Multi-Omics Analysis Reveals Synergistic Enhancement of Nitrogen Assimilation Efficiency via Coordinated Regulation of Nitrogen and Carbon Metabolism by Co-Application of Brassinolide and Pyraclostrobin in Arabidopsis thaliana

Abstract

Improving nitrogen (N) assimilation efficiency without yield penalties is important to sustainable food security. The chemical regulation approach of N assimilation efficiency is still less explored. We previously found that the co-application of brassinolide (BL) and pyraclostrobin (Pyr) synergistically boosted biomass and yield via regulating photosynthesis in Arabidopsis thaliana. However, the synergistic effect of BL and Pyr on N metabolism remains unclear. In this work, we examined the N and protein contents, key N assimilatory enzyme activities, and transcriptomic and metabolomic changes in the four treatments (untreated, BL, Pyr, and BL + Pyr). Our results showed that BL + Pyr treatment synergistically improved N and protein contents by 56.2% and 58.0%, exceeding the effects of individual BL (no increase) or Pyr treatment (36.4% and 36.1%). Besides synergistically increasing the activity of NR (354%), NiR (42%), GS (62%), and GOGAT (62%), the BL + Pyr treatment uniquely coordinated N metabolism, carbon utilization, and photosynthesis at the transcriptional and metabolic levels, outperforming the effects of individual BL or Pyr treatments. These results revealed that BL + Pyr treatments could synergistically improve N assimilation efficiency through improving N assimilatory enzyme activities and coordinated regulation of N and carbon metabolism. The identified genes and metabolites also informed potential targets and agrochemical combinations to enhance N assimilation efficiency.

Keywords: brassinolide; metabolome; nitrogen assimilation efficiency; pyraclostrobin; transcriptome.

Figure 1

Determination of leaf nitrogen and protein content. The concentrations (%) of nitrogen (A) and protein (B) of leaves, and the total fixed weight (mg) of nitrogen (C) and protein (D) in leaves per plant. Leaves were sampled at 27-, 31-, and 39-day-old seedlings in the untreated group (CK), Pyr group (treated with 3 μM Pyr), BL + Pyr group (treated with 1 μM BL and 3 μM Pyr), and BL group (treated with 1 μM BL), respectively. Data are presented as the mean ± SD of three separate replicate experiments. Different letters indicate significant differences between the four treatments based on t-test comparisons at p < 0.05.

Figure 2

Activities of nitrogen assimilation key enzymes. The enzyme activities of NR (A), NiR (B), GS (C), and GOGAT (D). Leaves were sampled at 27-, 31-, and 39-day-old seedlings in the untreated group (CK), Pyr group (treated with 3 μM Pyr), BL + Pyr group (treated with 1 μM BL and 3 μM Pyr), and BL group (treated with 1 μM BL), respectively. Data are presented as the mean ± SD of three separate replicate experiments. Different letters indicate significant differences between the 4 treatments based on t-test comparisons at p < 0.05.

Figure 3

The identification of differentially expressed genes (DEGs) based on RNA-Seq: (A), hierarchical cluster analysis according to the FPKM of genes; (B), number of DEGs in separate comparison pairs; (C), Venn diagrams showing the overlapping and non-overlapping DEGs between the three groups (BL + Pyr, BL, and Pyr) and the untreated group; (D), Venn diagrams showing the overlapping and non-overlapping DEGs between the other three groups (untreated, BL, and Pyr groups) and the BL + Pyr group. The red triangle denoted the region that contained the DEGs specific to BL + Pyr.

Figure 4

Nitrogen metabolism pathways tagged with DEGs. The heat map close to the enzyme (green elliptical box) showed the expression level of the gene encoding the corresponding enzyme. “*” in the heat map represented a significant difference of BL, Pyr, and BL + Pyr-treated group versus the untreated group, with redder colors in the heatmap representing higher expression.

Figure 5

The GO and KEGG enrichment analysis of DEGs induced by BL + Pyr rather than by BL or Pyr. (A) Enrichment circular plot for the key significantly enriched GO terms of DEGs (the number of up and down DEGs in each GO term was represent at the right table); (B) bar graph of the top 30 significantly enriched KEGG pathways (The horizontal axis is the percentage of genes and the vertical axis is the details of KEGG terms, and the depths of the colors in the graph represent enrichment significance based on p-value, with p-value decreasing from dark to light. The number after the bars represented the number of DEGs, followed by the p-value in brackets).

Figure 6

The identification of differentially accumulated metabolites (DAMs) based on metabolomic analysis. (A) PCA analysis according to the accumulation of metabolites in the leaves; (B) number of DAMs in separate comparison pairs; (C) Venn diagrams showing the overlapping and non-overlapping DAMs between the three groups (BL + Pyr, BL, and Pyr) and the untreated group; (D) Venn diagrams showing the overlapping and non-overlapping DAMs between the other three groups (untreated, BL and Pyr groups) and the BL + Pyr group. The red triangle denoted the region that contained the DAMs specific to BL + Pyr.

Figure 7

The accumulation of nitrogen metabolism pathway intermediates based on metabolomic analysis. The accumulation of Glu (A), Gln (B), Asp (C), Asn (D), Ala (E), and α-Ketoglutarate (F). Data are presented as the mean ± SD of three separate biological repetitions. Different letters indicate significant differences between 4 treatments based on t-test comparisons at p < 0.05.

Figure 8

The KEGG enrichment analysis of metabolites differentially accumulated by BL + Pyr rather than by BL or Pyr. The left side was an enrichment bubble chart showing the enriched KEGG pathways, while the right side presented the BL + Pyr -unique DAMs annotated in the pathways.

Figure 9

The co-enrichment analysis conducted on BL + Pyr-specific DEGs and DAMs within KEGG pathways. The enriched KEGG pathways are plotted along the X-axis, while the Y-axis signifies the enrichment factor. Triangles represent DEGs, circles represent DAMs. The bubble size correlates with the count of DAMs or DEGs, and the bubble color indicates the −log10 (p-value), providing a measure of statistical significance.