High-Throughput Analysis of Non-Photochemical Quenching in Crops Using Pulse Amplitude Modulated Chlorophyll Fluorometry

Summary

The protocol introduces a high-throughput method for measuring the relaxation of non-photochemical quenching by pulse amplitude modulated chlorophyll fluorometry. The method is applied to field-grown Glycine max and can be adapted to other species to screen for genetic diversity or breeding populations.

Abstract

Photosynthesis is not optimized in modern crop varieties, and therefore provides an opportunity for improvement. Speeding up the relaxation of non-photochemical quenching (NPQ) has proven to be an effective strategy to increase photosynthetic performance. However, the potential to breed for improved NPQ and a complete understanding of the genetic basis of NPQ relaxation is lacking due to limitations of oversampling and data collection from field-grown crop plants. Building on previous reports, we present a high-throughput assay for analysis of NPQ relaxation rates in Glycine max (soybean) using pulse amplitude modulated (PAM) chlorophyll fluorometry. Leaf disks are sampled from field-grown soybeans before transportation to a laboratory where NPQ relaxation is measured in a closed PAM-fluorometer. NPQ relaxation parameters are calculated by fitting a bi-exponential function to the measured NPQ values following a transition from high to low light. Using this method, it is possible to test hundreds of genotypes within a day. The procedure has the potential to screen mutant and diversity panels for variation in NPQ relaxation, and can therefore be applied to both fundamental and applied research questions.

Introduction

Photosynthesis consists of light absorption, primary electron transfer, energy stabilization, and the synthesis and transport of photosynthetic products¹. Understanding each step is vital to guide efforts to increase crop photosynthetic efficiency. Light affects the rate of photosynthesis, requiring balancing energy supply, in the form of photons, with demand for reducing equivalents. When supply exceeds demand, for example under high-light or during reduced CO₂ fixation caused by stomatal closure, build-up of reducing power increases the probability of reactive oxygen species formation with the potential to damage the photosynthetic....

Protocol

1. Seed planting

1. Choose a field site with fertile, well-drained, but not sandy soil, and with a pH of nearly 6.5. Mark out 1.2 m row plots with 0.75 m spacing by scoring the ground with a hoe.
2. Plant 50 seeds/m of G. max cv. IA3023 at 3 cm depth along each plot at the beginning of the growing season when soil temperatures are between 25 to 30 °C.
   NOTE: For the purpose of screening genetic diversity, it is expected that multiple different genotypes are grown and compared. Plant 2-5 rows per genotype arranged in a random block design. Consideration should be given to whether the climatic conditions suit the grow....

Results

Figure 1A depicts a typical measurement of NPQ in field-grown soybean. Plants were grown in Urbana, IL (latitude 40.084604°, longitude -88.227952°) during summer 2021, with seeds planted on June 5th. 2021. The leaf discs were sampled after 30 days of planting seeds, and measurements were made with the protocol provided (Table 1). F_v/F_m and NPQ values were calculated for each leaf disk (Supplementary Table 4) and NPQ relaxation par.......

Discussion

Careful choice and handling of leaf disks are critical to obtain reliable measurements of NPQ. First, damage to the tissue, such as rough handling with tweezers, will introduce stress, resulting in low values for the maximum quantum efficiency of photosynthesis. Non-stressed plants typically have F_v/F_m values of around 0.83¹⁸, with significant declines indicating a reduction in photosynthetic performance⁹. However, plants grown under .......

Materials

Name	Company	Catalog Number	Comments
24 well tissue culture plate	Fisher Scientific	FB012929	Country of Origin: United States of America
96 well tissue culture plate	Fisher Scientific	FB012931	Country of Origin: United States of America
Aluminum foil	Antylia Scientific	61018-56	Country of Origin: United States of America
Black marker pen	Sharpie	SAN30001	Country of Origin: United States of America
CF imager	Technologica Ltd.	N/A	chlorophyll fluorescence imager Country of Origin: United Kingdom
Cork-borer, 7mm	Humboldt Mfg Co	H9665	Country of Origin: United States of America
FluorImager V2.305 Software	Technologica Ltd.	N/A	imaging software Country of Origin: United Kingdom
iHank-Nose 100-Pack of Premium Nasal Aspirator Hygiene Filters	Amazon	B07P6XCTGV	Country of Origin: United States of America
Marker stakes	John Henry Company	KN0151	Country of Origin: United States of America
Paper scissors	VWR	82027-596	Country of Origin: United States of America
Parafilm	Bemis Company Inc.	S3-594-6	Semi -transparent flexible film Country of Origin: United States of America
Solid rubber stoppers	Fisher Scientific	14-130M	Country of Origin: United States of America