Genome-Wide Urea Response in Rice Genotypes Contrasting for Nitrogen Use Efficiency

Abstract

Rice is an ideal crop for improvement of nitrogen use efficiency (NUE), especially with urea, its predominant fertilizer. There is a paucity of studies on rice genotypes contrasting for NUE. We compared low urea-responsive transcriptomes of contrasting rice genotypes, namely Nidhi (low NUE) and Panvel1 (high NUE). Transcriptomes of whole plants grown with media containing normal (15 mM) and low urea (1.5 mM) revealed 1497 and 2819 differentially expressed genes (DEGs) in Nidhi and Panvel1, respectively, of which 271 were common. Though 1226 DEGs were genotype-specific in Nidhi and 2548 in Panvel1, there was far higher commonality in underlying processes. High NUE is associated with the urea-responsive regulation of other nutrient transporters, miRNAs, transcription factors (TFs) and better photosynthesis, water use efficiency and post-translational modifications. Many of their genes co-localized to NUE-QTLs on chromosomes 1, 3 and 9. A field evaluation under different doses of urea revealed better agronomic performance including grain yield, transport/uptake efficiencies and NUE of Panvel1. Comparison of our urea-based transcriptomes with our previous nitrate-based transcriptomes revealed many common processes despite large differences in their expression profiles. Our model proposes that differential involvement of transporters and TFs, among others, contributes to better urea uptake, translocation, utilization, flower development and yield for high NUE.

Keywords: QTLs; networks; nitrogen use efficiency; rice; transcriptome; urea.

Figure 1

Transcriptomic analyses of low urea response in contrasting NUE genotypes. Nidhi and Panvel1 indica rice genotypes were grown in nutrient depleted soil in greenhouse under normal (15 mM) and low (1.5 mM) urea conditions [10]. Urea-responsive differentially expressed transcripts are shown as volcano plots for Nidhi (A) and Panvel1 (B). Each dot on the plot represents the transcript and horizontal dashed line corresponds to p value cut−off (p = 0.05). Up-regulated transcripts are shown as red scattered dots, whereas green scattered dots represent down-regulated transcripts under low−urea treatment. (C) Bar graph represents the up− or down−regulated genes detected in Nidhi and Panvel1. (D) TreeMap shows top 10 statistically significant biological processes (p < 0.05) observed in Nidhi and Panvel1. The colour of the box is according to the p value (−log₂).

Figure 2

Validation of expression profile of urea-responsive genes by RT−qPCR. Relative change in the gene expression was calculated by comparative Ct value method and actin gene was used for data normalization. The control values were taken as zero and the test values are shown as average of three technical and two independent biological replicates (+SE). Each sub−figure compares gene expression of RT−qPCR and microarray for Nidhi versus Panvel1 for gene OsVDAC6 (A), DTH2 (B), UGT703A2 (C), ASCAB-9A (D), NF-YB (E), psaN (F), OSSIIb (G) and PRR95 (H). Test of significance between bars has been shown on RT−qPCR data of Nidhi versus Panvel1. * p  <  0.05, ** p  <  0.01.

Figure 3

Venn selection for NUE genes (N-responsive and yield-related genes) in (A) Nidhi urea and (B) Panvel1 urea. Representative figures for physical location of NUE candidates located on chromosomes 1, 3 and 9 in (C–E) Nidhi urea and (F–H) Panvel1 urea. Gene ID is given on the right side and the physical location of genes is given on the left side of the map (in Mb).

Figure 4

Validation of efficiencies derived associated with the biological processes. Validation was carried out using Licor instrument 6400XT (LI-COR, Lincoln, NE, USA) on 21−days-old grown plants. Plants were grown in nutrient-depleted soil and fertilized with Arnon Hoagland medium having urea as sole source of N with 15 mM N concentration as control, while 1.5 mM was used as test. Measurement was carried out in four biological replicates. Efficiencies were derived from the standard formulas as described here. (A) Photosynthetic efficiency was measured in terms of µ mol CO₂m⁻²s⁻¹/µ mol l⁻¹, (B) transpiration efficiency was measured in terms of µ mol CO₂m⁻²s⁻¹/mmol (H₂O)/m⁻²s⁻¹ and (C) internal water use efficiency was measured in terms of µ mol CO₂ m⁻²s⁻¹/mol(H₂O) m⁻²s⁻¹). The test of significance has been shown as star (* p < 0.05) between the bars of low urea and normal urea, while NS represents non-significance.

Figure 5

Field validation of the performance of rice genotypes. Field evaluation of the genotypes Nidhi and Panvel1 was conducted at National Rice Research Institute (NRRI-ICAR) Cuttack, Odisha, India in N0, N50 and N100 N kg of added urea/ha. Mean data of the effect of urea dose on the genotypes are shown for panicle weight, yield per panicle, 1000 grain weight, grain yield per hectare, uptake efficiency, fertilizer use efficiency, partial factor productivity, nitrogen transport efficiency and utilization efficiency. The significance levels are shown between the bars of genotype Nidhi and Panvel1 for each of the urea dose being compared (* p  <  0.05, ** p  <  0.01 and NS denotes non-significant).

Figure 6

Common and specific nitrate− and urea−responsive genes and pathways in Nidhi and Panvel1. Venn diagrams represent the specific and overlap of DEGs (A) and all the assigned pathways (B) in low nitrate versus low urea responses in both the genotypes. Combined N−responsive genes and their top enriched pathways annotations are shown for nitrate (C) and urea (D). (E) Hierarchical clustering of common N−responsive genes from Nidhi shows distinct pattern in Panvel1. Each column represents a single N (nitrate or urea) treatment in a particular genotype (Nidhi or Panvel1), whereas each row represents a DEG. Stacked bar graph represents the relative percentage of top 10 enriched transporters (F) and transcription factors (G) responsive to nitrate [16] and urea (current study). The X−axis represents the nitrate or urea treatment in Nidhi or Panvel1. Details of genes and pathways are given in Supplementary Table S16. DEGs−NN, DEGs−Nidhi nitrate; DEGs−PN, DEGs−Panvel1 nitrate; DEGs−NU, DEGs−Nidhi urea; DEGs−PU, DEGs−Panvel1 urea; Path−NN, Pathways−Nidhi nitrate; Path−PN, Pathways−Panvel1 nitrate; Path−NU, Pathways−Nidhi urea; Path−PU, Pathways−Panvel1 urea.

Figure 7

Hypothetical model depicting the important classes of genes and associated pathways differentially regulated in low−(Nidhi) and high−(Panvel1) NUE genotypes. Blue and red colour denote the differential regulation of gene classes/pathways in low− and high−NUE genotypes, respectively. Up− and down−ward arrows represent the upregulation and downregulation, respectively, in a particular class/pathways. Both the arrows together represent mixed regulation of gene classes/pathways. TFs, transcription factors.