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 light. The reaction is very fast, taking place in the order of ms to s3, leading to recognizable chlorophyll accumulation within minutes after etiolated seedling irradiation4,5,6. More time (from hours to days) is required for chloroplast biogenesis to establish a fully functional photosynthetic apparatus3.
Various methods exist to analyze chlorophyll content, including high-performance liquid chromatography (HPLC) or spectrophotometry. Usually, these techniques demand the destruction of plant tissue4,5,6, restricting the determination of changes in chlorophyll levels over time. Methods allowing non-invasive chlorophyll kinetics establishment may open a whole new perspective to study plants in diverse aspects ranging from fundamental research questions, such as analyzing the process of chlorophyll synthesis in time and space, to more practical applications, such as assessment of stress tolerance or effect of biostimulants on the chlorophyll kinetics. Considering this, we introduced a system for monitoring chlorophyll formation, iReenCAM7. It incorporates a CCD camera, emission filters, light sources, and a pipeline for automated fluorescence analysis (Figure 1). The main feature of the developed device is high spatial and temporal resolution, outperforming in the parameters used in current approaches, and sufficient sensitivity and specificity when compared with standard analytical methods7.
The non-invasive procedure described here requires minimum reagents and comprises simple steps, allowing to obtain a chlorophyll kinetics profile in living Arabidopsis seedlings during very early stages of de-etiolation. The protocol can be useful for the study of highly dynamic process of chlorophyl synthesis influenced by number of factors, both exogenous (salt, drought, biostimulants, heavy metals, etc.) and endogenous (typically associated with changes in the gene activity) in origin without a need to detach any plant tissue, thus avoiding additional stress.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
This work was supported from the European Regional Development Fund-Project SINGING PLANT (No. CZ.02.1.01/0.0/0.0/16_026/0008446). This project has received funding through the MSCA4Ukraine project (ID 1233580), which is funded by the European Union. We are grateful to Lenka Sochurkova for the graphical design of Figure 1.
Materials
| 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 |
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