## 2 Results and Analysis
### P. eucalypti FBS135 Modulates Its Gene Transcription Adapting to Nitrogen Scarcity  
The gene expression levels of _P. eucalypti_ FBS135 were calculated based on the number of sequencing reads aligned to each transcript, and their distribution was normalized using FPKM. The results showed that the overall expression level of genes decreased when there \begin{table}
\begin{tabular}{c c c c c c c c} \hline
**Sample** & **Raw** & **Clean** & **Clean** & **Error** & **Q** & **Q** & **GC** \\
**Name** & **Reads** & **Reads** & **Bases** & **Rate** & **20** & **30** & **Content** \\ \hline AN\_WT\_1 & 7,797,428 & 7,673,596 & 1.16G & 0.02 & 98.27 & 94.73 & 55.31 \\ AN\_WT\_2 & 7,208,796 & 7,091,792 & 1.07G & 0.02 & 98.37 & 94.99 & 53.84 \\ AN\_WT\_3 & 7,801,876 & 7,697,252 & 1.16G & 0.02 & 98.48 & 95.19 & 52.76 \\ A\_WT\_1 & 7,637,382 & 7,550,282 & 1.14G & 0.02 & 98.14 & 94.33 & 50.37 \\ A\_WT\_2 & 7,858,328 & 7,767,674 & 1.17G & 0.02 & 98.16 & 94.42 & 51.5 \\ A\_WT\_3 & 7,435,342 & 7,338,626 & 1.11G & 0.02 & 98.36 & 94.94 & 51.66 \\ \hline \end{tabular}
\end{table} 
Table 1: Sample sequencing data quality summary of _P. eucalypti_ FBS135. Figure 1: Growth of _P. eucalypti_ FBS135 on solid media with different concentrations of nitrogen source. (**A**) LB (control with organic nitrogen); (**B**) nitrogen-free Ashby medium; (**C**) Ashby medium +0.2 g/L NH4 Cl; (**D**) Ashby medium +1 g/L NH4 Cl; (**E**) Ashby medium +8 g/L NH4 Cl; and (**F**) Ashby medium +16g/L NH4 Cl. was no NH\({}_{4}\)+ addition (Figure 2A), possibly indicating that many genes would not be expressed or under-expressed in an environment lacking nitrogen. Using \(p_{\text{adj}}\) < 0.05 and \(\mid\)FoldChange \(\mid\)\(>\) 2 as the screening criteria, we identified the differentially expressed genes (DEGs) using the negative binomial distribution model of DESeq. A total of 1936 genes were differentially expressed in _P. eucalypt_ FBS135 (Figure 2B) (Supplementary Table S1). After adding NH\({}_{4}\)Cl, the expression of 1023 genes was upregulated, while the expression of 913 genes was downregulated (Figure 2B). Among the top five  
Figure 2: Effect of NH\({}_{4}\)+ addition on the general gene expression of _P. eucalypt_FBS135. (**A**) Distribution of gene expression levels of FBS135 without (**A**) and with (AN) NH\({}_{4}\)+ addition. (**B**) Volcano diagram of differentially expressed genes (DEGs) between the A condition and the AN condition. Upregulated genes in the A condition are indicated by red dots, while downregulated genes in the A condition are indicated by green dots. (**C**) qPCR verification of the DEGs of FBS135 detected via the transcriptome sequencing. (**D**) Hierarchical clustering of the DEGs under the A and the AN conditions. upregulated genes, three encode proteins for phosphonate metabolism with a 200-900 fold change, while among the top five downregulated genes, four encode for cold-shock proteins with a 60-160 fold change. We selected nine DEGs and verified their expression with qPCR, which gave a consistent fold change as the sequencing result (Figure 2C), indicating the transcriptome data were reliable. The genes were classified into two groups according to their expression levels in the different samples via hierarchical clustering analysis (Figure 2D). Three samples belonged to the group with the NH4 Cl addition, while the other three belonged to the group with no NH4 Cl addition. The expression of over 80% of genes varied significantly between groups but remained stable within one group, indicating a significant effect of nitrogen limitation on the gene transcription of FBS135. Using the hierarchical clustering method, the 1936 DEGs were divided into four clusters, each of which had specific expression patterns among the six samples (Figure 3). Cluster 1 contained 832 genes, which were highly expressed in the presence of NH4\({}^{+}\) (group AN) and lowly expressed in the absence of NH4\({}^{+}\) (group A). The gene expression in either group showed a similar level at the three time points (36 h, 48 h, and 60 h) (Figure 3A).