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Study Links Heavy Metal Exposure to Altered microRNA Expression in Tobacco

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A common pigment used in food additives, paints, and cosmetics has been shown to impact the growth of tobacco while affecting microRNA expression, suggesting that environmental contaminants can influence non-coding RNAs in plants, according to new research out of East Carolina University.

The work focused on titanium dioxide, or TiO2, a whitening agent used so widely that an estimated 4 million metric tons of it is produced globally each year, according to a paper appearing in Functional and Integrative Genomics.

Due to its heavy use, TiO2 residues are released into the environment, and TiO2 nanoparticles are increasingly becoming a source of concern. For instance, exposure to the nanoparticles was recently classified as a carcinogen, and animal research has linked them to epithelial cell injury and oxidative DNA damage.

Still, little work has been done to examine the effect of TiO2 nanoparticles on plants, and no studies have looked at how they might influence regulatory RNAs.

To address this knowledge gap, a team from East Carolina University used tobacco as a model species to study the how TiO2 affect the growth, development, and miRNA expression of agricultural plants.

Tobacco seeds were sterilized and sowed in a nutritional medium on Petri dishes and then treated with different concentrations of TiO2 nanoparticles. Some seeds were left untreated to serve as controls.

After three weeks of growth, the germination rate of all TiO2-treated and control seeds was recorded, as was the number of leaves per seedling per plate. The seedlings were then removed from the plates so that their root lengths and biomass could be measured.

Total RNA was then isolated from the seedlings, and qRT-PCR was used to detect miRNA expression levels. The scientists focused on 11 miRNAs — miR-156, miR-159, miR-162, miR-167, miR-169, miR-172, miR-393, miR-395, miR-396, miR-398, and miR-399 — which are known to be aberrantly expressed during abiotic stress or play key roles in plant growth and development.

The expression levels of two stress-related genes — alcohol dehydrogenase and alcohol peroxidase — were also evaluated, as were two housekeeping genes for use as reference genes to normalized expression values.

After three weeks of planting, the researchers wrote in their paper, seeds began to germinate with no obvious effects from the TiO2 treatment at low concentrations. However, at higher concentrations, the nanoparticles "significantly affected" the germination rates of the seeds.

At the highest level tested, 5 percent of TiO2 nanoparticles in the medium, germination was completely absent.

"Although tobacco seeds germinated with a higher percentage after exposure to 2.5 percent [of the nanoparticles], it was hard for these seeds to produce roots and fully develop cotyledons," and the seedlings died in about a week, the team noted.

After three weeks, there was a "significant obvious decrease" in the growth and development of plants exposed to the nanoparticles as evidenced by decreases in root length, leaf numbers, and overall biomass.

Roots were the most sensitive to TiO2, according to the paper, with reductions in length at 0.1 and 1 percent concentrations and complete inhibition of appearance and elongation at 2.5 percent.

"An interesting phenomenon is that TiO2 nanoparticle exposure influenced differential root patterns," the investigators wrote in their paper. "Tobacco is a dicot; generally speaking, one seed only produces one major root. However, after exposure to 1.0 percent TiO2, a majority of tobacco seedlings produced two roots, although both were very short."

Leaf size was also impacted, growing much smaller on treated plants versus normal controls, although this effect was mostly observed in plants exposed to higher concentrations of TiO2 nanoparticles. The effects of exposure on biomass, however, were seen at all concentrations.

In terms of the effect of TiO2 on miRNA expression, the researchers found that most were upregulated with increasing nanoparticle concentrations — miR-169, miR-393, miR-395, miR-399, miR-172, and miR-396 — while some were downregulated, including miR-159 and miR-156. Three miRNAs — miR-162, miR-167, and miR-398 — were mostly unaffected as TiO2 nanoparticle concentrations increased.

According to the paper, miR-395 and miR-399 were the most impacted by TiO2, suggesting that they may play important roles in tobacco seedling facing nanoparticle or heavy metal stress. Notably, both have been found by other groups to be upregulated during environmental abiotic stress including drought and salinity.

The addition of TiO2 nanoparticles was also found to upregulate levels of alcohol dehydrogenase and alcohol peroxidase in a concentration-dependent manner.

Overall, the study shows that TiO2 nanoparticles have a "negative impact on tobacco growth and development, and that miRNAs may play an important role in tobacco response to heavy metals/nanoparticles by regulating gene expression," the research team concluded.

And while the study implicates certain conserved miRNAs, additional experimentation with high-throughout deep sequencing or direct cloning will be needed to uncover novel mIRNAs involved in the stress-response mechanism.

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