Chlorophyll analysis has been conducted in numerous studies due to the importance of this pigment in the physiology of plants. Chlorophyll is involved in the absorption and transfer of light energy, and electron transfer, all of which are vital processes in photosynthesis. Chlorophyll content can change in response to biotic and abiotic stresses such as pathogen infection [1], and light stress [2, 3]. Thus, quantification of chlorophyll provides important information about the effects of environments on plant growth [4-8].
Historically, spectroscopic methods have been most frequently used for chlorophyll measurement because they provide a quick, accurate and inexpensive estimation of chlorophyll concentration [9-11]. However, conventional spectroscopic methods, where bulk photosynthetic pigments are measured in the same cuvette, have limitations in their ability to simultaneously measure multiple photosynthetic pigments due to the overlapping absorption spectra of these pigments. For this reason, it has become more common to separate photosynthetic pigments by high-pressure liquid chromatography (HPLC) prior to spectrophotometric analysis [12, 13]. When separating pigments by HPLC, extra care must be taken since HPLC analysis is prone to the artifactual modification of pigments. In particular, cleavage of the phytol chain of chlorophyll molecules readily occurs with the use of common extraction solvents such as 80% acetone [14]. The products of chlorophyll hydrolysis are chlorophyllide and free phytol. Since chlorophyllide has the same absorption spectra as chlorophyll in the visible light spectrum and phytol does not, cleavage of the phytol chain does not affect the values obtained using conventional spectroscopic methods of chlorophyll determination when samples are extracted with organic solvents. However, due to the polar nature of chlorophyllide, it is readily separated from chlorophyll with HPLC, and thus the artifactual formation of chlorophyllide can result in erroneous data using HPLC-based determination of chlorophyll concentration.
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Conversion of chlorophyll to chlorophyllide induced by the extraction agent reduces the apparent concentration of chlorophyll in samples. It is usually difficult to distinguish whether or not the chlorophyllide detected during HPLC analysis is an artifact or a natural product. In fact, chlorophyllide has been considered a natural product in leaves without examining the basis of its formation [15, 16]. In order to avoid possible misinterpretation of chlorophyll levels, it is essential to employ extraction methods that result in a minimal amount of conversion of chlorophyll to chlorophyllide.
It has been reported that the hydrolase enzyme, chlorophyllase (CLH) catalyzes the formation of chlorophyllide during pigment extraction [17] (Figure 1A). This enzyme is unusually stable in high concentrations of organic solvents such as 50-70% aqueous acetone [18, 19]. Higher plants contain one or two isoforms of this enzyme [20] and Arabidopsis has two CLH isoforms encoded by CLH1 and CLH2 genes, respectively [21]. CLH1 encodes the isoform of CLH that accounts for the majority of CLH in Arabidopsis leaves. CLH1 gene expression is significantly upregulated by methyl-jasmonate (MeJA), a phytohormone mediating various biotic and abiotic signaling pathways [22]. In contrast, CLH2 is constitutively expressed and only represents a minor fraction of CLH activity [23]. In the present study, we assessed how much chlorophyllide is formed during pigment extraction compared to the amount that naturally occurs in leaves. In a subsequent analysis, we then examined whether or not CLH is involved in chlorophyllide formation during extraction by comparing its formation in leaves of wild-type and an Arabidopsis mutant which is deficient in CLH activity. Collectively, these experiments indicated that the majority of chlorophyllide detected in extracts obtained using 80% acetone or pure acetone is produced during pigment extraction through the reaction catalyzed by CLH. We also compared three different methods of pigment extraction that were previously reported in literature. Bacon and Holden [17] reported that CLH activity could be suppressed by boiling leaves for a period of 5 min. They also indicated, however, that the boiling treatment also removes Mg2+ from chlorophyll [17]. We found that, in the case of Arabidopsis leaves, CLH can be inactivated and Mg2+ removal from chlorophyll can be reduced when samples were boiled for only 5 sec. In the method of Schenk et al. [23], leaves were first ground into powder in liquid nitrogen and pigments were subsequently extracted in buffered acetone cooled to −20°C. We found this method is very efficient when processing a relatively small number of samples. Finally, we tested the use of N, N’-dimethylformamide (DMF) as an extraction agent to eliminate the formation of chlorophyllide during sample preparation. Although Moran and Porath [24] reported that chlorophyll is stable in this solvent, they did not characterize the effect of DMF on chlorophyllide formation. In our study, DMF was capable of extracting pigments without enabling the conversion of chlorophyll to chlorophyllide in Arabidopsis, however, for the other species which we have tested in this study, DMF cannot completely suppress the activity of CLH. Collectively, all three methods (boiling leaf sample, freezing leaf samples in liquid nitrogen with the use of pre-cooled acetone, and the use of DMF as an extraction agent) were superior to the methods only using 80% or pure acetone for the extraction of photosynthetic pigments. It is important to understand advantages and disadvantages of each method and choose an appropriate one for each plant species and for the purpose of pigment analysis.
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