Autoluminescent Plant Technology By Harley Pairan Autoluminescence in

Autoluminescent Plant Technology By: Harley Pairan

Autoluminescence in Bacteria – Bioluminescent Bacteria are light producing bacteria found primarily in water based sources. – Traditional Purposes of Biolumenescence – Attraction of mates – Defense against predators – Warning signals – Bacterial Use of Bioluminescence – Distribution

Biochemistry of bioluminescence – The enzyme luciferase is responsible for catalyzing the chemical reaction of bioluminescence. – The basic reaction for resulting blue/green light: – FMNH 2 + O 2 + R-CHO -> FMN + H 2 O + R-COOH + Light (~ 495 nm) – The lux operon and subsequent lux gene sequences control the synthesis of aldehydes for the reaction

Luminescent Plants of the Past – Most “luminescent” plants have only ever been capable of their luminescence because of an exogenous application of luciferins or Salicylic acid – Long term however these plants saw fading of the luminescence and a need for reapplication if wanting to achieve the desired effect again.

New Technology Development – Dr. Alex Kirchevsky and his team took on the challenge of creating autoluminescent plants – Use two different lines of the Nicotiana tabacum transplastomic plants the lux operon from Photobacterium leiognathi was introduced. – One line: Integration into the rps 12/Trn. V locus of the chloroplast – Second line: Integration into the Trnl/Trn. A locus – The outcome? Those lines with chloroplast integration show autoluminescent phenotypes

Several steps in creating these integrated gene lines. Creation of the p. CAS 3 series vector • The Ca. MV 35 S promoter of p. SAT 4 -MCS replaced by a Prrn promoter • Cloned as Agel/Ncol PCR fragment • Amplified with N. tabacum plastid genomic DNA as the template • Resulting in the p. CAS 3 vector Similar procedures resulted in the ultimate p. CAS 3 -aad. A-LUX vector which was integrated directly into the loci Integrating the vector • Corresponding Homologous Recombination sequences to the rps 12/Trn. V or Trnl/Trn. A loci were amplified • Insertion to flank the lux operon expression cassette Verification for all steps done by DNA sequencing How It Works

Results?

Future Potential – Plants that are capable of autoluminescence have a big future ahead of them – Use for lighting country backroads – Use for cosmetic purposes of lighting yards similar to lawn ornaments without the burden of using electricity – This could however lead to a shift in how predation functions in nature. Is it ethical?

References – (1) Krichevsky, A. ; Meyers, B. ; Vainstein, A. ; Maliga, P. ; Citovsky, V. Autoluminescent plants. PLo. S One 2010, 5 (11), 1– 7. – (2) Meighen EA (1991) Molecular biology of bacterial luminescence. Microbiol Rev 55: 123– 142. – (3) Wani, S. H. ; Sah, S. K. ; Sági, L. ; Solymosi, K. Transplastomic plants for innovations in agriculture. A review. Agron. Sustain. Dev. 2015, 35 (4), 1391– 1430. – (4) Krichevsky, A. Bioluminescent Plants Comprising Bacterial Lux Operon and Methods of Making Same. – (5) Krichevsky, A. (2013). U. S. Patent No. US 20130074221 A 1. Washington, DC: U. S. Patent and Trademark Office.