A Functional Analysis of microRNAs in Nicotiana tabacum

MicroRNAs (miRNAs) are a newly discovered class of endogenous post-transcriptional gene regulators that are typically 20-22 nucleotides in length that do not code for proteins. MiRNAs regulate gene expression by either inhibiting protein translation, or by targeting messenger RNAs (mRNAs) for cleavage. MiRNAs are highly evolutionarily conserved and have been found in many plants such as corn, soybeans, rice, and Arabidopsis. Nicotiana tabacum, or cultivated tobacco, is an important economic, agricultural, and research crop that provided approximately $4.4 billion dollars to the United States economy in 2007. Although much genetic research has already been dedicated to tobacco, hardly any research regarding the role of miRNAs has been performed. In this project, the primary objective was to provide a functional analysis of miRNAs in Nicotiana tabacum. First, the expression profiles of miRNAs and their targets were generated for different organs through the use of quantitative real time PCR (qRT-PCR). It was found that all the miRNAs and targets that were tested were differentially expressed throughout different tissue types in tobacco. In particular, miR159 was found to be expressed the highest in all tissue types tested. Secondly, very few reference gene analyses have been performed in tobacco, therefore a reference gene analysis in tobacco was conducted. In this analysis, 12 housekeeping genes were tested for their effectiveness in serving as reference genes for gene expression analyses in tobacco. QRT-PCR was used to quantify the amount of expression of each candidate reference gene, and a new comprehensive reference gene analysis tool, RefFinder, was used to rank the candidate reference genes based on the stability of their expression. The housekeeping genes that show the least amount of variability in expression were deemed appropriate reference genes for use. Through the use of RefFinder, GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and PP2A (protein phosphatase 2A) were identified as being the most relaible reference genes for use in tobacco gene expression studies. Finally, microRNAs have been identified that mediate stress responses in plants to abiotic factors, but there have been few studies conducted on the effects nanoparticles may have on tobacco growth, development, and microRNA expression. To study this, tobacco seedlings were exposed to varying concentrations of aluminum oxide nanoparticles and tested to see which miRNAs were significantly changed in expression. It was found that the growth and development of tobacco seedlings was significantly adversely affected by increasing concentrations of aluminum oxide nanoparticles. In addition, several miRNAs were identified that may play a significant role in mediating plant responses to nanoparticles stress due to the magnitude of up regulation in expression. Overall, by providing a functional analysis of miRNAs in tobacco, these results will further help scientists to understand how plants react to their environment and will allow the further use of miRNA-mediated biotechnology to further improve crop yield and quality.M.S

Effects of Aluminum Oxide Nanoparticles on the Growth, Development, and microRNA Expression of Tobacco (Nicotiana tabacum)

Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al2O3 nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al2O3 nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al2O3 nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al2O3 nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al2O3 nanoparticles in the environment

A Functional Analysis of microRNAs in Nicotiana tabacum

MicroRNAs (miRNAs) are a newly discovered class of endogenous post-transcriptional gene regulators that are typically 20-22 nucleotides in length that do not code for proteins. MiRNAs regulate gene expression by either inhibiting protein translation or by targeting messenger RNAs (mRNAs) for cleavage. MiRNAs are highly evolutionarily conserved and have been found in many plants such as corn soybeans rice and Arabidopsis. Nicotiana tabacum or cultivated tobacco is an important economic agricultural and research crop that provided approximately $4.4 billion dollars to the United States economy in 2007. Although much genetic research has already been dedicated to tobacco hardly any research regarding the role of miRNAs has been performed. In this project the primary objective was to provide a functional analysis of miRNAs in Nicotiana tabacum. First the expression profiles of miRNAs and their targets were generated for different organs through the use of quantitative real time PCR (qRT-PCR). It was found that all the miRNAs and targets that were tested were differentially expressed throughout different tissue types in tobacco. In particular miR159 was found to be expressed the highest in all tissue types tested. Secondly very few reference gene analyses have been performed in tobacco therefore a reference gene analysis in tobacco was conducted. In this analysis 12 housekeeping genes were tested for their effectiveness in serving as reference genes for gene expression analyses in tobacco. QRT-PCR was used to quantify the amount of expression of each candidate reference gene and a new comprehensive reference gene analysis tool RefFinder was used to rank the candidate reference genes based on the stability of their expression. The housekeeping genes that show the least amount of variability in expression were deemed appropriate reference genes for use. Through the use of RefFinder GAPDH (glyceraldehyde 3-phosphate dehydrogenase) and PP2A (protein phosphatase 2A) were identified as being the most relaible reference genes for use in tobacco gene expression studies. Finally microRNAs have been identified that mediate stress responses in plants to abiotic factors but there have been few studies conducted on the effects nanoparticles may have on tobacco growth development and microRNA expression. To study this tobacco seedlings were exposed to varying concentrations of aluminum oxide nanoparticles and tested to see which miRNAs were significantly changed in expression. It was found that the growth and development of tobacco seedlings was significantly adversely affected by increasing concentrations of aluminum oxide nanoparticles. In addition several miRNAs were identified that may play a significant role in mediating plant responses to nanoparticles stress due to the magnitude of up regulation in expression. Overall by providing a functional analysis of miRNAs in tobacco these results will further help scientists to understand how plants react to their environment and will allow the further use of miRNA-mediated biotechnology to further improve crop yield and quality

Average miRNA fold changes in expression in response to aluminum oxide nanoparticles (control, 0.1%, 0.5%, and 1%) that do not fall within the 95% confidence interval.

<p>Average miRNA fold changes in expression in response to aluminum oxide nanoparticles (control, 0.1%, 0.5%, and 1%) that do not fall within the 95% confidence interval.</p

Nanoparticles effect the growth and development of three week old tobacco seedlings.

<p>All physical measurements were averaged for each treatment (control, 0.1% Al₂O₃, 0.5% Al₂O₃, and 1% Al₂O₃). Each average is stated with the standard error associated with that treatment and the significance levels are denoted with the letters, a–d. Different letters dentote statistical significance within the physical measurement.</p

Phenotype of aluminum oxide treated tobacco seedlings.

<p>From left to right, control, 0.1%, 0.5%, and 1% aluminum oxide treated seedlings.</p

Statistically significant average fold changes of microRNA expression levels in tobacco plants exposed to differing treatments of aluminum oxide nanoparticles.

<p>(control, 0.1%, 0.5%, and 1%). Statistical significance is denoted by letters a–d. Different letters signify statistical significance.</p

Effects of aluminum oxide nanoparticles on the growth, development, and microrna expression of tobacco (nicotiana tabacum)

Nanoparticles are a class of newly emerging environmental pollutions. To date, few experiments have been conducted to investigate the effect nanoparticles may have on plant growth and development. It is important to study the effects nanoparticles have on plants because they are stationary organisms that cannot move away from environmental stresses like animals can, therefore they must overcome these stresses by molecular routes such as altering gene expression. microRNAs (miRNA) are a newly discovered, endogenous class of post-transcriptional gene regulators that function to alter gene expression by either targeting mRNAs for degradation or inhibiting mRNAs translating into proteins. miRNAs have been shown to mediate abiotic stress responses such as drought and salinity in plants by altering gene expression, however no study has been performed on the effect of nanoparticles on the miRNA expression profile; therefore our aim in this study was to classify if certain miRNAs play a role in plant response to Al2O3 nanoparticle stress. In this study, we exposed tobacco (Nicotiana tabacum) plants (an important cash crop as well as a model organism) to 0%, 0.1%, 0.5%, and 1% Al2O3 nanoparticles and found that as exposure to the nanoparticles increased, the average root length, the average biomass, and the leaf count of the seedlings significantly decreased. We also found that miR395, miR397, miR398, and miR399 showed an extreme increase in expression during exposure to 1% Al2O3 nanoparticles as compared to the other treatments and the control, therefore these miRNAs may play a key role in mediating plant stress responses to nanoparticle stress in the environment. The results of this study show that Al2O3 nanoparticles have a negative effect on the growth and development of tobacco seedlings and that miRNAs may play a role in the ability of plants to withstand stress to Al2O3 nanoparticles in the environment