A team from the National Centre for Biological Sciences (NCBS), Bengaluru, has found that the rich colour in fruits and leaves of plants are indirectly controlled by specific micro RNAs — miR828 and miR858.
Grape plants bear fruits having colours that can be deep purple or green. This colour is due to compounds called anthocyanins and flavonols, both of which are present in grape fruits.
When the grape plant has a high amount of anthocyanin as compared to flavonol, the fruits are deep purple. When the reverse is true, the grapes are not brightly coloured. The relative abundance of anthocyanin and flavonol is controlled by genes known as the MYB transcription factors. Also referred to as activators, when present in large amounts, they result in dark purple grape, as in the Bangalore Blue variety, and absence correlates with lack of bright colour but high incidence of flavonols as in the Dilkhush grape variety.
Repressor target
“Researchers knew microRNAs can regulate MYBs, but they did not know why such a regulation takes place. They were mostly working with Arabidopsis model where one might not see coloured fruits,” says P V Shivaprasad of the Epigenetics lab in NCBS, where the work was done.
The team found that the microRNAs miR828 and miR858 were also found in abundance when the grapes had dark colour. Hence they figured out that there must be an intermediary repressor which was what the miRNA targeted.
“The microRNA was targeting something that is competing with the activator MYB. This [competing factor] is a repressor of the anthocyanin pathways,” says Varsha Tirumalai, PhD student at NCBS and first author of the paper published in the Journal of Experimental Botany.
Micro RNAs are regulators of gene expression, acting like switches. They decide which protein should be made and how much in a given cell or tissue or an organism. They are tiny, having some 20 to 22 digits of RNA. The miRNA inhibit target RNAs by cutting them into two bits in plants. The miRNAs partner with a protein called Argonaute to do this regulation.
Two experiments
The researchers did two sets of experiments with tobacco plants (Nicotiana tabacum). First, they injected through the stomata in the leaves of the plants, an Agrobacterium culture by which overexpression of the gene in question was achieved. This method of feeding the culture led to a local effect in the particular leaf only. Also the cells in the leaves were not damaged as the injection was done through the stomata. The leaves also overexpressed the gene and changed colour to a reddish shade.
They also did another experiment in which instead of pushing the culture through the stomata they infected an “ex plant”— a scientific term for a piece of the plant from which the whole plant can be grown. They found that the plants grown in this manner had reddish leaves and abundant anthocyanin.
“This was very interesting. I have never seen red-leaved tobacco plants earlier. Abundant flavonols were not produced in tobacco earlier,” says Varsha.
“Anthocyanins and flavonols remove reactive oxygen species that damage DNA, RNA and proteins. Reactive oxygen species are involved in most human diseases,” explains Dr Shivaprasad.
Biofortification
Plants having anthocyanin and flavonol can be generated by controlling the microRNAs affecting them. “We can make more of them in tobacco, which can be extracted easily and used as supplements,” he adds. The genes identified here can be used in biofortification.
The group is trying to patent flavonol engineering. Their next step is to find out how the MYB transcription factor works to make the specific enzymes needed to make flavonols.
