Rosette leaves (40–60 μg) were harvested and weighed to determine the fresh weight (FW) of plants grown under standard conditions. In contrast, rosette leaves from plants exposed to heat stress treatment were harvested, frozen, ground in liquid nitrogen, and lyophilized to determine the dry weight (DW) of leaf materials. TBS intermediates and end-products were extracted from frozen or lyophilized leaf powders in 300–600 μL of ice-cold pigment-extraction buffer (acetone: 0.2 M NH4OH, 9:1, v/v) at −20 °C for at least 1 h. After centrifugation (14,000 g, 20 min, and 4 °C), the supernatant was subjected to high-performance liquid chromatography (HPLC). After removal of the supernatant, non-covalently bound heme was extracted from the pellet using AHD buffer (acetone: hydrochloric acid: dimethylsulfoxide, 10:0.5:2, v/v/v), and centrifuged at 14,000 g for 20 min at room temperature. HPLC analyses were conducted using the Agilent 1100 or 1290 HPLC system equipped with a diode array and fluorescence detectors (Agilent Technologies), essentially as described previously42 (link).
Diode array and fluorescence detectors
Manufactured by Agilent Technologies
Diode array and fluorescence detectors are analytical instruments used in various laboratory applications. They are designed to detect and measure the presence and concentration of specific compounds or analytes in a sample. These detectors utilize principles of diode array spectroscopy and fluorescence emission to provide sensitive and selective detection capabilities.
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2 protocols using diode array and fluorescence detectors
1
Quantification of Tetrapyrrole Intermediates
2
HPLC Analysis of Porphyrin Pigments
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Porphyrins, Pchlide and Chl were extracted in alkaline acetone (9:1, 100% acetone:0.2 M NH 4 OH, v/v) and analyzed by HPLC as described before (Czarnecki et al., 2011; Papenbrock et al., 1999; Richter et al., 2010) . Leaf samples for Pchlide analysis, which were harvested from dark-and light-exposed seedlings, were fixed with steam for 2 min prior to extraction (Koski and Smith, 1948) . Leaf tissues were harvested, frozen, ground in liquid nitrogen and lyophilized to determine the dry weight (DW). Mg porphyrins, Pchlide, Chlide and Chl were extracted from frozen tissue or tissue lyophilized in alkaline acetone at 4°C. After centrifugation, the pellet was resuspended with AHD buffer (acetone:hydrochloric acid:dimethylsulfoxide, 10:0.5:2, v/v/v) and centrifuged at 14 000 ×g for 20 min at room temperature to isolate non-covalently bound heme (Richter et al., 2019) . The extracts were eventually separated and quantified by HPLC using an Agilent 1100 or 1290 HPLC system equipped with a diode array and fluorescence detectors (Agilent Technologies).
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