Arabidopsis Transcriptome Analysis Reveals Key Roles of Melatonin in Plant Defense Systems

Melatonin is a ubiquitous molecule and exists across kingdoms including plant species. Studies on melatonin in plants have mainly focused on its physiological influence on growth and development, and on its biosynthesis. Much less attention has been drawn to its affect on genome-wide gene expression. To comprehensively investigate the role(s) of melatonin at the genomics level, we utilized mRNA-seq technology to analyze Arabidopsis plants subjected to a 16-hour 100 pM (low) and 1 mM (high) melatonin treatment. The expression profiles were analyzed to identify differentially expressed genes. 100 pM melatonin treatment significantly affected the expression of only 81 genes with 51 down-regulated and 30 up-regulated. However, 1 mM melatonin significantly altered 1308 genes with 566 up-regulated and 742 down-regulated. Not all genes altered by low melatonin were affected by high melatonin, indicating different roles of melatonin in regulation of plant growth and development under low and high concentrations. Furthermore, a large number of genes altered by melatonin were involved in plant stress defense. Transcript levels for many stress receptors, kinases, and stress-associated calcium signals were up-regulated. The majority of transcription factors identified were also involved in plant stress defense. Additionally, most identified genes in ABA, ET, SA and JA pathways were up-regulated, while genes pertaining to auxin responses and signaling, peroxidases, and those associated with cell wall synthesis and modifications were mostly down-regulated. Our results indicate critical roles of melatonin in plant defense against various environmental stresses, and provide a framework for functional analysis of genes in melatonin-mediated signaling pathways

Schematic overview of genes exhibiting a change of 2-fold or more in response to melatonin associated with stress response and signaling.

<p>Fold change in transcript levels is represented by the color scale with darkest blue indicating genes with the greatest increase in transcript levels (4-fold or greater) and darkest red indicating genes with the greatest decrease in transcript levels (at least 4-fold). Each gene involved in stress responses is represented once.</p

Gene Ontology classification into molecular function of all genes significantly (p<0.05) differentially expressed in response to 1 mM and 100 pM melatonin.

<p>Genes were classified into at least one category using AmiGO GO slimmer. The proportion of genes that fall into each category were calculated by dividing the number of genes assigned to a category by the total number of genes that could be classified for each treatment.</p><p>*Indicates Molecular function is unknown.</p

Scatter plots show genomic scale reproducibility.

<p>The scatter plots comparing the clean reads of two biological duplicates from control (A), 100 pM melatonin (B) and 1 mM melatonin (C) treatments. Genes are represented by dots. For each gene, the RNA expression level in one rep is given on the x axis and the same gene in the other rep is given on the y axis.</p

Redox-related genes and peroxidases affected by 1 mM melatonin.

<p>Redox-related genes and peroxidases affected by 1 mM melatonin.</p

Cell wall related genes with at least a 2 fold change in expression levels in response to 1 mM Melatonin.

<p>Cell wall related genes with at least a 2 fold change in expression levels in response to 1 mM Melatonin.</p

Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.

<p>Receptors, kinases, and genes involved in calcium signaling whose expression levels were affected by 1 mM melatonin by at least 2 fold.</p

Venn Diagram representing the overlap of the molecular responses to low (100 pM) and high (1 mM) melatonin treatments.

<p>The number in the parenthesis indicates the number of genes altered by melatonin for each treatment.</p

Effect of melatonin on paraquat-induced oxidative stress.

<p>Arabidopsis leaves were detached and floated in solution containing 0, 10(top row) or presence (bottom row) of 1 mM melatonin. After 48 hours, leaves exposed to paraquat in the absence of melatonin were photobleached while leaves incubated with melatonin during exposure to paraquat remained green.</p

qRT-PCR analysis confirming overall results of RNA-seq experiments.

<p>The fold changes in transcript levels identified in RNA-seq experiments for 15 selected genes are graphed in A (up-regulated) and C (down-regulated). qRT-PCR was performed using the same samples for RNA-seq experiments with primers for the selected genes showing up-regulated (B) or down-regulated (D) by 1 mM melatonin. All q-RT-PCR were repeated four times. * p<0.05, ** p<0.01, ***p<0.001.</p