A fast, adaptable piecewise gradient method for high-throughput quantification of leaf carotenoids using RP-HPLC-PDA

Abstract

Lutein and b-carotene are the major carotenoids in plant leaves, playing crucial roles in photosynthesis.These pigments also have important applications in human and animal health; therefore, it is essential to accurately determine their quantities in crops. A high-throughput RP-HPLC technique was developed for quantifying the levels of these carotenoids in leaf tissue using a piecewise gradient system. The analysis time was reduced to only 10 min, including column re-equilibration, through the use of a short, efficientPoroshell column with an HPLC-PDA instrument. The method was successfully validated, resulting in sub-mg mL À1 quantification limits for both analytes. To demonstrate the adaptability of the method, application to both saponified and unsaponified leaf samples was performed. The piecewise gradient allows for the customizability of the mobile phase selectivity towards specific sample components and can potentially be adapted to screen multiple tissues of plant varieties and mutants with more extensive carotenoid profiles.

Alfalfa response to heat stress is modulated by microRNA156

“…Leaf chlorophyll was extracted thrice from 50 mg of ground leaf tissue using 2:1:1 hexane:acetone:ethanol and reconstituted in 5:4:1 acetonitrile:dichloromethane:methanol with 0.5% butylated hydroxytoluene (BHT) after evaporation under a stream of nitrogen (Kormendi et al ). Non‐enzymatic antioxidants were extracted from 100 mg leaf tissue for the Trolox equivalent antioxidant capacity (TEAC) assay twice using 1:1 methanol:water and evaporated by vacuum (Li et al ).…”

“…To determine chlorophyll content, 5 μl of reconstituted chlorophyll/carotenoid extracts was filtered into 2‐ml high‐performance liquid chromatography (HPLC) vials and analyzed by HPLC using an Agilent 1200 Series HPLC, and separated on a Poroshell 120, EC‐C18, 4.6 × 75 mm, 2.7 μm particle size column with a Poroshell 120 Fast guard column (Agilent Technologies, Mississauga, Canada). Chlorophyll HPLC mobile phase gradients were run according to Kormendi et al (). Chlorophylls were monitored at 645 and 663 nm, and concentrations were determined based on peak area ratios (Ni et al ).…”

COP9 signalosome subunit 5A affects phenylpropanoid metabolism, trichome formation and transcription of key genes of a regulatory tri-protein complex in Arabidopsis

“…Carotenoids extracted in triplicate batches from Arabidopsis seed or 14-d seedling leaves were analysed as described by Kormendi et al [ 46 ] with minor modifications. Briefly, 50 mg frozen seeds or leaf tissues were ground using RETCH-mill (Retsch Gmbh, 42,787 Haan, Germany) and extracted twice using 750 μL of extraction solvent (2:1:1 hexanes:acetone:ethanol).…”