrate e in the plants under different nitrate conditions. f Enzyme activities of nitrate reductase in the plants under different nitrate conditions. g Nitrate uptake activity assay. DW, dry weight. To further assess the role of NLP7 in N assimilation in plant, we performed qRT-PCR to investigate the expression of some genes related with nitrate assimilation and signalling in 3 days N-starved seedlings and seedlings resupplied with nitrate for 0.

As showed in Fig. However, after nitrate resupplied, almost all the genes were highly induced in WT and NLP7 -overexpressing plants, but the induction levels were much lower in the nlp These results indicate that NLP7 plays a vital role in nitrate assimilation and signalling, which is consistent with the recent reports In addition, we checked the expression levels of all these genes in 7-day old plants grown on MS medium and observed that 11 of the 13 tested genes, except NRT1.

On the other hand, 10 out of the 13 genes showed prominent higher expression levels in the NLP7 -overexpressing plants Supplementary Fig. UBQ5 was used as an internal control. NRT, nitrate transporter; GS2, glutamine synthetase 2; NIA, nitrate reductase; NIR1, nitrite reductase 1; NLA, nitrogen limitation adaptation; LBD, lateral organ boundary domain; AFB3, auxin signaling F-box 3.

N metabolism is known to coordinate with photosynthesis and C metabolism In this study, we found nlp plants displayed N-starved phenotypes with a much smaller and pale rosette when grown in soil Fig.

Further analysis found chlorophyll content was remarkably reduced in the leaves of nlp plants, whereas increased in the NLP7 -overexpressing plants Fig. Photosynthesis rate was enhanced in the NLP7 -overexpressing plants and decreased in the nlp plants Fig.

However, compared with WT, total C content reduced by 2. Total N content was found decreased in nlp while markedly increased in the NLP7 -overexpressing plants compared with WT under both nitrate -rich and -deficient conditions Fig. a Image of 5-week-old NLP7 -overexpressing, nlp and WT plants grew in N rich soil.

b Chlorophyll contents of the rosette leaves of the 5-week-old plants. c Comparisons of photosynthesis rate in the 5-week-old NLP7 -overexpressing, nlp and WT plants.

Photosynthesis rate was measured as described in Experimental procedures. Two measurements were made for each plant, and eight plants were used for each line. g—i Expression levels of the PEPC genes AtPPC1 AT1G g , AtPPC2 AT2G h , AtPPC3 At3G i.

We also measured some C metabolism-related compounds. These data suggest that NLP7 may affect C metabolism. In addition, we found that NLP7 affected the expression level of AtPPC genes, which encode the PEPC. NLP7 transgenic plants showed higher transcript levels of AtPPC1 in response to nitrate re-addition Fig.

Expression of AtPPC2 and AtPPC3 were induced by nitrate in both the WT and NLP7 -overexpressing plants, but more dramatically in the NLP7 -overexpressing plants. However, expression of these two genes did not change much in the nlp mutant after nitrate re-addition Fig.

These data indicate NLP7 may affect PEPC activities. Indeed, further analyses showed that PEPC activities increased noticeably in the NLP7 -overexpressing plants while reduced in the nlp plants, especially under N-limiting conditions Fig.

Meanwhile, NLP7 -overexpressing plants had higher expression levels of genes encoding cytosolic isocitrate dehydrogenase ICDH , mitochondrial ICDH and peroxisomal ICDH after 3 days N-starvation and 0.

These results suggest that NLP7 expression level influenced not only N assimilation but also C assimilation. In order to investigate whether NLP7 can similarly modulate N assimilation in different plant species, we generated NLP7 -ovexpressing transgenic tobacco plants.

Figure 6a—c showed that the transgenic tobacco plants exhibited growth advantages with increased FW compared with WT under different nitrate conditions.

The NLP7 -overexpressing tobacco plants also had much longer PRs Fig. In addition, we assayed the N use level of the transgenic tobacco using hydroponic culture method. Moreover, compared with WT, five of the six tested genes in the N assimilation pathway showed significant higher transcript levels in the transgenic plants compared with the WT plants Fig.

These results suggest that the strategy with NLP7 may be applicable to improve N use in other plant species. a Identification of the NLP7 transgenic tobacco by qRT-PCR. Ubiquitin-conjugating enzyme E2 NtUbc2 , accession number AB was used as an internal control b Images of the day-old WT and NLP7 transgenic tobacco plants grown on medium with different concentrations of nitrate.

c,d FW c and PR length d of the day-old WT and transgenic tobacco plants. i—n NLP7 up-regulated the expression levels of N assimilation related genes in tobacco. Expression levels of six tobacco genes were quantified by qRT-PCR: nitrate transporter NtNRT2.

NtUbc2 was used as an internal control. Decreasing fertilizer N inputs by improving plant NUE is an important strategic goal for global agriculture. However, traditional breeding strategies to improve NUE in some crop plants have experienced a plateau 6.

Although genetic engineering for improving N use has been broadly studied in recent decades, the success has been limited because of focusing on single gene manipulation.

Most of these studies attempted to improve plant N use by individually constitutive overexpression of the genes involved in N uptake or metabolism 2.

Unfortunately, most of these transgenic plants did not show increased NUE, and some even showed negative pleiotropic effects, indicating the notions of single-point rate-limiting regulation being oversimplified 5 , 6 , In addition, unnecessary accumulation of metabolic intermediates may affect the plant development.

Therefore, it should be much better to engineer the plant metabolism by enhancing a few steps cooperatively instead of one step in a metabolic pathway to avoid these weaknesses.

The introduction of the ZmDof1 gene into Arabidopsis and rice highlighted the great utility of TFs in engineering N and C metabolisms in plants 21 , In this study, we found another TF, NLP7, could simultaneously coordinate many processes in N utilization and signaling pathway as well as C fixation and metabolism pathway, resulting in improved N use and plant growth.

Recently, NLPs had proved to play a central role in nitrate signaling in Arabidopsis. One mechanism of NLPs modulates nitrate-induced gene expression probably through post-translational regulation The underlying mechanisms for how inactive form of NLPs been converted into active form by nitrate signalling are not clear.

However, according to the ATH1 chip data by Scheible et al. Transcription of OsNLP4 is repressed by several abiotic stress treatments and induced by low phosphate availability Konishi and Yanagisawa 27 found all the NLPs were not significantly induced by nitrate re-addition for one hour.

However, this result can not eliminate the possibility that the expression of different NLPs being regulated by nitrate at other time points. Moreover, constitutive overexpression of NLP6-VP16 using the 35S promoter up-regulated the expression of nitrate-inducible genes NIR1 and NIA2 in both the N-starved seedlings and seedlings resupplied with nitrate All these results imply that not only post-translational but also transcriptional regulation of NLPs probably plays roles in the regulation of N signalling and assimilation.

Based on this hypothesis, it is possible for us to improve plant N use by change the transcriptional levels of NLPs. NLP7 was reported to orchestrate the early response to nitrate 28 , However, the expression of NLP7 is not induced by the N source or nitrate 29 , but the localization of NLP7 protein is regulated by nitrate via a nuclear retention mechanism Therefore, it is reasonable to speculate that if we overexpressed NLP7 in plant, more NLP7 protein would be produced and accumulated in the nucleus in response to nitrate availability.

This hypothesis could be proved by the results of Marchive et al. S4 of the paper. In this figure, less NLP7-GFP protein was accumulated in both the cytosol and the nucleus of root cells of N-starved pNLP7:NLP7-GFP plantlets compared with that of N-starved p35S:NLP7-GFP plantlets.

Once nitrate was re-supplied, within minutes, noticeably more NLP7-GFP protein was accumulated in the nucleus of root cells in p35S:NLP7-GFP plants compared with that of pNLP7:NLP7-GFP plantlets. As a result, the more NLP7 protein accumulated in nuclear would enhance the expression of N assimilation genes and thus improve the N use ability of the NLP7 -overexpressing plants.

Indeed, our results support this hypothesis that the overexpression of NLP7 significantly enhanced the plant N use by enhancing N assimilation efficiency. Overexpression of NLP7 in Arabidopsis led to higher shoot and root biomass under both N-rich and -deficient conditions Figs 1 and 2 , Supplementary Figs S3 and S5 , with remarkable rise in multiple N metabolites and N content, an elevation in enzyme activities of N metabolism Figs 3 and 5.

On the contrary, the nlp mutant had impaired N use ability and showed more severe N-deficient phenotypes, even under N rich conditions Figs 1 and 2. In sharp contrast to the nlp , overexpression of NLP7 resulted in up-regulation of a range of genes involved in nitrate transport NRT1.

Increased transcript levels of these genes would lead to enhanced N uptake and metabolism as revealed in Fig. On the other hand, the up-regulated TFs would produce a broad range of regulatory outcomes.

These coordinated regulations by NLP7 enable plants to rapidly adapt to N availability and maintain plant N homeostasis. Notably, transcript level of NLA , a positive regulator of plant adaptation to N limitation 38 , was also found up-regulated in NLP7 -overexpressing plants Fig.

S7 , contributing to better performances of the transgenic plants under N-deficient conditions. Moreover, ectopic expression of Arabidopsis NLP7 in tobacco also had similar effects as in Arabidopsis Fig.

All these data indicate that overexpression of NLP7 can improve plant N use ability by coordinately regulating N metabolism, transport and signalling pathways. Root is the most important organ for sensing N availability and morphological adaptation to N supply Agreed with the previous study 29 , our results also found that NLP7 was highly expressed in leaves, central cylinder of roots and LR primordia at different stages Supplementary Fig.

S9 , implying a possible function of this gene in root development. In fact, our results revealed that overexpression of NLP7 conferred increased LR density and PR length under both high and low nitrate conditions by vertical growth assay Fig.

The expression of many genes involved in root development in response to N availability, such as NRT1. The up-regulation of these genes in the NLP7 -overexpressing plants probably contributes to the root architecture changes to some extent.

Overall, these morphological changes in the root system of NLP7 -overexpressing plant enhanced its N acquisition ability to match its high-efficient N metabolism.

Plants have the ability to optimize biomass partitioning to maximize whole-plant growth rate according to the external environment These results implied that NLP7 -overexpressing plant has higher N use ability, thus leading to more tolerance to the N deficiency and more N allocated to the shoot to maximize its relative growth rate.

N and C assimilation processes are closely linked and tightly co-regulated NUE is not only dependent on N assimilation, and manipulating C metabolism was useful in some cases in improving NUE Therefore, it is reasonable to improve both the C and N utilization efficiencies simultaneously to optimize plant growth and yield.

Interestingly, we found such cooperation between N and C assimilations in the NLP7 -overexpressing plants. In addition to the increased N content Fig. Overexpression of NLP7 conferred significant higher photosynthesis rate Fig.

The changed chlorophyll contents of the NLP7 -overexpressing and mutant plants Figs 1c,d and 5b may have direct influences on C fixation Fig. Moreover, expression levels of PEPC genes and ICDH genes were found up-regulated in the NLP7 -overexpressing plants after nitrate induction Fig.

Consequently, the NLP7 -overexpressing plants had much higher PEPC activities under both N-rich and -deficient conditions Fig. According to the ChIP-chip data by Marchive et al.

It has been well documented that PEPC, a key enzyme in photosynthesis, also acts as a key player in N storage and C fixation and as a crosstalk point between C and N metabolisms 21 , ICDH genes encode the key enzymes to provide 2OG necessary for ammonium assimilation In conclusion, we have demonstrated that NLP7 is potentially a promising candidate for improving plant N use ability.

The localization of NLP7 is controlled by nitrate via a nuclear retention mechanism Amount of NLP7 protein in the cytosol and nucleus may maintain a dynamic balance in response to the nitrate availability. Constitutively overexpression of NLP7 in the plant might break the normal balance of NLP7 localization between the cytosol and nucleus and promoted NLP7 protein relocation to the nucleus, especially under N-rich condition.

Being activated by nitrate signalling 27 , the nuclear accumulated NLP7 would enhance N assimilation by cooperatively modulating a number of genes related to N metabolism, transport and signalling.

Consequently, the overexpression of NLP7 conferred better growth under both N-deficient and -sufficient conditions. Moreover, overexpression of NLP7 also improved C assimilation simultaneously. Our results also imply that NLP7 -mediated nitrate regulation is not only through post-translational mechanisms, probably also through translational levels.

It is conceivable that NLP7 can be used to enhance N use ability and increase crop yield. Homozygous mutant plants were confirmed by RT-PCR using the primers NLP7 RT-PCR LP and RP.

For pNLP7:NLP7—GFP construct, a fragment containing NLP7 promoter and coding region amplified by genomic PCR with primers NLP7 -attb-LP and NLP7 -attb-RP2 was cloned into pMDC to fuse with GFP The NLP7 -overexpressing transgenic Arabidopsis were obtained by Agrobacterium -mediated floral-dip method 52 and identified by qRT-PCR with specific primers NLP7 -qPCR LP and RP.

The transgenic tobacco plants were generated as previously described 53 and identified by RT-PCR with the primers NLP7 RT-PCR LP and RP. All the primers used are listed in Table S1. N-limited soil was the soil had been used once for Arabidopsis growth.

qRT-PCR was performed with a StepOne Plus Real Time PCR System by using a TaKaRa SYBR Premix Ex Taq II reagent kit. All the primers used are shown in Table S1.

The metabolite analyses were performed on the seedlings of day-old plants grown on agar medium with different concentrations of nitrate. Total soluble protein was measured using the Bradford Protein Assay Kit Sangon Biotech, Shanghai, China and total amino acid according to Rosen Enzymes were extracted from day-old plants grown on agar medium with different concentrations of nitrate.

The maximum in vitro activities of NR was assayed as described previously GS enzyme activities was measured according to Cai et al. Photosynthesis rates were measured using a portable photosynthesis system LI-COR LIXT in the morning to AM under constant light in the greenhouse as described by Yu et al.

The roots of the tobacco seedling were wrapped with sponge and then grown on a support made of thick polystyrene foam board with holes to allow the root systems of the plants to grow into the hydroponic solution.

Nutrient solution was changed every 5 days. How to cite this article : Yu, L. et al. Overexpression of Arabidopsis NLP7 improves plant growth under both nitrogen-limiting and -sufficient conditions by enhancing nitrogen and carbon assimilation.

Nosengo, N. Fertilized to death. Certain helpful microbes break down fibers into short chain fatty acids, which have been shown to stimulate immune cell activity. These fibers are sometimes called prebiotics because they feed microbes.

Therefore, a diet containing probiotic and prebiotic foods may be beneficial. Probiotic foods contain live helpful bacteria, and prebiotic foods contain fiber and oligosaccharides that feed and maintain healthy colonies of those bacteria.

Animal studies have found that deficiencies in zinc , selenium , iron , copper, folic acid , and vitamins A , B6 , C , D , and E can alter immune responses.

Epidemiological studies find that those who are poorly nourished are at greater risk of bacterial, viral, and other infections. Eating a good quality diet, as depicted by the Healthy Eating Plate, can prevent deficiencies in these nutrients.

However, there are certain populations and situations in which one cannot always eat a variety of nutritious foods, or who have increased nutrient needs. In these cases a vitamin and mineral supplement may help to fill nutritional gaps.

Studies have shown that vitamin supplementation can improve immune responses in these groups. The elderly are a particularly high-risk group. The immune response generally declines with increasing age as the number and quality of immune cells decreases.

This causes a higher risk of poorer outcomes if the elderly develop chronic or acute diseases. In addition, about one-third of elderly in industrialized countries have nutrient deficiencies. Diet variety may also be limited due to budget constraints or lower interest in cooking for one person; poor dentition; mental impairment; or lack of transportation and community resources to obtain healthy food.

Megadose supplements many times the RDA do not appear justified, and can sometimes be harmful or even suppress the immune system e. Remember that vitamin supplements should not be considered a substitute for a good diet because no supplements contain all the benefits of healthful foods.

Several herbal supplements have been suggested to boost immune function. What does the research say? Diet Review: Anti-Inflammatory Diet. Food Safety, Nutrition, and Wellness during COVID Ask the Expert: The role of diet and nutritional supplements during COVID The contents of this website are for educational purposes and are not intended to offer personal medical advice.

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Skip to content The Nutrition Source. The Nutrition Source Menu. Search for:. Home Nutrition News What Should I Eat? What Is Our Immune System? These barriers include: Skin that keeps out the majority of pathogens Mucus that traps pathogens Stomach acid that destroys pathogens Enzymes in our sweat and tears that help create anti-bacterial compounds Immune system cells that attack all foreign cells entering the body Adaptive or acquired immunity is a system that learns to recognize a pathogen.

Other conditions that trigger an immune response Antigens are substances that the body labels as foreign and harmful, which triggers immune cell activity. What factors can depress our immune system? Older age: As we age, our internal organs may become less efficient; immune-related organs like the thymus or bone marrow produce less immune cells needed to fight off infections.

Aging is sometimes associated with micronutrient deficiencies, which may worsen a declining immune function. Environmental toxins smoke and other particles contributing to air pollution, excessive alcohol : These substances can impair or suppress the normal activity of immune cells.

Excess weight: Obesity is associated with low-grade chronic inflammation. Fat tissue produces adipocytokines that can promote inflammatory processes.