Simple extraction methods that prevent the artifactual conversion of chlorophyll to chlorophyllide during pigment isolation from leaf samples

Abstract

BackgroundWhen conducting plant research, the measurement of photosynthetic pigments can provide basic information on the physiological status of a plant. High-pressure liquid chromatography (HPLC) is becoming widely used for this purpose because it provides an accurate determination of a variety of photosynthetic pigments simultaneously. This technique has a drawback compared with conventional spectroscopic techniques, however, in that it is more prone to structural modification of pigments during extraction, thus potentially generating erroneous results. During pigment extraction procedures with acetone or alcohol, the phytol side chain of chlorophyll is sometimes removed, forming chlorophyllide, which affects chlorophyll measurement using HPLC.ResultsWe evaluated the artifactual chlorophyllide production during chlorophyll extraction by comparing different extraction methods with wild-type and mutant Arabidopsis leaves that lack the major isoform of chlorophyllase. Several extraction methods were compared to provide alternatives to researchers who utilize HPLC for the analysis of chlorophyll levels. As a result, the following three methods are recommended. In the first method, leaves are briefly boiled prior to extraction. In the second method, grinding and homogenization of leaves are performed at sub-zero temperatures. In the third method, N, N’-dimethylformamide (DMF) is used for the extraction of pigments. When compared, the first two methods eliminated almost all chlorophyllide-forming activity in Arabidopsis thaliana, Glebionis coronaria, Pisum sativum L. and Prunus sargentii Rehd. However, DMF effectively suppressed the activity of chlorophyllase only in Arabidopsis leaves.ConclusionChlorophyllide production in leaf extracts is predominantly an artifact. All three methods evaluated in this study reduce the artifactual production of chlorophyllide and are thus suitable for pigment extraction for HPLC analysis. The boiling method would be a practical choice when leaves are not too thick. However, it may convert a small fraction of chlorophyll a into pheophytin a. Although extraction at sub-zero temperatures is suitable for all plant species examined in this study, this method might be complicated for a large number of samples and it requires liquid nitrogen and equipment for leaf grinding. Using DMF as an extractant is simple and suitable with Arabidopsis samples. However, this solvent cannot completely block the formation of chlorophyllide in thicker leaves.

Reexamination of Chlorophyllase Function Implies Its Involvement in Defense against Chewing Herbivores

“…This and previous studies (Hu et al, 2013) show that disruption of plant cells renders chlorophyll accessible to CLH, which immediately begins to hydrolyze the pigment into chlorophyllide. Because chlorophyll hydrolysis is known to occur commonly in the gut of insect herbivores (Park et al, 2003;Badgaa et al, 2014), it is plausible that CLH continues to produce chlorophyllide as the ingested leaf tissue passes through the insect guts.…”

“…gov/ij/). Chlorophyll was extracted from leaf tissue by homogenization with acetone, which was precooled at -30°C (Hu et al, 2013). Extracts were subsequently centrifuged for 5 min at 20,000g at 4°C, and the supernatant was analyzed by HPLC using a symmetry C8 column (150-mm length and 4.6-mm i.d.…”

Simultaneous regulation of antenna size and photosystem I/II stoichiometry in Arabidopsis thaliana

“…Leaves were weighed and ground in pure acetone cooled at -20°C using a Shaker Master (Biochemical Science) (Hu et al 2013). The vessel used for grinding was also pre-cooled in liquid nitrogen.…”

Ekstraksi Dan Karakterisasi Klorofil Dari Daun Suji (Pleomele Angustifolia) Sebagai Pewarna Pangan Alami

“…Selain itu sediaan pewarna yang dihasilkan dari proses ekstraksi berbentuk konsentrat cair yang memiliki kelemahan umur simpan yang pendek (Tama et al, 2014). Ekstraksi klorofil umumnya menggunakan solvent berbasis alkohol seperti aseton (El-Mouhty and ElNagar, 2014; Kong et al, 2014;Hu et al, 2013;Putri et al, 2012;Wu et al, 2002), metanol, etanol, DMF (Hosikian et al, 2010) dan etanol (Yuniwati et al, 2012). Penggunaan solven yang mengandung alkohol dapat menimbulkan keraguan bagi masyarakat muslim di Indonesia.…”