evaluation-photosynthetic-behaviors-simultaneous-measurements-leaf

Evaluation of Photosynthetic Behaviors by Simultaneous Measurements of Leaf Reflectance and Chlorophyll Fluorescence Analyses

Chlorophyll a fluorescence analysis is widely used to measure photosynthetic behaviors in intact plants, and has resulted in the development of many ...

Tohoku University

Radish ( L. var. ), a widely cultivated root vegetable crop, possesses a large sink organ (the root), implying that photosynthetic activity in radish can be enhanced by altering both the source and sink capacity of the plant. However, since radish is a self-incompatible plant, improved mutation-breeding strategies are needed for this crop. TILLING (Targeting Induced Local Lesions IN Genomes) is a powerful method used for reverse genetics. In this study, we developed a new TILLING strategy involving a two-step mutant selection process for mutagenized radish plants: the first selection is performed to identify a BCM line, that is, progenies of M plants crossed with wild-type, and the second step is performed to identify BCM individuals with mutations. We focused on Rubisco as a target, since Rubisco is the most abundant plant protein and a key photosynthetic enzyme. We found that the radish genome contains six  genes and one pseudogene encoding small Rubisco subunits. We screened 955 EMS-induced BCM lines using our newly developed TILLING strategy and obtained six mutant lines for the six  genes, encoding proteins with four different types of amino acid substitutions. Finally, we selected a homozygous mutant and subjected it to physiological measurements.

Mutant selection in the self-incompatible plant radish ( L. ) using two-step TILLING.

Non-photochemical quenching (NPQ) is the most important photoprotective system in higher plants. NPQ can be divided into several steps according to the timescale of relaxation of chlorophyll fluorescence after reaching a steady state (i.e., the fast phase, qE; middle phase, qZ or qT; and slow phase, qI). The dissipation of excess energy as heat during the xanthophyll cycle, a large component of NPQ, is detectable during the fast to middle phase (sec to min). Although thermal dissipation is primarily investigated using indirect methods such as chlorophyll a fluorescence measurements, such analyses require dark adaptation or the application of a saturating pulse during measurement, making it difficult to continuously monitor this process. Here, we designed an unconventional technique for real-time monitoring of changes in thylakoid lumen pH (as reflected by changes in xanthophyll pigment content) based on the photochemical reflectance index (PRI), which we estimated by measuring light-driven leaf reflectance at 531 nm. We analyzed two Arabidopsis thaliana mutants, npq1 (unable to convert violaxanthin to zeaxanthin due to inhibited violaxanthin de-epoxidase [VDE] activity) and npq4 (lacking PsbS protein), to uncover the regulator of the PRI. The PRI was variable in wild-type and npq4 plants, but not in npq1, indicating that the PRI is related to xanthophyll cycle-dependent thermal energy quenching (qZ) rather than the linear electron transport rate or NPQ. In situ lumen pH substitution using a pH-controlled buffer solution caused a shift in PRI. These results suggest that the PRI reflects only xanthophyll cycle conversion and is therefore a useful parameter for monitoring thylakoid lumen pH (reflecting VDE activity) in vivo.

Article	Year
Mutant selection in the self-incompatible plant radish ( L. ) using two-step TILLING. Breeding science\| PubMed ID: 28744180	2017
Physiological validation of photochemical reflectance index (PRI) as a photosynthetic parameter using Arabidopsis thaliana mutants. Biochemical and biophysical research communications\| PubMed ID: 29501490	2018

Kaori Kohzuma

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