Protocol
Authors
Jose Ignacio Garcia Plazaola, Raquel Esteban
Author Affliations
Dpt. Plant Biology and Ecology, University of the Basque Country (UPV/EHU), Apdo 644, 48080 Bilbao, Spain
Overview
Changes in pigment stoichiometry are a key element in the acclimation of plants to fluctuating environmental conditions. The exact evaluation of the ecophysiological meaning of pigment composition requires the separation and quantification of all carotenoids and chlorophylls. This protocol provides a simple method for the extraction, separation and quantification of all major photosynthetic pigments (carotenoids and chlorophylls). It is based on an acetone extraction followed by reverse phase chromatography (HPLC) and photodiode array detection. It allows a good separation of lutein and its isomer zeaxanthin, and the runtime is relatively short (25 minutes).
Background
Carotenoids are robust molecules, relatively stable during the extraction procedure. Carotenoids are lipophilic molecules, and a non-polar solvent is required for their extraction. A certain amount of water (1-5%) favours the extraction of some xanthophylls. Chlorophylls are not so stable, and high light during the extraction produces phototransformations (standard laboratory illumination is enough to prevent photodegradation of chlorophylls) while the presence of acid traces leads to the formation of pheophytin after the replacement of the Mg++ from the chlorophyll ring by H+. The quantitation of total chlorophylls and carotenoids can be achieved by spectrophotometry, but the separate identification and quantitation of all plant pigments requires the use of a HPLC system. The first technique is used in studies in which the physiological roles of individual carotenoids are not important, while the second one is required whenever carotenoid composition is relevant for the purpose of the research projet. HPLC separates molecules by pumping a mobile phase through a densely packed column. After separation, detection is performed by a photodiode array (PDA) detector that scans the absorbance of the eluent at a range of wavelenghts. Each compound (pigment in this case) is detected as a peak of absorbance over the baseline of the chosen wavelenght.
Materials/Equipment
Mortar and pestle •
Liquid nitrogen •
Liquid nitrogen containers •
Ice or containers to maintain the samples cold •
Acetone HPLC grade with 0.5g/l of CaCO3 •
Absolute ethanol •
Eppendorf tubes •
Epperdorf centrifuge •
1 mL HPLC vials and caps •
0.22 μm PTFT filters and syringes •
HPLC system: two pumps, autosampler, PDA detector, Integration software •
HPLC solvents (ethylacetate, methanol, water, tris buffer, acetonitrile) •
Xanthophyll standards. See figure 1 (some material and equipment picture).
Fig. 1.Some material and equipment for the extraction. See details below in step 2 (figure created by B. Olascoaga)
Units, terms, definitions
HPLC, high-performance liquid chromatography
PDA, photodiode array detector. Detection system able to scan a range of wavelenghts symultaneously
PTFT, Hydrophobic polytetrafluoroethylene filters, acetone resistants.
Procedure
In the following lines a protocol for the carotenoids extraction in leaves of a typical annual mesophyte is described. However, the exact procedure of extraction must be adapted to each species, amount of plant material, and mode of storage.
Step 1
SAMPLE COLLECTION AND STORAGE.
In general, the universal mode of collection of biological samples implies the use of liquid nitrogen to freeze them and the subsequent storage at -80 °C until analysis. An essential requirement is to maintain the cold chain all the time until the chemical compounds are stabilised with the extraction medium. If samples melt, even for a few seconds, irreversible chemical modifications may occur, altering the chemical composition. Alternatively, frozen samples in liquid nitrogen can be lyophilized and stored at room temperature. When liquid nitrogen is not available (as in many remote areas), samples can be desiccated and stored in silica as it is described in Esteban et al (2009). Conservation protocols generate two main types of samples: desiccated or frozen (and hydrated). Both types differ in chemical terms by the presence of water. And this water is very important to facilitate the extraction of some xanthophylls, so when water is not present in samples, a small amount is added to the extraction medium. Thus, for frozen samples, which contain water, extraction is performed with pure acetone (buffered with calcium carbonate), but lyophilized or desiccated samples should be extracted with acetone 95-99%. As an indication, 1 mL of extraction medium is adequate to obtain a good extract of 20 mm2 of leaf tissue (Fig. 2).
Fig. 2. HPLC vials of oak (A), spinach (B), dandelion (C) and seeds (D, E). Note the colour difference between extracts. The adequate colour for a good quantification should be as A, B and C, which corresponds with 28 mm2 in 1 mL.
Detailed procedure:
Step 2
EXTRACTION
– Chill the mortar with liquid nitrogen and immerse the samples.
-Homogenise to powder with pestle. Note that the mortar always must contain liquid nitrogen until acetone is added.
-Add 1 mL of acetone and homogenise with the sample, the mixture is collected in a 2 mL eppendorf tube. 1 mL of acetone is used to clean the mortar and pool with the extract, the volume in the eppendorf is adjusted to 2 mL.
– Centrifuge the tube 5 minutes at a speed of at least 5000 rpm, and collect the supernatant. Pellet can be re-extracted if it contains visible chlorophyll and pooled together with the first extraction.
-Filter extract through a 0.22 μm PTFE filter. The first drops that pass the filter should be discarded to avoid contamination. Fill the HPLC vial and close with a cap. It can be stored in the freezer for some days but it is better to immediately inject the sample in the HPLC.
– Clean the mortar, pestle, syringes, filters and other materials with ethanol and dry them with nitrogen gas.
Step 3
CHROMATOGRAPHY
Several methods have been described for carotenoid analyses. In the present protocol we use the method described by Garcia-Plazaola and Becerril (1999), with the modifications described by García-Plazaola and Becerril (2001), which is optimized for a Waters HPLC system, but can be successfully implemented in any other cromatograph. We advice users to follow the specific instructions of each equipment and to get a minimum training on chromatographic techniques and principles. In Fig. 3 an HPLC system and its components are shown.
Fig. 3. A typical HPLC system and its components. The system is controlled by a computer.
Basic chromatography is carried out on Spherisorb ODS-1 reversed phase column (5-μm particle size; 4.6 × 250 mm) (Waters, Ireland) proceeded by a Nova-Pak C-18 (4 μm; 3.9 ×20 mm) guard column (Waters, Ireland). The column must be preconditioned before first use by flushing with methanol 50: water 50 at a flow rate of 0.5–1 ml min–1 for 24 h. Detection is performed with a PAD 996 detector. in the range 250-700 nm.
To validate the success of the implementation of the protocol it is necessary to obtain a good separation of the isomers lutein and zeaxanthin, which are fundamental molecules in the photosynthetic apparatus (Fig. 5).
Chromatographic conditions are as follows: HPLC solvents:
The mobile phase consists of two components:
- solvent A, acetonitrile: methanol: Tris buffer (0.1 M pH 8) (84:2:14)
- solvent B, methanol: ethyl acetate (68:32) (Polle et al., 2001).
The solvents must be HPLC grade or vacuum filtered through a nylon membrane of 0.2 μm pore size before use (as for the Tris buffer in solvent A) in order to avoid problems in the accuracy of the system. Pigments are eluted using a linear gradient from 100% A to 100% B for the first 12 min, followed by an isocratic elution with 100% B for the next 6 min. This is followed by a 1 min linear gradient from 100% B to 100% A and an isocratic elution with 100% A for a further 6 min to allow the column to re-equilibrate with solvent A prior to the next injection (Fig. 4). The solvent flow rate is 1.2 ml min-1 with working pressures below 1000 psi. The injection volume is 15 μl.
Fig. 4. Solvent A (continuous line) and B (non-continuous line) evolution during the injection.
After pigments separation, a first tentative identification can be done on the basis of the absorption spectra, but the correct identification and quantification requires the use of standards of known concentration. Calibration should be repeated periodically, and it is advisable to add an internal standard (lutein for example) in all sample sets to detect changes in the sensitivity of the detection method. Typically, peaks are detected and integrated at 445 nm for carotenoid and chlorophyll content (Fig. 5).
Fig. 5. HPLC chromatograms showing a typical pattern of pigments in an extract from leaves of a typical mesophyte (Taraxacum officinale) extracted at 445 nm. The name of each pigment is also shown next to the peak: Neo, neoxanthin; Vio, violaxanthin; Ant, antheraxanthin; Lut, lutein; Zea, zeaxanthin; Chl b, chlorophyll b; Chl a, chlorophyll a, ; β-C, β-carotene.
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Table 1. Retention times and spectral maxima for pigments detected in extracts from plants under the chromatographic system described above.
Fig. 6. The spectrum of each carotenoid is shown.
Notes and troubleshooting tips
Time estimation.
The estimated preparation time for the extraction of 24 samples (step 2) is of ~3 hours and ~12 h for the chromatography (step 3)
Notes and trouble shooting tips.
“Normal” pigment values. In a typical non-stressed mesophyte leaf, chlorophyll a+b concentrations should be in the range 200-700 µmol m-2. Carotenoid to chlorophyll ratios should be 30-60 for neoxanthin, 100-220 for lutein, 40-80 for VAZ cycle pigments, 50-100 for α-+β-carotene. If the results fall out of these ranges there is probably a calibration mistake.
Usual problems:
-Samples melted before the extraction. No solution, sample again.
-Samples are too tough. Add a hint of sand to the mortar.
-Lutein and zeaxanthin elute together. Try a slight modification of the amount of water (i.e. Tris buffer) in solvent A.
-There is a high amount of pheophytin (a peak that elutes after β-Car, its spectrum has two maxima at 410 and 660 nm). This means that there are acid traces in the acetone or pigment degradation during storage.
Unusual chromatogram profiles. Most plants show the pattern reported in this protocol, but in some cases chromatograms may differ substantially from that shown in Fig 5, which corresponds to a mesophyte growing in a garden (Taraxacum officinale). Main exceptions to this pattern are:
- Plants or leaves growing in deep shade: may present two unusual features: trans-Neo (whose presence would generate a double peak in Neo), lutein epoxide (detected as a peak close to antheraxanthin) and α-Car (detected as a peak which elutes immediately before β-Car).
- Stressed leaves with red carotenoids. Leaf reddening is usually due to the presence of anthocyanins, which are not extracted with acetone. However in some groups reddening is due to some unusual carotenoids, such as rhodoxanthin in Cryptomeria japonica (Han et al 2003) or Aloe vera (Díaz et al., 1990), and escholtzxanthin in Buxus sempervirens (Ida et al., 1995). These molecules can be easily detected by the presence of unusual peaks in the chromatograms which have their maxima around 500 nm (contrasting with yellow xanthophylls whose peaks are at 450 nm). Red fruits of yew (Taxus baccata) are a good source of rhodoxanthin.
- Presence of lactucaxanthin, unusual xanthophyll which replaces partially Lut in some species (Demmig-Adams and Adams, 1996) generally when grown in shady positions. Lettuce leaves contain significant amounts of this xanthophyll and can be used to confirm its presence in a suspicious chromatogram.
- Senescing leaves and fruits contain large amounts of esterified xanthophylls. This generates chromatograms with dozens of overlapping peaks. Esterification with fatty acids does not alter visible spectrum, but it moves the retention time because of the decrease in the degree of polarity. Saponification with KOH breaks the ester releasing xanthophylls that can be analyzed as described in this paper, but the only way to characterize xanthophylls esters is with a mass detector
- Absence of Neo. This xanthophyll is ubiquitous in all plants and green algae, except in some parasitic plants (e.g. Cuscuta reflexa) (Bungard et al. 1999) and in fruits of some species (Esteban et al, 2010).
- Algae. Characterisation of pigment profiles of algae is not directly addressed by this protocol, but at least some easy tips can be mentioned to identify pigments in macroalgae: Green algae present the same profile as plants, red algae may lack some xanthophylls (this varies depending on the species) and always lack Chl b and Neo, brown algae lack Chl b and Neo and contain fucoxanthin and Chlorophyll c.
Links to resources and suppliers
Pigment standards (DHI): https://c14.dhigroup.com/productdescriptions/phytoplanktonpigmentstandards
Chomatography (Waters): http://www.waters.com
Solvents (Sigma): http://www.sigmaaldrich.com/chemistry/solvents/products.html
Literature references
Bungard RA, Ruban AV, Hibberd JM, Press MC, Horton P, Scholes JD. 1999. Unusual carotenoid composition and a new type of xanthophyll cycle in plants. PNAS, USA 96: 1135-1139.
Demmig-Adams B, Adams WW 1996. Chlorophyll and carotenoid composition in leaves of Euonymus kiatschovicus acclimated to different degrees of light stress in the field. Aust. J. Plant Physiol. 23:649-659.
Diaz M, Ball E, Lüttge U 1990. Stress-induced accumulation of the xanthophyll rhodoxanthin in leaves of Aloe vera. Plant Physiol. Biochem. 28:679-682.
Esteban R, Balaguer L, Manrique E, Rubio de Casas R, Ochoa R, Fleck I, Pintó-Marijuan M, Casals I, Morales D, Jiménez MS, Lorenzo R, Artetxe U, Becerril JM, García-Plazaola JI. 2009. Alternative mehods for sampling and preservation of photosynthetic pigments and tocopherols in plant material from remote locations. Photosynth Res. 101: 77-88.
Esteban R, Olano JM, Castresana J, Fernandez-Marín B, Hernandez A, Becerril JM, García-Plazaola JI. Distribution and evolutionary trends of photoprotective isoprenoids (xanthophylls and tocopherols) within the plant kingdom. Physiol. Plantarum. 135: 379-389.
Esteban R, Olascoaga B, Becerril JM, García-Plazaola JI.2010. Insights into carotenoid dynamics in non-foliar photosynthetic tissues of avocado. Physiol. Plantarum 140: 69-78.
Han Q, Katahata S, Kakubari Y, Mukai Y. 2004. Seasonal changes in the xanthophyll cycle and antioxidants in sun-exposed and shaded parts of crown of Cryptomeria japonica in relation to rhodoxanthin accumulation during cold acclimation. Tree Physiol. 24:609-616.
Ida K, Masamoto K, Maoka T, Fujiwara Y, Takeda S, Hasegawa E. 1995. The leaves of the common box, Buxus sempervirens (Buxaceae), become red as the level of a red carotenoid, anhydroeschscholtzxanthin, increases. J. Plant Res. 108:369-376.
García-Plazaola, JI, Becerril, JM 1999. A rapid HPLC method to measure lipophilic antioxidants in stressed plants: simultaneous determination of carotenoids and tocopherols. Phytochemical Analysis 10, 307-313.
García-Plazaola JI, Becerril, JM 2001. Seasonal changes in photosynthetic pigments and antioxidants in beech (Fagus sylvatica) in a Mediterranean climate: implications for tree decline diagnosis. Australian Journal of Plant Physiology 2, 225-32
Polle JEW, Niyogi KK, Melis A. 2001. Absence of lutein, violaxanthin and neoxanthin affects the functional chlorophyll antenna size of photosystem-II but not that of photosystem-I in the green alga Chlamydomonas reinhardtii. Plant Cell Physiol. 42: 482-491.
Health, safety & hazardous waste disposal considerations
Follow the usual safety rules for handling of liquid nitrogen.
The organic solvents used in the method have the following risks and hazards:
– aceton, acetonitrile, ethyl acetate: highly flammable, harmful
– methanol: highly flammable, toxic
Wear safety glasses and suitable gloves during pigment extraction and solvent preparation.
Extraction should be done udner a fume hood.
Eluent and waste of organic solvents should be collected in a container and disposed of according to the local waste regulations.
Store the solvents and waste in firesafe cabinets or in a storage room for such solvents.