Transcriptome and Proteomics Analysis of Wheat Seedling Roots Reveals That Increasing NH4+/NO3– Ratio Induced Root Lignification and Reduced Nitrogen Utilization

Abstract

Wheat growth and nitrogen (N) uptake gradually decrease in response to high NH4+/NO3– ratio. However, the mechanisms underlying the response of wheat seedling roots to changes in NH4+/NO3– ratio remain unclear. In this study, we investigated wheat growth, transcriptome, and proteome profiles of roots in response to increasing NH4+/NO3– ratios (Na: 100/0; Nr1: 75/25, Nr2: 50/50, Nr3: 25/75, and Nn: 0/100). High NH4+/NO3– ratio significantly reduced leaf relative chlorophyll content, Fv/Fm, and ΦII values. Both total root length and specific root length decreased with increasing NH4+/NO3– ratios. Moreover, the rise in NH4+/NO3– ratio significantly promoted O2– production. Furthermore, transcriptome sequencing and tandem mass tag-based quantitative proteome analyses identified 14,376 differentially expressed genes (DEGs) and 1,819 differentially expressed proteins (DEPs). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis indicated that glutathione metabolism and phenylpropanoid biosynthesis were the main two shared enriched pathways across ratio comparisons. Upregulated DEGs and DEPs involving glutathione S-transferases may contribute to the prevention of oxidative stress. An increment in the NH4+/NO3– ratio induced the expression of genes and proteins involved in lignin biosynthesis, which increased root lignin content. Additionally, phylogenetic tree analysis showed that both A0A3B6NPP6 and A0A3B6LM09 belong to the cinnamyl-alcohol dehydrogenase subfamily. Fifteen downregulated DEGs were identified as high-affinity nitrate transporters or nitrate transporters. Upregulated TraesCS3D02G344800 and TraesCS3A02G350800 were involved in ammonium transport. Downregulated A0A3B6Q9B3 is involved in nitrate transport, whereas A0A3B6PQS3 is a ferredoxin-nitrite reductase. This may explain why an increase in the NH4+/NO3– ratio significantly reduced root NO3–-N content but increased NH4+-N content. Overall, these results demonstrated that increasing the NH4+/NO3– ratio at the seedling stage induced the accumulation of reactive oxygen species, which in turn enhanced root glutathione metabolism and lignification, thereby resulting in increased root oxidative tolerance at the cost of reducing nitrate transport and utilization, which reduced leaf photosynthetic capacity and, ultimately, plant biomass accumulation.

Integrated physiological and weighted gene co-expression network analysis reveals the hub genes engaged in nitrate-regulated alleviation of ammonium toxicity at the seedling stage in wheat (Triticum aestivum L.)

“…Ca(NO 3 ) 2 , NH 4 Cl, and NH 4 NO 3 were used to set these ratios. The NH 4 + /NO 3 - ratios were determined according to our previous study ( Yang et al., 2022 ). The NH 4 + /NO 3 - ratios preparation according to the following method: 6 mmol L -1 NH 4 Cl and 4 mmol L -1 CaCl 2 for N a ; 4.5 mmol L -1 NH 4 Cl, 0.75 mmol L -1 Ca(NO 3 ) 2 and 3.25 mmol L -1 CaCl 2 for N r1 ; 3 mmol L -1 NH 4 NO 3 and 4 mmol L -1 CaCl 2 for N r2 ; 1.5 mmol L -1 NH 4 Cl, 2.25 mmol L -1 Ca(NO 3 ) 2 and 1.75 mmol L -1 CaCl 2 for N r3 ; 3 mmol L -1 Ca(NO 3 ) 2 and 1 mmol L -1 CaCl 2 for N n .…”

“…Root RNA extraction, cDNA library construction, Illumina sequencing, read mapping, and differential gene expression analyses have been described previously ( Yang et al., 2022 ). Gene co-expression networks were constructed using the WGCNA package in R software.…”

“…(2022) revealed that NRT1.1 and SLAH3 could form a functional unit to regulate nitrate-dependent alleviation of ammonium toxicity by regulating NO 3 - transport and balancing rhizosphere acidification. In our previous study, we reported that a high NH 4 + /NO 3 - ratio enhances the expression of genes and proteins involved in lignin biosynthesis, leading to root lignification, thereby resulting in increased root oxidative tolerance at the cost of reducing nitrate transport and utilization ( Yang et al., 2022 ). These results suggest that increasing nitrate levels may alleviate ammonium toxicity.…”

Wheat Proteomics for Abiotic Stress Tolerance and Root System Architecture: Current Status and Future Prospects

“…However, the first cell wall proteomics of wheat ( Figure 2 ) revealed that upregulation of chitinase under flooding stress helped in flooding tolerance; however, the downregulation of β-glucanase, β-glucosidase, methionine synthase, and glyoxalase inhibited cell wall elongation in wheat seedling roots, significantly reducing the rooting depth and fresh mass [ 71 ]. A recent study found that lignin biosynthesis (peroxidase enzyme and phenylalanine ammonia-lyase) played an important role in reducing total root length and specific root length under high NH 4 + /NO 3 − ratios [ 171 ]. However, lignification helps to protect the roots from oxidative stress due to ROS by restricting excess nitrogen supply.…”

“…However, lignification helps to protect the roots from oxidative stress due to ROS by restricting excess nitrogen supply. Roots were also protected by upregulating GST and phenylpropanoid biosynthesis pathway associated proteins, which played an important role in root development [ 171 ].…”