An in vivo plant platform to assess genes encoding native and synthetic enzymes for carotenoid biosynthesis

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

This chapter provides an overview of how to test enzyme function in plants using candidate genes encoding carotenoid biosynthetic enzymes and/or engineered enzyme versions for synthetic biology applications. A step-by-step guide explains transformation and screening of Agrobacterium tumefaciens containing a binary vector, preparation of inoculum, and transient expression of candidate or retooled enzymes in Nicotiana benthamiana leaf tissue. In planta expression is especially useful for screening candidate carotenoid biosynthetic enzymes because many of the substrates that these enzymes would use are already present in the leaf. In this assay pigments are extracted from leaf discs expressing transgenes and changes in pigment profiles resulting from the activity of these introduced enzymes are scored using High Performance Liquid Chromatography (HPLC). If a transgene product modifies a carotenoid substrate, differences are easily revealed by comparison against wildtype pigment profiles. Additionally, this in planta method presents the researcher with an opportunity to evaluate candidate carotenoid biosynthetic enzyme function in the existing context of N. benthamiana photosynthetic chloroplasts. Photosynthetic phenotypes that arise from changes in pigment composition can be determined by Pulse Amplitude Modulated (PAM) fluorescence measurements, if desired.

Original languageEnglish (US)
Title of host publicationCarotenoids
Subtitle of host publicationCarotenoid and apocarotenoid biosynthesis metabolic engineering and synthetic biology
EditorsEleanore T. Wurtzel, Eleanore T. Wurtzel
PublisherAcademic Press Inc.
Pages489-509
Number of pages21
ISBN (Print)9780323913539
DOIs
StatePublished - Jan 2022

Publication series

NameMethods in Enzymology
Volume671
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Keywords

  • Agrobacterium tumefaciens
  • Carotenoid-modifying enzymes
  • In planta transient gene expression
  • Nicotiana benthamiana
  • Photosynthesis engineering
  • Synthetic biology

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

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