Bio Patrika interviews Dr. Sharma on his thoughts about “CRISPR/CAS9 and regulation of flavonoid biosynthesis in plants”

Mr. Ashish Sharma’s interview with Bio Patrika hosting “Vigyan Patrika”, a series of author interviews. Mr. Sharma is currently working as CSIR-Senior Research Fellow at CSIR-National Botanical Research Institute, Lucknow, Uttar Pradesh. He has completed his PhD under the supervision of Dr. Prabodh Kumar Trivedi, the Director, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow and is waiting to defend his thesis. Mr. Sharma published paper titled “Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis” as the first author in Nature Plants journal (2020).

How would you explain your paper’s key results to the non-scientific community?

Our research published in Nature Plants reports the identification and functional characterization of microRNA encoding peptide (miPEP858a) in model plant Arabidopsis thaliana. Besides, this study also demonstrated the possibility of using CRISPR/Cas9 as an innovative tool for the functional analysis of single members of miRNA families in plants. These peptides have significant potential to control flavonoid biosynthesis. Flavonoids are a class of ubiquitous plant secondary metabolites with numerous health-promoting properties including anti-cancer, anti-inflammatory and anti-viral activities. These are the vital components of the human diet.

“[…] CRISPR/Cas9 as an innovative tool for the functional analysis of single members of miRNA families in plants.”

Nobel Prize 2020 in Chemistry for the Discovery of ‘Genetic Scissors’ called CRISPR/Cas9 awarded to Emmanuelle Charpentier and Jennifer A. Doudna.

To explore the detailed role of these peptides in plants, we generated mutant plants defective in miPEP858a along with mutants of both the members of miR858 family through the CRISPR/Cas9 approach. Interestingly, hindering miR858a activity by editing miPEP858a resulted in the up-regulation of genes involved in flavonoid (flavonols and anthocyanins) biosynthesis at the cost of lignin production.

We also developed transgenic lines overexpressing miPEP858a, which elucidated that this peptide also promotes plant growth and development in association with auxin. We performed complementation studies using synthetic miPEP858a peptide to gain a more in-depth insight into its function. Exogenous application of this peptide to miPEP mutant plants was able to complement the lost function and rescue the growth phenotypes of the peptide, confirming its vital role in regulating miR858a expression. This study adds a new layer of regulation of miRNA and confirms the role of miPEPs in plant growth and development.

Biogenesis of miPEP. Schematic representation of biogenesis of miPEP, miRNA gene transcribed by RNA pol II to form pri-miRNA, which is subsequently processed to form the mature miRNA. A part of pri-miRNA which have functional ORF, encodes small peptide known as microRNA-encoded peptide (miPEP), which increases the transcription of miRNA gene.

What are the possible consequences of these findings for your research area?

Till recently, the biological roles of miRNAs were explained either by over-expressing the miRNA under the control of a strong promoter like CaMV35S or through down-regulating/sequester the miRNA with RNAi/target mimicry approach and studying the phenotypic and molecular analysis of these transgenic plants.

As miPEPs are anticipated to be functional only in cells where the specific encoding miRNA is expressed, this would avoid non-specific phenotypes associated with ectopic expression of miRNAs, which is one of the major disadvantages of the approaches mentioned above. Thus, the application of synthetic miPEPs or their transgenic overexpression will help in functional analysis of individual members of the miRNA families.

The findings of our study also suggest that the exogenous application of miPEPs can evade the tedious and complicated procedures involved in the production of transgenic plants and is also easy to implement. The possibility to use miPEPs in exogenous treatments owing to their specificity provides a new and powerful tool to study the role of miRNAs and can significantly modulate key plant developmental processes with high agronomical important crops like rice, wheat, maize and tomato etc. These findings provide an additional layer of miRNA regulation.

“[…] the exogenous application of miPEPs can evade the tedious and complicated procedures involved in the production of transgenic plants and is also easy to implement.”

What was the exciting moment (eureka moment) during your research?

Establishment of CRISPR/Cas9 in the lab by mutating AtPDS gene (a carotenoid pathway gene), which upon successful editing displayed striking albino phenotype in leaf and over-all plant, that for me was the most exciting moment. Later, the miR858 family and miPEP858a were mutated, and these edited plants displayed remarkable pinkish/purple colour due to enhanced accumulation of anthocyanin (which has medicinal properties) was the most exciting moment. 

What do you hope to do next?

This study elucidated the in-depth role of non-coding RNAs demonstrating that they are capable of producing regulatory peptides that possess significant biological functions, mostly associated with agronomical traits like plant height, seed size, plant growth and development and synthesis of secondary metabolites (flavonoids and anthocyanin).

Our next big aim is to elucidate the in-depth regulatory mechanisms of miPEPs and its cross-talk with other regulatory molecules to gain further insight into how this small peptide regulates miRNAs as well as the crucial biological processes in plants. We are also looking forward to the identification of newer miPEPs and other regulatory small peptides with high agronomical values. 

Where do you seek scientific inspiration?

After the successful editing of PDS for the establishment of CRISPR/Cas9, I felt that I am moving in the right direction, and later, we edited different members of the miR858 family in Arabidopsis thaliana. During that time, research on discoveries of small plant peptides and the mighty roles they play was the new interest to scientists worldwide. These few amino-acids accounts for a drastic change in plant physiology and adaptation motivated me to look for miRNA encoded peptide for miR858. I was incredibly fortunate to identify a functional miPEP and then carried out its detailed analysis. Throughout my research, I was lucky enough to be continuously inspired by my supervisor, Dr. Prabodh Kumar Trivedi, who always taught me that to conduct adequate research; we ever need to have a good imagination, persistence and patience.

How do you intend to help Indian science improve?

This peptide signifies an additional and excellent example of peptides derived from previously known as non-coding RNA with powerful biological functions whose encoding sequences were previously hidden in the genome. The identification of miPEPs unlocks new means of studying the corresponding miRNA families and therefore, would help in improving desired traits and yields in agronomical crops.

Also, exogenous application of synthetic miPEPs stimulates the expression of the corresponding miRNA owing to its specificity. The miPEPs, in this regard, can be used as an alternative tool to enhance the crucial agronomical traits of crops. However, the application of synthetic peptides in fields would be costly but beneficial for plants, which are way difficult to be manipulated and thus avoiding the need for genetic modifications/ cloning and other complex processes.

Applications and potential of miPEPS in crop improvement. miPEPs could be effectively used in crops to improve traits, such as yield, plant architecture and stress tolerance.

Reference

Sharma A, Badola PK, Bhatia C, Sharma D, Trivedi PK. Primary transcript of miR858 encodes regulatory peptide and controls flavonoid biosynthesis and development in Arabidopsis. Nature Plants. 2020. doi: 10.1038/s41477-020-00769-x.

Ashish Sharma email: sharmaashish23march@gmail.com

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