The authors have declared that no competing interests exist.
Conceived and designed the experiments: SR SW YG. Performed the experiments: SR SW GB. Analyzed the data: NZ SW XZ SR GN CF. Wrote the paper: SR SW.
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.
Melatonin (
Since the relatively recent discovery of melatonin in plants, investigations to elucidate its function in plants has been driven by what is known in animals. One such area of focus is the involvement of melatonin in modulating circadian rhythms and photoperiod-dependent processes. While melatonin levels do appear to be affected by light/dark cycles in some plants, the pattern varies among species, tissues, and organs
Melatonin has been studied extensively as an antioxidant in mammals. Many studies demonstrate the ability of melatonin to protect against many human diseases, including those linked to oxidative stress
The structure of melatonin is another feature that has driven investigations into its function in plants. Melatonin is structurally similar to the plant hormone indole-3-acetic acid (IAA) and has many features that make it a candidate for a functional auxin
While much of the work conducted on melatonin in plants has focused on its physiological influence on growth and development, and on its biosynthesis, little work has focused on its affect on gene expression. Microarray analysis using endogenous melatonin-rich transgenic rice identified several hundred genes that are up- or down- regulated by elevated melatonin levels
After melatonin treatment, total RNA was extracted using the QIAGEN RNeasy Mini Kit according to the manufacturer's instructions (QIAGEN). Purification of mRNA from total RNA was conducted using an Oligotex mRNA Mini Kit (QIAGEN). The mRNA was then used to construct cDNA libraries using the mRNA-Seq Sample Preparation Kit™ (Illumina) following standard protocols. Briefly, the mRNA was fragmented by exposure to divalent cations at 94°C and the fragmented mRNA was converted into double stranded cDNA. The cDNA ends were polished, the 3′-hydroxls extended with A bases, and ligated to Illumina-specific adapter-primers. The adaptor ligated DNA was amplified by 15 cycles of PCR followed by purification using Qiagen™ PCR purification kit to obtain the final library for sequencing. The DNA yield and fragment insert size distribution of the library were determined on the Agilent Bioanalyzer. Library quantifications were performed by qPCR assays using the KAPA Library Quant Kit™ following the manufacturer's instructions.
All six constructed libraries were loaded on the Illumina flow cell at the appropriate concentration and bridge amplified to create millions of individual clonal clusters. The flow cells were sequenced on the HiSeq2000 sequencing instrument using 50 b single end protocols at the Center for the Study of Biological Complexity of the Virginia Commonwealth University.
After high throughput sequencing performance, short prematurely terminated sequences and those with low quality, as well as reads with any ambiguous bases were removed from raw sequence reads. Clean reads were aligned to the Arabidopsis genome assembly TAIR10 (as a reference) using TopHat (v1.3.1) BWA software
Gene Ontology classification of all genes exhibiting significant changes in transcript levels was conducted using AmiGO online tool
A total of 60 genes were selected for verification of mRNA-seq data using qRT-PCR. Primer pairs for each selected gene were presented in
The ability of melatonin to attenuate paraquat-induced oxidative stress was examined using detached Arabidopsis leaves. Seedlings were grown under short day conditions (10/14 hour light/dark photoperiod) to promote vegetative growth for four weeks. Leaves were then detached and incubated in 0 mM, 10 mM or 50 mM paraquat in the presence or absence of 1 mM melatonin under 16/8 hour light/dark photoperiod. After 48 hours, leaves were analyzed for oxidative stress as visualized by photobleaching.
To elucidate the roles of melatonin in regulating genome-wide gene expression, we conducted mRNA-seq analysis using Arabidopsis after 16 hours melatonin treatment. Because melatonin may function as a hormone at low concentration, and an antioxidant at high concentrations
SAMPLE | CLEAN READS | MAPPED READS | % MAPPED | TOTAL BASES | MAPPED BASES | TRANSCRIPTOME COVERAGE |
Control 1 | 16777955 | 14617162 | 87.1 | 855675705 | 745475262 | 14.3× |
100 pM Mel 1 | 17302219 | 15224369 | 88.0 | 882413169 | 776442819 | 14.7× |
1 mM Mel 1 | 12395079 | 10782224 | 87.0 | 632149029 | 549893424 | 10.5× |
Control 2 | 12383964 | 10938665 | 88.3 | 631582164 | 557871915 | 10.5× |
100 pM Mel 2 | 18378365 | 16281804 | 88.6 | 937296615 | 830372004 | 15.6× |
1 mM Mel 2 | 17452335 | 15331176 | 87.9 | 890069085 | 781889976 | 14.8× |
Sequences were mapped to the Arabidopsis TAIR10 genome using TopHat.
To examine the variability among our RNA-seq experiments, all clean reads from melatonin treated and control sets were plotted for all possible pairs of independent experiments. Scatter plots of these data for control plants, 100 pM and 1 mM melatonin-treated plants are shown in
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.
Although the results indicated that our experiments are highly reproducible, we further examined the reliability of the observed changes between treatments. qRT-PCR experiments were performed for a total of 60 genes exhibiting expression changes in response to melatonin treatments in the mRNA-seq analysis. The transcript levels were measured using the same RNAs used for the RNA-seq transcriptome analysis. The full list of 60 genes and their qRT-PCR validation is included in
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.
The Gene Ontology (GO) classification was conducted for all genes exhibiting a significant change in transcript levels using the AmiGO Slimmer tool and TAIR database. The Slimmer tool assigned general parent (GO slim) terms to the genes according to biological process, cellular component, and molecular function. The most striking difference between up and down regulated genes are those involved in response to stress and responses to endogenous and biotic stimuli. Of the up-regulated genes that were classified to a GO slim term, approximately 42% were involved in response to stress, 25% in response to endogenous stimulus, 24% in response to biotic stimulus, and 22% in response to signal transduction (
1 mM Melatonin | 100 pM Melatonin | ||||
GO Slim ID | Biological Process | up-regulated | down-regulated | up-regulated | down-regulated |
GO:0009987 | cellular process | 57.73 | 57.40 | 43.33 | 55.32 |
GO:0008152 | metabolic process | 50.72 | 51.59 | 50.00 | 53.19 |
GO:0006950 | response to stress | 42.27 | 16.74 | 30.00 | 29.79 |
GO:0009058 | biosynthetic process | 28.60 | 35.82 | 23.33 | 31.91 |
GO:0009719 | response to endogenous stimulus | 25.00 | 9.13 | 23.33 | 12.77 |
GO:0009607 | response to biotic stimulus | 23.92 | 6.64 | 13.33 | 14.89 |
GO:0007154 | cell communication | 23.56 | 8.85 | 6.67 | 14.89 |
GO:0006810 | transport | 23.20 | 14.38 | 10.00 | 12.77 |
GO:0007165 | signal transduction | 21.94 | 6.50 | 3.33 | 10.64 |
GO:0009628 | response to abiotic stimulus | 21.76 | 17.43 | 23.33 | 17.02 |
GO:0008150 | Biological process |
18.88 | 23.79 | 23.33 | 21.28 |
GO:0016043 | cellular component organization | 15.65 | 17.15 | 3.33 | 17.02 |
GO:0019538 | protein metabolic process | 12.59 | 11.62 | 3.33 | 19.15 |
GO:0009056 | catabolic process | 12.05 | 10.79 | 20.00 | 6.38 |
GO:0007275 | multicellular organismal development | 11.87 | 17.29 | 10.00 | 12.77 |
GO:0008219 | cell death | 10.61 | 1.94 | 3.33 | 8.51 |
GO:0019748 | secondary metabolic process | 8.99 | 7.61 | 10.00 | 4.26 |
GO:0006139 | nucleobase-containing compound metabolic process | 8.45 | 22.13 | 13.33 | 14.89 |
GO:0006464 | cellular protein modification process | 7.37 | 7.05 | 0 | 10.64 |
GO:0009605 | response to external stimulus | 6.83 | 4.01 | 3.33 | 6.38 |
GO:0006629 | lipid metabolic process | 5.58 | 9.68 | 13.33 | 8.51 |
GO:0000003 | reproduction | 4.86 | 5.39 | 0 | 4.26 |
GO:0009791 | post-embryonic development | 4.86 | 9.68 | 3.33 | 4.26 |
GO:0005975 | carbohydrate metabolic process | 4.32 | 13.69 | 3.33 | 4.26 |
GO:0009653 | anatomical structure morphogenesis | 4.14 | 10.93 | 3.33 | 4.26 |
GO:0009991 | response to extracellular stimulus | 3.60 | 1.94 | 3.33 | 6.38 |
GO:0040007 | growth | 3.60 | 5.81 | 0 | 4.26 |
GO:0016049 | cell growth | 2.70 | 3.46 | 0 | 2.13 |
GO:0030154 | cell differentiation | 2.34 | 5.39 | 3.33 | 2.13 |
GO:0019725 | cellular homeostasis | 1.98 | 2.49 | 3.33 | 0 |
GO:0006091 | generation of precursor metabolites and energy | 1.62 | 8.16 | 3.33 | 0 |
GO:0009908 | flower development | 1.62 | 3.04 | 0.00 | 4.26 |
GO:0009790 | embryo development | 1.26 | 1.52 | 0.00 | 2.13 |
GO:0015979 | photosynthesis | 1.26 | 9.68 | 6.67 | 2.13 |
GO:0007049 | cell cycle | 1.08 | 1.80 | 0 | 2.13 |
GO:0006259 | DNA metabolic process | 0.90 | 2.21 | 0 | 0 |
GO:0009838 | abscission | 0.54 | 0.14 | 0 | 2.13 |
GO:0009856 | pollination | 0.54 | 0.41 | 0 | 0 |
GO:0040029 | regulation of gene expression, epigenetic | 0.36 | 1.11 | 0 | 2.13 |
GO:0006412 | translation | 0.18 | 1.80 | 0 | 4.26 |
GO:0009606 | tropism | 0.18 | 0.69 | 0 | 0 |
GO:0007267 | cell-cell signaling | 0 | 0.28 | 0 | 0 |
GO:0009875 | pollen-pistil interaction | 0 | 0.14 | 0 | 0 |
Genes were classified using AmiGO GO slimmer. The proportion of genes assigned to each category was calculated by dividing the number of genes assigned to a category by the total number of differentially expressed genes for each treatment. Genes may be classified into one or more GO Slim Term.
*Indicates biological process is unknown.
The biological processes that trended towards down-regulation in response to 1 mM melatonin included biosynthetic processes, metabolism of carbohydrates and nucleobase-containing compounds, development, cellular organization, morphogenesis, photosynthesis, and generation of precursor metabolites and energy.
The cellular components associated with genes down-regulated in response to 1 mM melatonin included cytoplasm, extracellular region, and, consistent with the down-regulation of photosynthesis associated genes, the thylakoid and plastids (
1 mM Melatonin | 100 pM Melatonin | ||||
GO Slim ID | Cellular Component | up-regulated | down regulated | up-regulated | down regulated |
GO:0005623 | cell | 80.04 | 77.04 | 80.00 | 63.83 |
GO:0005622 | intracellular | 66.19 | 67.77 | 73.33 | 53.19 |
GO:0005737 | cytoplasm | 44.78 | 51.87 | 60.00 | 34.04 |
GO:0005634 | nucleus | 27.16 | 20.89 | 16.67 | 17.02 |
GO:0016020 | membrane | 25.36 | 26.83 | 20.00 | 12.77 |
GO:0005886 | plasma membrane | 20.32 | 11.34 | 3.33 | 10.64 |
GO:0005576 | extracellular region | 13.67 | 20.19 | 10.00 | 23.40 |
GO:0009536 | plastid | 12.77 | 30.71 | 43.33 | 17.02 |
GO:0005739 | mitochondrion | 10.07 | 8.44 | 6.67 | 6.38 |
GO:0005575 | Cellular component |
8.63 | 9.54 | 13.33 | 2.13 |
GO:0005829 | cytosol | 6.65 | 5.26 | 3.33 | 4.26 |
GO:0005794 | Golgi apparatus | 6.12 | 2.07 | 3.33 | 4.26 |
GO:0005618 | cell wall | 5.94 | 5.39 | 3.33 | 6.38 |
GO:0030312 | external encapsulating structure | 5.94 | 5.39 | 3.33 | 6.38 |
GO:0005773 | vacuole | 5.04 | 4.56 | 20.00 | 4.26 |
GO:0005783 | endoplasmic reticulum | 2.52 | 2.21 | 3.33 | 0 |
GO:0005768 | endosome | 0.72 | 0.55 | 0 | 0 |
GO:0005730 | nucleolus | 0.54 | 0.69 | 3.33 | 2.13 |
GO:0005777 | peroxisome | 0.54 | 0.83 | 0 | 0 |
GO:0009579 | thylakoid | 0.54 | 11.48 | 10.00 | 0 |
GO:0005654 | nucleoplasm | 0.36 | 0.00 | 0 | 2.13 |
GO:0005635 | nuclear envelope | 0.18 | 0.28 | 0 | 0 |
GO:0005615 | extracellular space | 0 | 0.14 | 0 | 0 |
GO:0005764 | lysosome | 0 | 0 | 0 | 0 |
GO:0005840 | ribosome | 0 | 1.38 | 3.33 | 0 |
GO:0005856 | cytoskeleton | 0 | 0.41 | 0.00 | 0 |
The proportion of genes in each category were calculated by dividing the number of genes assigned to a category by the total number of genes that were classified in a treatment by the AmiGO GO slimmer.
1 mM Melatonin | 100 pM Melatonin | ||||
GO Slim ID | Molecular Function | up-regulated | down regulated | up-regulated | down regulated |
GO:0003824 | catalytic activity | 37.41 | 32.23 | 43.33 | 46.81 |
GO:0005488 | binding | 25.72 | 21.85 | 23.33 | 25.53 |
GO:0003674 | Molecular function |
21.04 | 24.76 | 30.00 | 21.28 |
GO:0016740 | transferase activity | 16.73 | 9.82 | 13.33 | 21.28 |
GO:0016787 | hydrolase activity | 12.05 | 10.93 | 23.33 | 10.64 |
GO:0005515 | protein binding | 11.33 | 7.05 | 13.33 | 8.51 |
GO:0016301 | kinase activity | 7.73 | 3.18 | 0 | 6.38 |
GO:0003700 | sequence-specific DNA binding transcription factor activity | 6.83 | 5.39 | 0 | 2.13 |
GO:0005215 | transporter activity | 5.94 | 4.56 | 3.33 | 2.13 |
GO:0003676 | nucleic acid binding | 4.32 | 7.33 | 0 | 8.51 |
GO:0000166 | nucleotide binding | 3.96 | 2.21 | 0 | 8.51 |
GO:0003677 | DNA binding | 3.24 | 5.39 | 0 | 4.26 |
GO:0008289 | lipid binding | 1.44 | 1.80 | 0 | 0 |
GO:0019825 | oxygen binding | 1.26 | 0.97 | 0 | 2.13 |
GO:0030234 | enzyme regulator activity | 1.26 | 1.24 | 3.33 | 4.26 |
GO:0004871 | signal transducer activity | 0.72 | 1.11 | 0 | 0 |
GO:0030246 | carbohydrate binding | 0.72 | 0.55 | 0 | 0 |
GO:0004872 | receptor activity | 0.54 | 0 | 0 | 0 |
GO:0003723 | RNA binding | 0.36 | 1.52 | 0 | 2.13 |
GO:0004518 | nuclease activity | 0.36 | 0.41 | 3.33 | 0 |
GO:0005102 | receptor binding | 0.36 | 0.14 | 0 | 0 |
GO:0003682 | chromatin binding | 0 | 0 | 0 | 0 |
GO:0003774 | motor activity | 0 | 0.83 | 0 | 0 |
GO:0005198 | structural molecule activity | 0 | 1.80 | 0 | 0 |
GO:0008135 | translation factor activity, nucleic acid binding | 0 | 0.14 | 0 | 0 |
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.
*Indicates Molecular function is unknown.
Interestingly, expression of chlorophyllase (CLH1), a light regulated enzyme involved in chlorophyll degradation
Due to the classic function of melatonin as a “clock” hormone in animals, one might infer that it would play a central role in timing of events in plants, such as flowering. However, since less than 1% of the genes affected by melatonin were involved in flowering, but nearly 40% were involved in stress responses and signaling, it is tempting to deduce that the central role of melatonin in plants is more likely as an antioxidant involved in stress response and as a photo-protectant. However, since the melatonin treatment was a one-time event and flowering is a complex programmed event that is promoted over several days, we cannot completely rule out that fluctuations in melatonin levels over time may signal day length and promote flowering.
Much fewer genes were affected by low level (100 pM) of melatonin treatment with only 81 genes significantly altered their expression. In view of the biological processes, most of the identified genes involved in cellular process, cell communication, response to stress, transporters and signal transductions are down-regulated. Cell death associated genes identified were also mostly down-regulated by 100 pM melatonin.
Comparative analysis was conducted on genes affected by 100 pM melatonin and those by 1 mM melatonin. Results showed that not all genes down-regulated by low (100 pM) melatonin were down regulated by high (1 mM) melatonin. In fact, out of 51 of 100 pM down-regulated genes, only 18 genes were down regulated by 1 mM melatonin treatment, and 13 genes were up-regulated by 1 mM melatonin treatment. Similarly, out of 30 genes up-regulated by 100 pM melatonin, only 5 were also up-regulated by 1 mM melatonin and 8 were down regulated by 1 mM melatonin. Seventeen genes were uniquely up-regulated and 20 were uniquely down-regulated by low (100 pM) melatonin treatment (
The number in the parenthesis indicates the number of genes altered by melatonin for each treatment.
One of the advantages of mRNA-seq technique over microarray is that it can discover novel genes that were not previously annotated
Given that a large group of genes altered by melatonin are involved in plant stress tolerance, we further categorized these genes according to their specific roles in plant defense. A schematic of the plants response to biotic and abiotic stresses to include key events and players involved in perception, signaling, and counter-attack that were affected by melatonin is depicted in
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.
When facing environmental stresses, plants first perceive stress signal and initiate a signal transduction cascade to defend against the pronounced stresses
Accession | Gene | Description | Fold Change |
AT4G01870 | AT4G01870 | TolB related protein | 3.18457 |
AT1G66090 | AT1G66090 | Disease resistance protein (TIR-NBS class) | 3.16883 |
AT1G57630 | AT1G57630 | Toll-Interleukin-Resistance domain family protein | 2.6298 |
AT4G14370 | AT4G14370 | TIR-NBS-LRR class disease resistance protein | 2.42877 |
AT2G32660 | RLP22 | Receptor like protein 22 | 2.23431 |
AT2G31880 | SOBIR1 | Leucine rich repeat transmembrane protein | 2.00079 |
AT1G66830 | AT1G66830 | Leucine-rich repeat receptor-like protein kinase | 3.02283 |
AT1G51830 | AT1G51830 | Leucine-rich repeat protein kinase | 2.84031 |
AT5G67080 | MAPKKK19 | Mitogen-activated protein kinase kinase kinase 19 | 2.67063 |
AT5G25930 | AT5G25930 | Leucine-rich repeat receptor-like protein kinase | 2.53548 |
AT4G11890 | ARCK1 | Osmotic-stress-inducible receptor-like cytosolic kinase 1 | 2.50129 |
AT2G05940 | RIPK | RPM1-induced protein kinase | 2.40356 |
AT1G01560 | MPK11 | MAP Kinase 11 | 2.30442 |
AT4G04490 | CRK36 | Cysteine-rich receptor-like protein kinase | 2.1158 |
AT1G21250 | WAK1 | Cell wall-associated kinase 1 | 2.05986 |
AT3G09010 | AT3G09010 | Protein kinase superfamily protein | 2.02544 |
AT2G41820 | AT2G41820 | Leucine-rich repeat protein kinase family protein | −2.06899 |
AT5G04190 | PKS4 | Phytochrome kinase substrate 4 | −2.76656 |
AT4G10390 | AT4G10390 | Receptor-like protein kinase | −3.10278 |
AT5G58300 | MCK7 | Leucine-rich repeat protein kinase family protein | Suppressed |
AT2G25090 | CIPK16 | CBL-INTERACTING PROTEIN KINASE 16 | 4.56701 |
AT1G21550 | AT1G21550 | Calcium-binding EF-hand family protein | 3.97178 |
AT1G74010 | AT1G74010 | Calcium-dependent phosphotriesterase superfamily | 3.74794 |
AT3G22910 | AT3G22910 | calcium-transporting ATPase 13 | 3.34891 |
AT1G66400 | CML23 | Calmodulin-like protein 23 | 3.34393 |
AT5G26920 | CBP60G | Calmodulin binding protein 60-like G | 3.26761 |
AT1G76650 | CML38 | Calmodulin-like 38 | 3.18083 |
AT3G50360 | CEN2 | Centrin2; Involved in calcium ion binding | 2.56703 |
AT3G47480 | AT3G47480 | Calcium-binding EF-hand family protein | 2.29581 |
AT2G33380 | RD20 | Encodes a calcium binding protein | 2.28141 |
AT5G63970 | AT5G63970 | Calcium-dependent phospholipid-binding protein | 2.25577 |
AT3G22930 | CML11 | CALMODULIN-LIKE 11 | 2.08638 |
AT5G45820 | CIPK20 | CBL-interacting protein kinase 20 | −3.86907 |
AT1G02900 | RALF1 | Similar to tobacco Rapid Alkalinization Factor (RALF) | −2.7376 |
Calcium signals are also a core regulator for plant cellular responses to environmental stimuli
A total of 53 transcription factors were identified with at least 2 fold changes by melatonin treatment (
Accession # | Gene | Description | Fold change | stress |
AT4G22070 | WRKY31 | WRKY DNA-binding protein 31 | 4.04382 | |
AT1G02450 | NIMIN1 | NIM1-Interacting 1 | 4.01997 | |
AT5G64810 | WRKY51 | WRKY DNA-binding protein 51 | 3.89444 | |
AT5G26170 | WRKY50 | WRKY DNA-binding protein 50 | 3.60762 | |
AT5G26920 | CBP60G | Calmodulin binding protein 60-like.g | 3.51898 | |
AT3G46080 | AT3G46080 | C2H2-type zinc finger family protein | 3.443 | |
AT3G46090 | ZAT7 | Zinc finger protein | 3.36216 | |
AT1G02580 | MEA | Maternal embryogenesis control protein | 3.23147 | |
AT1G01720 | NAC002 | NAC domain containing protein 2 | 3.07132 | |
AT1G01010 | NAC001 | NAC domain containing protein 1 | 3.03835 | |
AT3G56400 | WRKY70 | WRKY DNA-binding protein 70 | 2.74608 | |
AT4G01250 | WRKY22 | WRKY DNA-binding protein 22 | 2.73313 | |
AT2G38250 | AT2G38250 | Homeodomain-like superfamily protein | 2.6849 | |
AT5G63790 | NAC102 | NAC domain containing protein 102 | 2.60489 | |
AT3G50260 | CEJ1 | Cooperatively regulated by ethylene and jasmonate 1 | 2.57229 | |
AT5G13080 | WRKY75 | WRKY DNA-binding protein 75 | 2.53783 | |
AT1G73805 | SARD1 | SAR Deficient 1 | 2.41244 | |
AT5G59820 | RHL41 | Zinc finger protein | 2.39584 | |
AT1G62300 | WRKY6 | WRKY DNA-binding protein 6 | 2.37767 | |
AT5G01380 | AT5G01380 | Homeodomain-like superfamily protein | 2.28926 | |
AT2G16720 | MYB7 | MYB domain protein 7 | 2.26732 | |
AT3G19580 | AZF2 | Zinc finger protein 2 | 2.20418 | |
AT5G49450 | bZIP1 | Basic leucine zipper 1 | 2.1947 | |
AT1G52890 | NAC019 | NAC domain-containing protein 19 | 2.19063 | |
AT5G62020 | HSFB2A | Heat shock transcription factor B2A | 2.10743 | |
AT3G04070 | NAC047 | NAC domain containing protein 47 | 2.06633 | |
AT1G27730 | STZ | Salt tolerance zinc finger | 2.05565 | |
AT2G40750 | WRKY54 | WRKY DNA-binding protein 54 | 2.05047 | |
AT3G24500 | MBF1C | Multiprotein bridging factor 1C | 2.04727 | |
AT5G20240 | PI | Floral homeotic protein PISTILLATA | −2.00396 | |
AT3G02380 | COL2 | Zinc finger protein CONSTANS-LIKE 2 | −2.16211 | |
AT3G58120 | BZIP61 | Basic-leucine zipper transcription factor family protein | −2.18023 | |
AT1G76880 | AT1G76880 | Duplicated homeodomain-like superfamily protein | −2.24420 | |
AT1G35560 | TCP23 | TCP domain protein 23 | −2.26984 | |
AT3G10040 | AT3G10040 | Sequence-specific DNA binding transcription factor | −2.41108 | |
AT1G75240 | HB33 | Homeobox protein 33 | −2.51608 | |
AT5G05790 | AT5G05790 | Homeodomain-like superfamily protein | −2.57254 | |
AT1G14600 | AT1G14600 | Homeodomain-like superfamily protein | −2.60754 | |
AT1G09530 | PAP3 | Phytochrome-associated protein 3 | −2.67198 | |
AT4G32800 | AT4G32800 | Ethylene-responsive transcription factor ERF043 | −2.85108 | |
AT5G60890 | MYB34 | Myb-like transcription factor | −2.9674 | |
AT2G44940 | AT2G44940 | Ethylene-responsive transcription factor ERF034 | −3.01312 | |
AT4G17810 | AT4G17810 | C2H2 and C2HC zinc finger-containing protein | −3.05949 | |
AT2G42380 | BZIP34 | BZIP family of transcription factors | −3.10503 | |
AT1G19510 | ATRL5 | RAD-like 5 | −3.32387 | |
AT1G04250 | AXR3 | Auxin resistant 3 | −3.49394 | |
AT5G25190 | ESE3 | Ethylene-responsive transcription factor ERF003 | −3.49854 | |
AT5G56840 | AT5G56840 | myb-like transcription factor family protein | −3.52364 | |
AT1G04240 | SHY2 | Short hypocotyl 2 | −3.70592 | |
AT2G39250 | SNZ | AP2-like ethylene-responsive transcription factor | −4.03198 | |
AT5G61890 | AT5G61890 | Ethylene-responsive transcription factor ERF114 | −4.23551 | |
AT1G32360 | AT1G32360 | CCCH-type Zinc finger family protein | −4.39267 | |
AT2G18328 | RL4 | RAD-like 4 | −4.82888 |
Plant hormones and their cross-talk play important roles in both biotic and abiotic stress defense. Of these, abscisic acid (ABA) and ethylene (ET) are known for their abiotic stress defense functions
Two ACC synthases were also up-regulated in response to melatonin, suggesting that melatonin may induce ethylene biosynthesis. One of the ACC synthases identified is ACS8, an auxin inducible ACC synthase. Indeed, this response is similar to what was induced by 2,4-D
While the majority of auxin-responsive genes were down-regulated in response to 1 mM melatonin, most genes on ABA, SA, JA and ET pathways were up-regulated (
Genes with functions involving the cell wall, including modification and growth, were largely affected by melatonin. Of the 60 genes associated with the cell wall, 45 were down-regulated and 14 were up-regulated at least 2-fold, and one gene was completely suppressed (
Accession # | Gene | Description | Fold Change |
AT4G25810 | XTR6 | Xyloglucan endotransglycosylase 6 | 4.46599 |
AT5G57550 | XTH25 | Xyloglucan endotransglucosylase/hydrolase 25 | 4.05392 |
AT3G60140 | SRG2 | Beta-glucosidase 30 | 3.32985 |
AT2G45220 | AT2G45220 | Plant invertase/pectin methylesterase inhibitor superfamily | 2.85982 |
AT1G76470 | AT1G76470 | NAD(P)-binding Rossmann-fold superfamily protein | 2.75965 |
AT5G57560 | TCH4 | Xyloglucan endotransglucosylase/hydrolase 22 | 2.67655 |
AT4G30280 | XTH18 | Xyloglucan endotransglucosylase/hydrolase 18 | 2.57069 |
AT1G21670 | AT1G21670 | uncharacterized protein | 2.46195 |
AT4G01430 | UMAMIT29 | Nodulin MtN21-like transporter family protein | 2.42787 |
AT1G78770 | APC6 | Anaphase promoting complex 6 | 2.35509 |
AT3G45970 | ATEXPL1 | Expansin-like A1 | 2.12646 |
AT4G26470 | CML21 | Calmodulin-like protein 21 | 2.10667 |
AT1G05560 | UGT75B1 | UDP-glucosyltransferase 75B1 | 2.08708 |
AT2G43570 | CHI | Putative chitinase | 2.04539 |
AT2G37040 | PAL1 | Phenylalanine ammonia-lyase 1 | −2.04568 |
AT4G08685 | SAH7 | Allergen-like protein | −2.07476 |
AT2G39700 | EXPA4 | Alpha-Expansin 4 | −2.09564 |
AT5G14610 | AT5G14610 | DEAD box RNA helicase family protein | −2.10274 |
AT3G19450 | ATCAD4 | Cinnamyl alcohol dehydrogenase 4 | −2.11144 |
AT1G70370 | PG2 | Polygalacturonase 2 | −2.11182 |
AT4G33220 | PME44 | Pectin methylesterase 44 | −2.13826 |
AT2G33330 | PDLP3 | Plasmodesmata-located protein 3 | −2.15409 |
AT2G36870 | XTH32 | Xyloglucan endotransglucosylase/hydrolase 32 | −2.22347 |
AT3G26520 | SITIP | Gamma-tonoplast intrinsic protein 2 | −2.22602 |
AT1G03630 | PORC | Protochlorophyllide oxidoreductase C | −2.23808 |
AT2G45470 | FLA8 | Fasciclin-like arabinogalactan protein 8 | −2.26278 |
AT4G36360 | BGAL3 | Beta-galactosidase 3 | −2.27535 |
AT1G24100 | UGT74B1 | UDP-glucosyl transferase 74B1 | −2.28945 |
AT1G69530 | EXPA1 | Alpha-Expansin 11 | −2.31131 |
AT5G20540 | BRXL4 | Brevis radix-like 4 | −2.32922 |
AT5G47500 | PME5 | Pectin methylesterase 5 | −2.38968 |
AT4G12880 | ENODL19 | Early nodulin-like protein 19 | −2.40384 |
AT5G04360 | PU1 | Pullulanase 1 | −2.41719 |
AT1G05850 | ELP | Endo chitinase-like protein | −2.43396 |
AT3G44990 | XTH31 | Xyloglucan endo-transglycosylase | −2.44701 |
AT5G53090 | AT5G53090 | NAD(P)-binding Rossmann-fold superfamily protein | −2.68485 |
AT1G04680 | AT1G04680 | Pectin lyase-like superfamily protein | −2.68802 |
AT5G48900 | AT5G48900 | Pectin lyase-like superfamily protein | −2.69330 |
AT3G05900 | AT3G05900 | Neurofilament protein-related | −2.78189 |
AT2G28950 | EXPA6 | Alpha-Expansin 6 | −2.78667 |
AT3G16370 | AT3G16370 | GDSL-like Lipase/Acylhydrolase superfamily protein | −2.83692 |
AT5G03760 | ATCSLA09 | Cellulose synthase like A9 | −2.88537 |
AT4G39330 | CAD9 | Cinnamyl alcohol dehydrogenase 9 | −2.991 |
AT2G19590 | ACO1 | 1-aminocyclopropane-1-carboxylate oxidase 1 | −3.13848 |
AT2G34410 | RWA3 | Reduced wall acetylation 3 | −3.20576 |
AT3G29030 | EXPA5 | Alpha-Expansin 5 | −3.25293 |
AT1G04040 | AT1G04040 | HAD superfamily, subfamily IIIB acid phosphatase | −3.32924 |
AT2G37640 | EXP3 | Alpha-Expansin 3 | −3.40584 |
AT1G20190 | EXPA11 | Alpha-Expansin 11 | −3.47301 |
AT5G19730 | AT5G19730 | Pectin lyase-like superfamily protein | −3.62317 |
AT1G43800 | FTM1 | Floral transition at the meristem 1 | −3.98758 |
AT4G22610 | AT4G22610 | Protease inhibitor/seed storage/lipid transfer family protein | −4.01733 |
AT4G28250 | EXPB3 | Beta-expansin/allergen protein. | −4.03468 |
AT3G10340 | PAL4 | Phenylalanine ammonia-lyase | −4.14771 |
AT2G38120 | AUX1 | Auxin influx transporter | −4.28187 |
AT3G23090 | WDL3 | Targeting protein for Xklp2 protein family | −4.66044 |
AT2G40610 | EXPA8 | Alpha-Expansin Gene Family | −4.89447 |
AT4G02290 | GH9B13 | Glycosyl hydrolase 9B13 | −5.14512 |
AT1G26250 | AT1G26250 | Proline-rich extensin-like family protein | −7.28679 |
AT4G31850 | PGR3 | Pentatricopeptide repeat-containing protein | suppressed |
The results in the current study are significantly different from our previous study in cucumber roots showing that many of cell wall related genes, particularly pectinesterase genes, were up-regulated by melatonin
Genes and their protein products involved in various redox pathways are also used to protect cells from oxidative damage
Accession # | Gene | Description | Fold change | Stress response |
AT2G26400 | ATARD3 | Acireductone dioxygenase 3 | 4.84062 | |
AT5G56970 | CKX3 | Cytokinin oxidase 3 | 4.47185 | |
AT3G04000 | AT3G04000 | Aldehyde reductase | 4.39375 | |
AT2G37770 | ChlAKR | Chloroplastic aldo-keto reductase | 4.27787 | |
AT5G48450 | SKS2 | SKU5 Similar | 4.01997 | |
AT3G21460 | AT3G21460 | Glutaredoxin family protein | 3.82732 | |
AT1G04580 | AAO4 | Aldehyde oxidase 4 | 3.27115 | |
AT2G34500 | CYP710A1 | Cytochrome P450 710A1 | 3.09868 | |
AT5G07440 | GDH2 | Glutamate dehydrogenase 2 | 2.99839 | |
AT1G76470 | AT1G76470 | NAD(P)-binding Rossmann-fold superfamily protein | 2.75965 | |
AT1G30700 | AT1G30700 | FAD-binding Berberine family protein | 2.73673 | |
AT2G47130 | SDR3 | short-chain dehydrogenase/reductase 2 | 2.6147 | |
AT4G34120 | CDCP1 | Cystathione [Beta]-synthase domain-containing protein 1 | 2.52354 | |
AT1G54100 | ALDH7B4 | aldehyde dehydrogenase 7B4 | 2.42985 | |
AT4G20830 | AT4G20830 | FAD-binding Berberine family protein | 2.42483 | |
AT1G26390 | AT1G26390 | FAD-binding Berberine family protein | 2.40531 | |
AT4G20860 | AT4G20860 | FAD-binding Berberine family protein | 2.31804 | |
AT3G14620 | CYP72A8 | Cytochrome P450 72A8 | 2.24781 | |
AT4G37990 | ATCAD8 | Arabidopsis thaliana cinnamyl-alcohol dehydrogenase 8 | 2.23169 | |
AT1G09500 | AT1G09500 | Alcohol dehydrogenase-like protein | 2.1254 | |
AT1G30730 | AT1G30730 | FAD-binding Berberine family protein | 2.0479 | |
AT5G24530 | DMR6 | Downy mildew resistant 6 | 2.01769 | |
AT3G26210 | CYP71B23 | Cytochrome P45071B23 | 2.01045 | |
AT2G32720 | CB5-B | Cytochromes B5 | −2.11096 | |
AT3G19450 | ATCAD4 | Cinnamyl alcohol dehydrogenase 4 | −2.11144 | |
AT4G00360 | CYP86A2 | Cytochrome P450 86A2 | −2.16096 | |
AT1G14345 | AT1G14345 | NAD(P)-linked oxidoreductase superfamily protein | −2.16512 | |
AT2G46750 | GulLO2 | L -gulono-1,4-lactone oxidase 2 | −2.16775 | |
AT1G03630 | PORC | Protochlorophyllide oxidoreductase C | −2.23808 | |
AT5G43750 | NDH18 | NAD(P)H dehydrogenase 18 | −2.26847 | |
AT5G49730 | ATFRO6 | Ferric reduction oxidase 6 | −2.35762 | |
AT5G07460 | ATMSRA2 | Methionine sulfoxide reductase 2 | −2.38061 | |
AT1G14150 | PnsL2 | Photosynthetic NDH subcomplex L 2 | −2.39048 | |
AT1G17650 | GLYR2 | Glyoxylate reductase 2 | −2.46655 | |
AT1G07440 | AT1G07440 | NAD(P)-binding Rossmann-fold superfamily protein | −2.56359 | |
AT5G14200 | ATIMD1 | Arabidopsis isopropylmalate dehydrogenase 1 | −2.62532 | |
AT1G62540 | FMO GS-OX2 | Flavin-monooxygenase glucosinolate S-oxygenase 2 | −2.65092 | |
AT2G39470 | PPL2 | Photosynthetic NDH subcomplex L 1 | −2.6738 | |
AT5G53090 | AT5G53090 | NAD(P)-binding Rossmann-fold superfamily protein | −2.68485 | |
AT5G44410 | AT5G44410 | FAD-binding Berberine family protein | −2.79494 | |
AT4G25100 | FSD1 | FE-superoxide dismutase 1 | −2.79727 | |
AT4G39330 | CAD9 | Cinnamyl alcohol dehydrogenase 9 | −2.991 | |
AT5G58260 | NDHN | NADH Dehydrogenase-like complex N | −2.99281 | |
AT4G25600 | AT4G25600 | Oxoglutarate/iron-dependent oxygenase | −3.07505 | |
AT2G19590 | ACO1 | 1-aminocyclopropane-1-carboxylate oxidase 1 | −3.13848 | |
AT4G39510 | CYP96A12 | Cytochrome P450 96A12 | −3.2424 | |
AT1G65860 | FMO GS-OX1 | Flavin-monooxygenase glucosinolate S-oxygenase 1 | −3.3226 | |
AT1G06350 | ADS4 | Fatty acid desaturase family protein | −3.33981 | |
AT3G01440 | PnsL3 | Photosynthetic NDH subcomplex L 3 | −3.34393 | |
AT4G13770 | CYP83A1 | Cytochrome P450 83A1 | −3.3894 | |
AT4G25310 | AT4G25310 | 2-oxoglutarate and Fe(II)-dependent oxygenase | −3.84556 | |
AT4G19380 | AT4G19380 | Long-chain fatty alcohol dehydrogenase family protein | −3.84622 | |
AT1G43800 | FTM1 | Stearoyl-acyl-carrier-protein desaturase family protein | −3.98758 | |
AT1G16400 | CYP79F2 | Cytochrome P450 79F2 | −4.03821 | |
AT5G42800 | DFR | Dihydroflavonol 4-reductase | −4.19704 | |
AT4G12320 | CYP706A6 | Cytochrome P450 706A6 | −4.31681 | |
AT1G16410 | CYP79F1 | Cytochrome P450 79F1 | −4.38148 | |
AT1G30760 | AT1G30760 | FAD-binding Berberine family protein | −4.5319 | |
AT2G18030 | MSRA5 | Methionine sulfoxide reductase A5 | −5.04612 | |
AT5G39580 | AT5G39580 | Peroxidase superfamily protein | 3.33272 | |
AT1G14550 | AT1G14550 | Peroxidase superfamily protein | 2.66383 | |
AT1G14540 | PER4 | Peroxidase 4 | 2.46391 | |
AT3G49120 | PRXCB | Peroxidase CB | 2.45042 | |
AT4G33420 | AT4G33420 | Peroxidase superfamily protein | 2.2565 | |
AT3G21770 | AT3G21770 | Peroxidase superfamily protein | −2.21265 | |
AT4G08770 | AT4G08770 | Encodes a putative apoplastic peroxidase Prx37 | −2.21812 | |
AT3G26060 | PRXQ | Peroxiredoxin Q | −2.31392 | |
AT4G08780 | AT4G08780 | Peroxidase superfamily protein | −2.99013 | |
AT1G49570 | AT1G49570 | Peroxidase superfamily protein | −3.03705 | |
AT4G30170 | AT4G30170 | Peroxidase family protein | −3.79494 | |
AT1G05260 | RCI3 | Cold-inducible cationic peroxidase | −4.07505 | |
AT4G11290 | AT4G11290 | Peroxidase superfamily protein | −4.97627 |
Peroxidases comprise another group of genes related to oxidative stress and are used as biochemical markers for plant resistance to bacterial and fungal diseases
Since the majority of genes with altered expression levels are involved in plant stress defense and melatonin is a potent antioxidant, we further examined the potential of melatonin to alleviate paraquat induced oxidative stress. Four-week old detached Arabidopsis leaves were incubated in 0 mM, 10 mM or 50 mM paraquat in the presence or absence of 1 mM melatonin. Leaves treated with paraquat in the absence of melatonin were completely photobleached after 48 hours under 16/8 hour light/dark photoperiod (
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.
In conclusion, we report here the first comprehensive analysis of the effect of melatonin on genome-wide gene expression in Arabidopsis seedlings using RNA-seq technology. Given that Arabidopsis is an established plant model species and forward and reverse genetics methodologies are readily available, these datasets will provide fundamental information and serve as new tools to genetically dissect melatonin-mediated pathway(s) either common to both plants and animals, or unique to plants. Our transcriptome analysis reveals broader roles of melatonin in regulating plant growth and development. However, more importantly, melatonin may play critical role(s) in plant defense systems. Out of nearly 900 genes that were significantly up- or down- regulated by melatonin with at least 2 fold changes, almost 40% of the genes were related to plant stress defense, including many stress receptors, kinases, and transcription factors, as well as downstream genes encoding end products that were directly used for stress defense. Furthermore, the expression of many genes involved in different hormone signaling pathways such as auxin, ABA, SA, ET and JA, and linked to plant stress defense, was also altered in response to melatonin treatment. Concurrently, expression of many cell wall associated genes, and genes involved in redox pathways, particularly peroxidases were significantly changed by melatonin treatments. Taken together, our results suggest that melatonin plays a critical role in plant defense against environmental stresses, including both biotic and abiotic stresses. Further dissection of the melatonin mediated pathway may lead to the development of novel strategies for crop improvement in the face of ubiquitous environmental stresses.
Scatter plots between treatments.
(TIF)
Phylogenetic analysis of all clean RNA-seq data from six constructed cDNA libraries.
(TIF)
List of genes and their primer pairs used for qRT-PCR validation.
(DOCX)
List of genes that are significantly affected by 100 pM melatonin.
(DOCX)
List of genes that are significantly affected by 1 mM melatonin.
(DOCX)
qRT-PCR validation of RNA-seq data.
(DOCX)
Genes with changes in expression levels of at least 2 fold in response to 1 mM Melatonin involved in one or more hormone signaling pathways.
(DOCX)
Downstream genes in plant stress defense that are affected by melatonin and their fold changes.
(DOCX)
The Sequencing and analysis was performed in the Nucleic Acids Research Facilities and the Bioinformatics Computational Core Laboratories at Virginia Commonwealth University. The authors would also like to thank Myrna Serrano, Nihar U Sheth and Steven Bradley at the Virginia Commonwealth University for their excellent technical assistance in conducting mRNA-seq analysis. This article is a contribution of the Virginia State University, Agricultural Research Station (Journal series No. 315).