RNA-SEQ Reveals Transcriptional Level Changes of Poplar Roots in Different Forms of Nitrogen Treatments

Abstract

Poplar has emerged as a model plant for better understanding cellular and molecular changes accompanying tree growth, development, and response to environment. Long-term application of different forms of nitrogen (such as [Formula: see text]-N and [Formula: see text]-N) may cause morphological changes of poplar roots; however, the molecular level changes are still not well-known. In this study, we analyzed the expression profiling of poplar roots treated by three forms of nitrogen: S1 ([Formula: see text]), S2 (NH4NO3), and S3 ([Formula: see text]) by using RNA-SEQ technique. We found 463 genes significantly differentially expressed in roots by different N treatments, of which a total of 112 genes were found to differentially express between S1 and S2, 171 genes between S2 and S3, and 319 genes between S1 and S3. A cluster analysis shows significant difference in many transcription factor families and functional genes family under different N forms. Through an analysis of Mapman metabolic pathway, we found that the significantly differentially expressed genes are associated with fermentation, glycolysis, and tricarboxylic acid cycle (TCA), secondary metabolism, hormone metabolism, and transport processing. Interestingly, we did not find significantly differentially expressed genes in N metabolism pathway, mitochondrial electron transport/ATP synthesis and mineral nutrition. We also found abundant candidate genes (20 transcription factors and 30 functional genes) regulating morphology changes of poplar roots under the three N forms. The results obtained are beneficial to a better understanding of the potential molecular and cellular mechanisms regulating root morphology changes under different N treatments.

Keywords: RNA-SEQ; long-time nitrogen treatment; nitrogen forms; nitrogen metabolism; poplar.

Figure 1

Schematic representation of the experimental set-up: poplar plants were grown hydroponically in complete LA liquid medium (N⁺ medium) for 10 days, then transferred to nitrogen-free medium (N⁻ medium) for 3 days; subsets of plants were transferred back to complete medium (N resupply, S1 [${\text{NH}}_4^{+}$-N]; S2 [NH₄NO₃]; S3 [${\text{NO}}_3^{-}$-N]) for another 21 days. The total treatment period is 34 days.

Figure 2

Morphological parameters of poplar roots with different N forms for 21 days. Upper: The picture of the root under different N forms; Lower: Length of roots and the dry weighs of roots. Values are the mean of four replicates ± SE. a, b, and c indicate significant difference based on ANOVA analysis and Duncan's test (P < 0.05).

Figure 3

Change in gene expression profiles among different N forms. S1, ${\text{NH}}_4^{+}$ treatment; S2, NH₄NO₃; S3, ${\text{NO}}_3^{-}$. The number of up-regulated and down-regulated genes between S1 and S2, and between S2 and S3 are summarized. Between the S1 and S2 libraries, there are 33 up-regulated genes and 79 down-regulated genes, while 76 up-regulated genes and 95 down-regulated genes between the S3 and S2 libraries, and 130 up-regulated genes and 189 down-regulated genes between the S1 and S3 libraries.

Figure 4

Expression patterns clustering and gene annotation. Two-dimensional hierarchical clustering classifies 463 differential gene expression profiles into four expression cluster groups according to the similarity of their expression profiles.

Figure 5

Schematic representation of primary metabolism of poplar roots at different forms of nitrogen. Gray charts represent significantly differentially expressed genes in the metabolic pathways. White charts represent the genes without significantly different expression in the metabolic pathways. The left and right columns represent the S1/S2 and S3/S2 at the transcriptional level, respectively. Up- and down-facing triangles represent an increase and decrease in transcripts. The digital in columns represent the number of change genes. It can be found that there was no significantly difference gene in nitrogen metabolic pathway.