The In Vivo Pattern of Firefly Luciferase Expression in Transgenic Plants

Michel Schneider, David W. Ow, Stephen H. Howell

Plant Mol. Biol., Vol. 14 (1990)

Luciferase works fine in both peroxisomes and chloroplasts. They redirected luciferase to chloroplasts with a transit peptide and got the same light output as peroxisome-localized enzyme. The enzyme itself doesn't care which compartment it's in. But that was with flooded exogenous luciferin. When you're making luciferin endogenously, co-localization with luciferase in the same compartment becomes the whole point.
35S promoter is biased toward stems and roots, not leaves. Confirmed across multiple independent transformants (not position effect noise, real promoter behavior). For your agroinfiltration proof-of-concept this doesn't matter (you're injecting directly into leaves). But for eventual stable transgenics where you want visible leaf glow, you'd want to think about leaf-specific promoters like rbcS.
Promoter choice genuinely controls where the plant glows. Three different promoters gave three distinct patterns that were reproducible and matched mRNA levels. The expression pattern faithfully reflects transcription. Luciferase protein is stable and doesn't get selectively degraded in certain tissues. This means when you eventually optimize, you have real control over spatial expression through promoter selection.
Exogenous luciferin distorts the light pattern. Even though enzyme distribution was promoter-dependent, the actual glow pattern was skewed toward tissues in direct contact with the luciferin solution. Stems and roots glowed brighter than enzyme levels predicted because that's where the substrate physically was. This is the fundamental problem with exogenous delivery.
The co-compartmentalization argument. When substrate is flooding in from outside, it reaches peroxisomes and chloroplasts equally well — compartment doesn't matter. The advantage of targeting your whole pathway to peroxisomes only emerges with endogenous production, where local concentration at the enzyme matters. That's the design logic behind keeping luciferase in its native peroxisomal location alongside your biosynthetic enzymes.