Integrated mRNA and microRNA analysis identifies genes and small miRNA molecules associated with transcriptional and post-transcriptional-level responses to both drought stress and re-watering treatment in tobacco
Abstract:BackgroundDrought stress is one of the most severe problem limited agricultural productivity worldwide. It has been reported that plants response to drought-stress by sophisticated mechanisms at both transcriptional and post-transcriptional levels. However, the precise molecular mechanisms governing the responses of tobacco leaves to drought stress and water status are not well understood. To identify genes and miRNAs involved in drought-stress responses in tobacco, we performed both mRNA and small RNA sequenc… Show more
“…3 A). We detected two peaks at 21 and 24 nt in most samples, indicating that small RNAs of 21 and 24 nt are the two major size classes in tobacco, consistent with a previous study in this species 12 , 13 , 34 , 39 and with reports for Arabidopsis and rice 49 , 50 . Figure 3 Expression pattern of conserved and novel miRNAs.…”
Section: Resultssupporting
confidence: 92%
“…3A). We detected two peaks at 21 and 24 nt in most samples, indicating that small RNAs of 21 and 24 nt are the two major size classes in tobacco, consistent with a previous study in this species 12,13,34,39 and with reports for Arabidopsis and rice 49,50 . After removing small RNA reads originating from other small non-coding RNAs (including tRNA, rRNA, snRNA, and snoRNA), the left reads were aligned to the tobacco reference genome 30 .…”
Section: Resultssupporting
confidence: 92%
“…As development of sequencing techniques and improvement of tobacco genome [30][31][32][33] , several studies have used small RNA sequencing to profile miRNAs in tobacco 12,13,[34][35][36][37][38] . These studies have revealed insights including characterization of five miRNA families that exhibit differential accumulation profiles under drought conditions 39 , and a functional demonstration that miR164 targets mRNA transcripts of NtNAC-R1 and that such targeting increases upon topping and leads to multiple root-related phenotypes 40 .…”
tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Many nicotine biosynthetic genes have been identified and were known to be regulated by jasmonate-responsive transcription factors. As an important regulator in plant physiological processes, whether small RNAs are involved in nicotine biosynthesis is largely unknown. Here, we combine transcriptome, small RnAs and degradome analysis of two native tobacco germplasms YJ1 and ZY100 to investigate small RNA's function. YJ1 leaves accumulate twofold higher nicotine than ZY100. Transcriptome analysis revealed 3,865 genes which were differently expressed in leaf and root of two germplasms, including some known nicotine and jasmonate pathway genes. By small RNA sequencing, 193 miRNAs were identified to be differentially expressed between YJ1 and ZY100. Using in silico and degradome sequencing approaches, six nicotine biosynthetic genes and seven jasmonate pathway genes were predicted to be targeted by 77 miRNA loci. Three pairs among them were validated by transient expression in vivo. Combined analysis of degradome and transcriptome datasets revealed 51 novel miRNA-mRNA interactions that may regulate nicotine biosynthesis. The comprehensive analysis of our study may provide new insights into the regulatory network of nicotine biosynthesis. Around 20% of plants are known to produce alkaloids, a diverse class of more than 12,000 different N-containing natural product compounds which are important for plant defense and medical use 1,2. Cultivated tobacco (Nicotiana tabacum), as a natural allotetraploid, is possibly evolved from hybridization of two diploids N. tomentosiformis and N. sylvestris 3,4. Nicotine accounts for around 90% of total alkaloids in tobacco, which made tobacco as a model plant to study the biosynthesis, transportation, accumulation, and degradation of alkaloids. Extensive studies have established that nicotine is produced in roots before being transported to and accumulating in leaves 5-7. Nicotine functions as a defense toxin to many insect herbivores 8. Mechanical wounding and woundingelicited jasmonate signaling are known to increase the accumulation of nicotine 9-11. For instance, substantially increased nicotine synthesis and accumulation is known to result from the agricultural practice known as topping, wherein the early developing inflorescence organs are removed by producers to prevent seed production 12,13. Nicotine is synthesized from putrescine, which is derived from arginine or ornithine 14. Most of the enzymes involved in this pathway have been identified and characterized in the course of many decades of research, including ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT), aspartate oxidase (AO), quinolinate synthase (QS), quinolinate phosphoribosyl transferase (QPT), N-methylputrescine oxidase (MPO), and berberine bridge enzyme-like (BBL), among others 4,6,14. There has also been extensive research about the genetic control
“…3 A). We detected two peaks at 21 and 24 nt in most samples, indicating that small RNAs of 21 and 24 nt are the two major size classes in tobacco, consistent with a previous study in this species 12 , 13 , 34 , 39 and with reports for Arabidopsis and rice 49 , 50 . Figure 3 Expression pattern of conserved and novel miRNAs.…”
Section: Resultssupporting
confidence: 92%
“…3A). We detected two peaks at 21 and 24 nt in most samples, indicating that small RNAs of 21 and 24 nt are the two major size classes in tobacco, consistent with a previous study in this species 12,13,34,39 and with reports for Arabidopsis and rice 49,50 . After removing small RNA reads originating from other small non-coding RNAs (including tRNA, rRNA, snRNA, and snoRNA), the left reads were aligned to the tobacco reference genome 30 .…”
Section: Resultssupporting
confidence: 92%
“…As development of sequencing techniques and improvement of tobacco genome [30][31][32][33] , several studies have used small RNA sequencing to profile miRNAs in tobacco 12,13,[34][35][36][37][38] . These studies have revealed insights including characterization of five miRNA families that exhibit differential accumulation profiles under drought conditions 39 , and a functional demonstration that miR164 targets mRNA transcripts of NtNAC-R1 and that such targeting increases upon topping and leads to multiple root-related phenotypes 40 .…”
tobacco (Nicotiana tabacum) is considered as the model plant for alkaloid research, of which nicotine accounts for 90%. Many nicotine biosynthetic genes have been identified and were known to be regulated by jasmonate-responsive transcription factors. As an important regulator in plant physiological processes, whether small RNAs are involved in nicotine biosynthesis is largely unknown. Here, we combine transcriptome, small RnAs and degradome analysis of two native tobacco germplasms YJ1 and ZY100 to investigate small RNA's function. YJ1 leaves accumulate twofold higher nicotine than ZY100. Transcriptome analysis revealed 3,865 genes which were differently expressed in leaf and root of two germplasms, including some known nicotine and jasmonate pathway genes. By small RNA sequencing, 193 miRNAs were identified to be differentially expressed between YJ1 and ZY100. Using in silico and degradome sequencing approaches, six nicotine biosynthetic genes and seven jasmonate pathway genes were predicted to be targeted by 77 miRNA loci. Three pairs among them were validated by transient expression in vivo. Combined analysis of degradome and transcriptome datasets revealed 51 novel miRNA-mRNA interactions that may regulate nicotine biosynthesis. The comprehensive analysis of our study may provide new insights into the regulatory network of nicotine biosynthesis. Around 20% of plants are known to produce alkaloids, a diverse class of more than 12,000 different N-containing natural product compounds which are important for plant defense and medical use 1,2. Cultivated tobacco (Nicotiana tabacum), as a natural allotetraploid, is possibly evolved from hybridization of two diploids N. tomentosiformis and N. sylvestris 3,4. Nicotine accounts for around 90% of total alkaloids in tobacco, which made tobacco as a model plant to study the biosynthesis, transportation, accumulation, and degradation of alkaloids. Extensive studies have established that nicotine is produced in roots before being transported to and accumulating in leaves 5-7. Nicotine functions as a defense toxin to many insect herbivores 8. Mechanical wounding and woundingelicited jasmonate signaling are known to increase the accumulation of nicotine 9-11. For instance, substantially increased nicotine synthesis and accumulation is known to result from the agricultural practice known as topping, wherein the early developing inflorescence organs are removed by producers to prevent seed production 12,13. Nicotine is synthesized from putrescine, which is derived from arginine or ornithine 14. Most of the enzymes involved in this pathway have been identified and characterized in the course of many decades of research, including ornithine decarboxylase (ODC), putrescine N-methyltransferase (PMT), aspartate oxidase (AO), quinolinate synthase (QS), quinolinate phosphoribosyl transferase (QPT), N-methylputrescine oxidase (MPO), and berberine bridge enzyme-like (BBL), among others 4,6,14. There has also been extensive research about the genetic control
“…A total of 63.34%, 61.38%, 62.01%, 64.46%, 62.25%, and 66.7% of the valid reads in the CO-1, CO-2, CO-3, HTS-1, HTS-2, and HTS-3 libraries, respectively, mapped to the reference Nile tilapia genome. The somewhat low percentage of mapped reads may be attributed to the specificity of gene expression in different tissues and/or some of the unmapped reads may represent incompletely sequenced regions of the reference Nile tilapia genome 21 . We screened 15 and 26 DE miRNAs and mRNAs, respectively, involved in the GIFT response to heat stress and verified them by qRT-PCR.…”
Genetically improved farmed tilapia (GIFT, Oreochromis niloticus) are commercially important fish that are cultured in China. GIFT are highly susceptible to diseases when exposed to high temperatures in summer. Better understanding the GIFT regulatory response to heat stress will not only help in determining the relationship between heat stress signalling pathways and adaption mechanisms, but will also contribute to breeding new high-temperature tolerant strains of GIFT. In this study, we built control (28 °C) and heat-treated (37.5 °C) groups, and extracted RNA from the liver tissues for high-throughput next-generation sequencing to study the miRNA and mRNA expression profiles. We identified 28 differentially expressed (DE) miRNAs and 744 DE mRNAs between the control and heat-treated groups and annotated them using the KEGG database. A total of 38 target genes were predicted for 21 of the DE miRNAs, including 64 negative miRNA–mRNA interactions. We verified 15 DE miRNA–mRNA pairs and 16 other DE mRNAs by quantitative real-time PCR. Important regulatory pathways involved in the early response of GIFT to heat stress included organism system, metabolism, and diseases. Our findings will facilitate the understanding of regulatory pathways affected by acute heat stress, which will help to better prevent heat damage to GIFT.
“…qRT-PCR reactions were performed using FastStart Universal SYBR Green Master (Roche, China) on a BIOER FQD-96A instrument. The tobacco actin gene was used as reference gene for the normalization of reactions in the mRNA analysis 83 , and a tobacco U6 sequence was used as the reference for data normalization in the miRNA analysis 36 . The relative expression of each sample was calculated using the 2-ΔΔCT method 84 .…”
3-Acetonyl-3-hydroxyoxindole (AHO) induces systemic acquired resistance (SAR) in Nicotiana. However, the underlying molecular mechanism is not well understood. To understand the molecular regulation during SAR induction, we examined mRNA levels, microRNA (miRNA) expression, and their regulatory mechanisms in control and AHO-treated tobacco leaves. Using RNA-seq analysis, we identified 1,445 significantly differentially expressed genes (DEGs) at least 2 folds with AHO treatment. The DEGs significantly enriched in six metabolism pathways including phenylpropanoid biosynthesis, sesquiterpenoid and triterpenoid biosynthesis for protective cuticle and wax. Key DEGs including PALs and PR-10 in salicylic acid pathway involved in SAR were significantly regulated. In addition, we identified 403 miRNAs belonging to 200 miRNA families by miRNA sequencing. In total, AHO treatment led to 17 up- and 6 down-regulated at least 2 folds (Wald test, P < 0.05) miRNAs (DEMs), respectively. Targeting analysis implicated four DEMs regulating three DEGs involved in disease resistance, including miR156, miR172f, miR172g, miR408a, SPL6 and AP2. We concluded that both mRNA and miRNA regulation enhances AHO-induced SAR. These data regarding DEGs, miRNAs, and their regulatory mechanisms provide molecular evidence for the mechanisms involved in tobacco SAR, which are likely to be present in other plants.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
