Non-invasive Assay for Chlorophyll Biosynthesis Kinetics Determination during Early Stages of Arabidopsis De-etiolation

Summary

Here, we describe an advanced tool designed for chlorophyll biosynthesis monitoring during the early stages of Arabidopsis seedling de-etiolation. The novel methodology provides non-invasive real-time chlorophyll fluorescence imaging at high spatial and temporal resolution.

Abstract

Chlorophyll biosynthesis is a hallmark of de-etiolation, one of the most dramatic stages in the plant life cycle. The tightly controlled and highly dynamic process of chlorophyll biosynthesis is triggered during the shift from the dark to the light in flowering plants. At the moment when etiolated seedlings are exposed to the first traces of sunlight, rapid (in order of seconds) conversion of protochlorophyllide into chlorophyllide is mediated by unique light-accepting protein complexes, leading via subsequent metabolic steps to the production of fully functional chlorophyll. Standard techniques for chlorophyll content analysis include pigment extraction from detached plant tissues, which does not apply to studying such fast processes. To investigate chlorophyll kinetics in vivo with high accuracy and spatiotemporal resolution in the first hours after light-induced de-etiolation, an instrument and protocol were developed. Here, we present a detailed procedure designed for statistically robust quantification of chlorophyll in the early stages of Arabidopsis de-etiolation.

Introduction

De-etiolation represents the most dramatic phase in the plant life cycle, characterized by a number of morphological changes and complete rearrangement of plant metabolism (from hetero- to auto-tropic)¹. Chlorophyll biosynthesis is a hallmark of light-induced de-etiolation in plants and a very dynamic process. Formation of chlorophyll from dark-produced precursor protochlorophyllide must be tightly coordinated to avoid damage due to reactive byproducts². The protochlorophyllide reduction to chlorophyllide is catalyzed by light-dependent protochlorophyllide oxidoreductases (PORs), unique enzymes activated directly by ligh....

Protocol

1. Medium preparation

1. Prepare the cultivation medium by mixing 0.75 g of gelling agent with 50 mL of sterile deionized water in a glass bottle to achieve a 1.5% (w/v) concentration for one Petri plate (120 x 120 x 17 mm). Gently shake the mixture and then heat it in a microwave until boiling to dissolve the gelling agent (the solution becomes clear).
2. Allow the medium to cool down to 58-60 °C before proceeding to the next steps. All subsequent steps must be performed under steri.......

Discussion

Critical steps of the protocol and troubleshooting - no light and take care of the mask
As highlighted directly in the protocol description above, avoiding even the trace amounts of light both during cultivation of etiolated plants seedlings or just before starting the protocol is of critical importance¹¹. In our setup, we use a dedicated dark chamber located in the walk-in phytotron and separated from the rest of the phytotron with light-tight rotating door (Supplem.......

Materials

Name	Company	Catalog Number	Comments
6-benzylaminopurine	Duchefa Biochemie	B0904.0001
Aluminum foil	Merck	Z691577
Arabidopsis thaliana Col-0 seeds	NASC collection	N1092
Cultivation chamber	PSI		custom made
Dimethilsulfoxid	Thermo Fisher Scientific	042780.AK
Eppendorf single-channeled, variable (100-1000 μL)	Merck	EP3123000063
Gelrite	Duchefa Biochemie	G1101
iReenCAM device	PSI		custom made/prototype
Laboratory bottles, with caps (Duran), 100mL	Merck	Z305170-10EA
Laminar-flow box	UniGreenScheme	ITEM-31156
Linerless Rubber Splicing Tape, 19 mm width, black, Scotch	3M Science. Applied to Life	7000006085
Microcentrifuge tube, 2 mL with lid, PPT, BRAND	Merck	BR780546-500EA
Micropore tape	3M Science. Applied to Life	7100225115
Osram lumilux green l18w/66	Ovalamp	200008833
Petri plates - Greiner dishes, square, 120 x 120 x17mm, vented	Merck	Z617679-240EA
Pipet tips, 1000 μL, Axygen	Merck	AXYT1000B
The Plant Screen Data Analyzer software	PSI		delivered as a part of the iReenCAM