Genome-Scale Transcriptome Analysis in Response to Nitric Oxide in Birch Cells: Implications of the Triterpene Biosynthetic Pathway

<div><p>Evidence supporting nitric oxide (NO) as a mediator of plant biochemistry continues to grow, but its functions at the molecular level remains poorly understood and, in some cases, controversial. To study the role of NO at the transcriptional level in <i>Betula platyphylla</i> cells, we conducted a genome-scale transcriptome analysis of these cells. The transcriptome of untreated birch cells and those treated by sodium nitroprusside (SNP) were analyzed using the Solexa sequencing. Data were collected by sequencing cDNA libraries of birch cells, which had a long period to adapt to the suspension culture conditions before SNP-treated cells and untreated cells were sampled. Among the 34,100 UniGenes detected, BLASTX search revealed that 20,631 genes showed significant (E-values≤10⁻⁵) sequence similarity with proteins from the NR-database. Numerous expressed sequence tags (i.e., 1374) were identified as differentially expressed between the 12 h SNP-treated cells and control cells samples: 403 up-regulated and 971 down-regulated. From this, we specifically examined a core set of NO-related transcripts. The altered expression levels of several transcripts, as determined by transcriptome analysis, was confirmed by qRT-PCR. The results of transcriptome analysis, gene expression quantification, the content of triterpenoid and activities of defensive enzymes elucidated NO has a significant effect on many processes including triterpenoid production, carbohydrate metabolism and cell wall biosynthesis.</p></div

Relative expression levels of genes related to cell wall synthesis in the groups treated with 1 mM SNP for 6, 12, 24, and 48 h (left).

<p>Relative expression levels of genes related to cell wall synthesis in SNP, KFeCN, and SNP + cPTIO (S + cPTIO) groups (right). Different letters represent significant difference (P<0.05).</p

Changes in antioxidant enzyme activities, including POD and APX among the control (CK), SNP, KFeCN, and SNP + cPTIO (S + cPTIO) groups.

<p>Different letters represent significant difference (P<0.05).</p

Changes in oleanolic acid content among the control (CK), 0.01 mM, 0.1 mM, 1 mM, and 10 mM SNP groups at different times.

<p>Different letters represent significant difference (P<0.05).</p

GO categories for the birch cell UniGenes.

<p>The percentage and total number of UniGenes in each category is shown.</p

Relative expression levels of genes related to triterpene synthesis in the groups treated with 1 mM SNP for 6, 12, 24, and 48 h (left).

<p>Relative expression levels of genes related to triterpene synthesis in the SNP, KFeCN, and SNP + cPTIO (S + cPTIO) groups (right). Different letters represent significant difference (P<0.05).</p

Relative expression levels of antioxidant genes in the groups treated with 1 mM SNP for 6, 12, 24, and 48 h (left); relative expression levels of antioxidant genes in the SNP, KFeCN, and SNP + cPTIO groups (S + cPTIO) (right).

<p>Different letters represent significant difference (P<0.05).</p

Changes in total triterpene and oleanolic acid content in the control (CK), SNP, KFeCN, and SNP + cPTIO (S + cPTIO) groups.

<p>Different letters represent significant difference (P<0.05).</p

Relative expression levels of genes related to endogenous NO synthesis (<i>BpNR</i> and <i>BpNOA1</i>) and calcium signaling (<i>BpCALM</i>) in groups treated with 1 mM SNP for 6, 12, 24, and 48 h (left).

<p>The relative expression levels of genes related to endogenous NO synthesis (<i>BpNR</i> and <i>BpNOA1</i>) and calcium signaling pathways (<i>BpCALM</i>) in the SNP, KFeCN, and SNP + cPTIO (S + cPTIO) groups (right). Different letters represent significant difference (P<0.05).</p

Distribution of UniGenes among different plant species.

<p>Distribution of UniGenes among different plant species.</p