Genetic Engineering Approaches for Enhancing Macronutrient Use Efficiency in Crops

Introduction

Global agriculture must increase food production while confronting soil degradation, water scarcity, climate variability, and environmental pollution from excessive fertilizer use. Nitrogen, phosphorus, potassium, and sulfur fertilizers have driven yield gains since the Green Revolution, but a large proportion of applied nutrients is lost through leaching, runoff, volatilization, or immobilization in soil (Khan et al., 2018; Good et al., 2007) [2,1] . These losses contribute to groundwater contamination, eutrophication, greenhouse-gas emissions, and rising production costs. Nutrient-use efficiency (NUE) describes the capacity of a plant to acquire nutrients from soil and convert them into biomass or harvestable yield (Nieves-Cordones et al., 2020; Giehl and von Wirén, 2014) [4,3] . NUE is commonly partitioned into nutrient uptake efficiency (acquisition from soil) and nutrient utilization efficiency (assimilation, remobilization, and allocation to economic yield). Improving NUE is challenging because it involves multiple genes, metabolic pathways, and interactions with environmental factors. Genetic engineering and genome editing complement conventional breeding by enabling targeted modification of genes controlling nutrient transport, assimilation, signaling, root architecture and stress tolerance. This review focuses on transgenic and genome-editing strategies for improving N, P, K and S use efficiency in major crops. ). Nitrate uptake is mediated by NRT1/NPF (low-to medium-affinity) and NRT2 (high-affinity) transporters, often in association with NAR2/NRT3 proteins (Feng et al., 2011; Chiba et al., 2015) [6,5] . Ammonium uptake is mediated by AMT transporters, particularly important in rice (Ranathunge et al., 2014) [7] . Overexpression of OsNRT2.1 under the OsNAR2.1 promoter enhanced nitrate uptake and grain yield in rice (Chen et al., 2016) [8] . Expression of OsNRT2.3b, a pH-sensitive nitrate transporter, increased nitrogen uptake, agronomic NUE, and grain yield by approximately 40% under specific conditions (Fan et al., 2016) [9] . Overexpression of OsAMT1.1 improved ammonium uptake, nitrogen assimilation, and yield (Ranathunge et al., 2014) [7] . However, constitutive overexpression of some transporters reduced NUE, underscoring the importance of promoter choice and tissue-specific regulation.

Nitrogen-use efficiency

Nitrogen assimilation and metabolism

After uptake, ammonium is assimilated via the glutamine synthetase (GS)-glutamate synthase (GOGAT) pathway. Overexpression of GS and GOGAT genes improved nitrogen assimilation in several species, although responses varied with tissue, developmental stage, and nitrogen form (Cai et al., 2009; Oliveira et al., 2002) [10,11] . Alanine aminotransferase (AlaAT) links nitrogen and carbon metabolism. Expression of barley HvAlaAT in canola, rice, and sugarcane increased biomass and maintained yield under reduced nitrogen supply (Good et al., 2007; Snyman et al., 2015) [12] . The maize transcription factor ZmDof1 enhanced carbon skeleton production and nitrogen utilization under low-nitrogen conditions (Yanagisawa et al., 2004; Kurai et al., 2011) [14,13] .

Regulatory genes

Transcription factors provide opportunities to modify multiple NUErelated processes simultaneously. TaNAC2-5A in wheat increased root growth, nitrogen accumulation, and grain yield (He et al., 2015) [15] . OsMADS25, NLP7, DEP1, HY5, and MYB61 regulate nitrogen uptake, assimilation, root development, and yield (Yu et [16,17,18,19] . These regulators are attractive targets but require careful evaluation of pleiotropic effects.

Phosphorus-use efficiency

Phosphate uptake and transport

Phosphorus is absorbed mainly as inorganic phosphate (Pi), but availability is limited by immobilization in acidic and alkaline soils (Bar-Yosef, 1991; Holford, 1997) [20,21] . High-affinity phosphate uptake is mediated by PHT1 transporters, while PHO1 proteins facilitate longdistance transport to shoots (Hamburger et al., 2002; Liu et al., 2011) [22, 23] . Overexpression of OsPHT1 and OsPT6 increased phosphate accumulation, tiller number, and seed yield in rice and soybean (Seo et al., 2008; Yan et al., 2014) [24,25] . NtPT1 and TaPT2 enhanced phosphate content, photosynthesis, and biomass under phosphate deficiency (Park et al., 2010; Guo et al., 2014) [27,26] .

Organic-acid secretion and phosphatases

Plants can increase phosphate availability by secreting organic acids that solubilize soil phosphate. Overexpression of bacterial citrate synthase increased citrate secretion, phosphate acquisition, and biomass by 25-35% in tobacco (Lopez-Bucio et al., 2000) [28] . Enhanced malate secretion also improved phosphate uptake and tolerance to deficiency (Lu et al., 2012) [29] . A substantial fraction of soil phosphorus occurs in organic forms. Engineering acid phosphatases and phytases (e.g., PAP1, PAP3, PAP15, GmACP1) improved hydrolysis of organic phosphorus and increased biomass (Wang et [31,32,33,30] .

Potassium-use efficiency

Potassium is essential for enzyme activation, osmotic regulation, photosynthesis, and stress tolerance. Potassium-use efficiency (KUE) depends on efficient uptake, transport, and redistribution. Potassium uptake involves HAK/KUP/KT transporters, Shaker channels, tandem-pore channels (TPK), and regulatory kinases (Wang et al., 2013; He et al., 2017) [35,34] . The AKT1-CIPK23-CBL module is central to high-affinity potassium uptake under deficiency (Xu et al., 2006; Wang et al., 2011) [37,36] . Overexpression of AtCIPK23 improved potassium accumulation, root growth, and deficiency tolerance (Xue et al., 2016) [38] . AtHAK5 and OsHAK5 enhanced highaffinity potassium uptake and shoot translocation (Nieves-Cordones et al., 2010; Yang et al., 2014) [39,40] . Overexpression of OsTPKb improved potassium homeostasis and tolerance to potassium deficiency and drought (Ahmad et al., 2016) [41] .

Expression of ApKUP3 in rice increased potassium accumulation, chlorophyll content, photosynthesis, and drought tolerance (Song et al., 2014) [42] . These results highlight the close link between potassium nutrition and abiotic-stress resilience.

Sulfur-use efficiency

Sulfur is absorbed mainly as sulfate and is required for synthesis of cysteine, methionine, glutathione, and various secondary metabolites. Sulfur-use efficiency (SUE) depends on sulfate uptake, transport, assimilation, and remobilization. Sulfate uptake is mediated by SULTR transporters. SULTR1;1 and SULTR1;2 function in root uptake, while SULTR1;3 contributes to vascular transport (Yoshimoto et al., 2002, 2003; Rae and Smith, 2002) [43,44] . Overexpression of GmSULTR1;2b increased sulfate uptake, biomass, and seed weight in tobacco (Ding et al., 2016) [45] . Sulfate assimilation involves adenosine-5′-phosphosulfate reductase (APR) and sulfite reductase (SiR). Expression of bacterial APR increased cysteine and glutathione levels (Tsakraklides et al., 2002) [46] . Overexpression of maize SiR enhanced sulfite reduction and stress tolerance (Xia et al., 2018) [47] . Expression of the tomato sulfate transporter LeST1.1 in Brassica juncea improved sulfur status, chlorophyll, protein content, and biomass under sulfur deficiency (Abdin et al., 2011) [48] . Engineering sulfur-rich storage proteins can improve nutritional quality. Expression of a sulfur-rich sunflower seed albumin in Lupinus angustifolius increased seed methionine content (Molvig et al., 1997) [49] , although similar approaches in rice were less effective (Hagan et al., 2003) [50] . [52,53,51,54] .

Genome editing for macronutrient-use efficiency

Nitrogen

CRISPR/Cas9 knockout of MIR396ef in rice increased grain size, panicle branching, and biomass under nitrogen-deficient conditions (Zhang et al., 2020) [55] . Base editing of NRT1.1B introduced nucleotide substitutions associated with improved NUE (Lu et al., 2017) [56] . Editing of MYB61 clarified its role in nitrogen utilization and interaction with GRF4 (Gao et al., 2020) [16] .

Phosphorus

CRISPR-mediated modification of OsACS and OsDOF15 revealed their roles in ethylene biosynthesis, lateral-root development, and primary-root elongation under phosphate deficiency (Lee et al., 2019; Qin et al., 2019) [57,58] . These studies highlight the potential to modify root architecture to enhance phosphate acquisition.

Potassium

Genome editing has been used to investigate genes involved in potassium and phosphate efficiency, including OsHKT1;1, OsHKT1;4, OsHA1, and OsPRX2 (Mohammed, 2018; Karunarathne et al., 2020; Mao et al., 2018) [60,59] . Disruption of OsPRX2 impaired potassiumdeficiency tolerance, indicating its role in potassium nutrition and stress adaptation. 8. Opportunities and Challenges Opportunities Direct modification of genes with known functions in nutrient transport, assimilation, and regulation. Faster validation of candidate genes compared with conventional breeding alone. Potential to combine multiple traits (e.g., transporters, enzymes, transcription factors) through gene stacking.

Ability to generate nutrient-efficient varieties with reduced fertilizer requirements.

Precision modification of regulatory sequences using base editing and prime editing.

Integration with genomic selection, marker-assisted breeding, and precision agronomy.

Challenges

Strong genotype × environment interactions affecting trait stability.

Differences between controlled-environment and field performance.

Possible trade-offs between nutrient efficiency, yield, and quality.

Metabolic imbalances from excessive transporter or enzyme expression.

Transformation and regeneration constraints in many crops.

Regulatory requirements and public acceptance of genetically modified and genome-edited crops.

Potential ecological effects on soil microbes and nutrient cycling (Good et al., 2007; Khan et al., 2018) [1,2] . [61,62,63] .

Comparative summary of candidate genes

Conclusion

Transgenic and genome-editing approaches provide powerful tools for improving macronutrient-use efficiency in crops. Manipulation of nitrate and ammonium transporters, nitrogen-assimilatory enzymes, and regulatory genes can enhance nitrogen acquisition and utilization. Phosphorus-use efficiency can be improved through phosphate transporters, organic-acid secretion, acid phosphatases, and phytases.

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