Sodium Metabisulfite Suppresses Stoichiometry of Phenolic Compounds in Oil Palm Leaves for Flow Cytometric DNA Content Analysis

MATERIALS AND METHODS

Plant materials: One-year-old seedlings of oil palm cv. Hybrid Tenera (Suratthani 1, Deli x Calabar, 2: Deli x La Me and 7, Deli x EKONA) were obtained as plant samples from the Suratthani Oil Palm Research Center, Surat Thani Province, Thailand.

Extraction: Phenolic and tannin contents in unopened leaves (UOL) and fully opened leaves (FOL) of oil palm were extracted followed by Narayanaswamy and Balakrishnan¹¹. After the extraction, the elute was filtered using a Whatman No. 1 filter paper prior to evaporating until the crude extract was obtained.

Analysis of types and amounts of phenolics: Total phenolic content analysis of oil palm UOL and FOL extracts was done by the Folin-Ciocalteu (FC) Method¹². The results were expressed in gallic acid and tannic acid equivalents as milligrams per milliliter standard equivalents. Gallic acid (Sigma, G7384), tannic acid (Sigma, 403040), apigenin (Sigma, 42251) and luteolin (Sigma, 72511) were used as the standards. Folin-Ciocalteu reagent that is phenol reactive (Sigma-Aldrich, E9252) and sodium carbonate (J.T. Baker, 2024) were used.

High-Performance Liquid Chromatography (HPLC) analysis of phenolic content: Qualitative and quantitative data of phenolic components in UOL and FOL of oil palm were determined by using HPLC. Twenty milliliters of gallic acid (100 mg L^–1) and tannic acid (1,000 mg L^–1) were dissolved in methanol and used as standard. Chromatographic experiments were conducted on an Agilent 1200 series DAD HPLC system using a C18 Hypersil ODS (5 μm particle size) (Supelco) column (250×4.0 mm). The solvents used to develop the gradient were 0.1% of acetonitrile (A) and H₃PO₄ (Fisher Scientific) phosphoric acid (B). The solvent gradient in volumetric ratios of solvents A and B was as follows: 0-45 min, 8A/ 92B, 45-50 min, 22A/78B, 50 min and 8A/92B. Detection was performed using 280 nm excitation whereas the flow rate was set to 1.0 mL min^–1. Concentration of each phenolic compound was calculated based on the retention times and calibration constants for every wavelength.

Analysis of phenolic compound suppression: To inhibit phenolic compound effects in vitro, 5-20 mM sodium metabisulfite was used as chemical inhibitor added to UOL and FOL extracts. Then, quantitative data of entire phenolic contents in UOL and FOL extracts were collected using HPLC.

Applying sodium metabisulfite and flow cytometry to determine the DNA content and genome size of oil palm seedlings: Oil palm seedlings had been cultivated for three years. Seeds of maize (Zea may CE-777)¹³ for use as an external reference plant were kindly provided by Dr. Jaroslav Dolezel (Institute of Experimental Botany, Olomouc, Czech Republic).

For FCM, approximately 50 mg of oil palm leaves were prepared and chopped in 1 mL of LB01 buffer and in modified LB01 plus by 10-30 mM sodium metabisulfite. The following parameters were analyzed, the relative fluorescence intensity of PI-stained nuclei (FL), half G0/G1 peak coefficient of variation (%CV), debris factor (%DF) and yield intact nuclei factor (YF)⁷. The DNA contents and haploid genome of oil palm samples were estimated¹⁴.

Statistical analysis: The mean values were subjected to ANOVA using SPSS 11 software (IBM Corp., Armonk, New York, USA). Tukey’s Test was used for multiple means comparison at a 5% significance level. Phenolic content, all FCM parameters, DNA content and genome size are expressed as Mean±SD.

RESULTS AND DISCUSSION

Total phenolic content analysis of one-year old oil palm leaves: The UOL and FOL from one-year-old oil palm seedlings were compared using absorbance measurement at 765 nm. The comparison of total phenolic contents showed remarkable differences by cultivar and by stage of the leaves. The main group of phenolic compounds found in oil palm leaves was tannins. Total tannin content in oil palm leaves had the rank order FOL>UOL (Table 1). Phenolic compounds are reported to be present in oil palm leaves¹⁵ and consequently lead to further effects on stoichiometric oxidation in FCM for plant analysis by affecting the staining of nuclei with fluorescence dye¹⁶. According to our results, the total phenolic content in oil palm leaves differed between UOL and FOL. However, the types of and total phenolic components in oil palm leaf samples were comparatively low relative to prior studies. This might be due to organic solvents and methods for quantitative and qualitative analyses differing from those of earlier studies. In previous works, the tannic acid amount has been reported as 165 mg/g/DW accounting for 26.57% of quantitative analysis of oil palm leaves¹⁷. In this study, types and amounts of phenolic compounds in oil palm leaves (found to be tannins and tannic acid) ranged within 23-78 mg L^–1 of standard solution, respectively.

HPLC analyses of phenolic components in leaves of one-year-old oil palm: The results revealed that apigenin and luteolin flavonoids were not detected in extracts of UOL or FOL oil palm leaves. For gallic acid and tannic acid, the standard peaks were observed at 3 and 10 min into the chromatogram, respectively. Representative chromatograms of the standard gallic acid and tannic acid were shown in Fig. 1a-b. The 0.1% acetonitrile and phosphoric acid used in the ratio of 8:92 (v/v) was found to give sharp peaks of gallic acid and tannic acid standard. The HPLC chromatograms of UOL and FOL extracts of oil palm seedlings revealed the presence of tannic acid (t_R = 15.88 min for B and t_R = 16.39 min for C, respectively) (Figures not shown). Concentrations of the two phenolic compounds measured and calculated based on similar retention times as the standards were shown in Table 2. The HPLC is the preferred technique for the qualification and quantification of phenolic compounds¹⁸, while the chromatogram results depend on many factors. The HPLC coupled with other novel techniques gives high sensitivity and the outputs are more reliable than from HPLC only¹⁹. Tahir et al.²⁰ reported the quantities (as mg g^–1) in oil palm leaves of apigenin and luteolin derivatives using LC-ESI-MS/MS by series of types and amounts for both phenolic compounds. According to our results, no gallic acid, apigenin, or luteolin was found in the leaves of oil palm, but there was only tannic acid. Thus, other novel applications should be used that are simple, rapid, environmentally friendly and comprehensive.

For phenolic compounds suppression, as sodium metabisulfite level was increased, the observed concentration of tannic acid continuously decreased (Table 3). The overall tannic acid content was suppressed by up to 50%, especially in the FOL extract and three concentrations of sodium metabisulfite were subjected to further FCM analyses.

Fig. 1(a-b):

Typical HPLC chromatograms of (a) Gallic acid and (b) Tannic acid, using 0.1% phosphoric acid and acetonitrile in gradient flow

Analysis of flow cytometric histogram quality: We reasoned that if oil palm leaves contain phenolic compounds, adding sodium metabisulfite to the buffer could suppress the phenolic compounds’ effects, giving accurate and reliable histograms. The efficiency of the modified buffers was considered via the FL and YF values (larger is better) and the CV (smaller is better) (Table 4). Although DF was higher in the FCM histogram, this histogram quality did not affect DNA content measurements. The FL and CV seemed to affect G0/G1 histogram position more with a modified buffer having sodium metabisulfite than YF and DF (Fig. 2). The increased FL was suitable as a reliable parameter to distinguish histogram positions by a concentration of sodium metabisulfite and this is the main criterion for DNA content calculation.

Our results confirmed that sodium metabisulfite in WPB buffer influenced the lysis buffer to avoid browning in the FCM estimations in all Elaeis guineensis leaf samples¹⁰. Browning of lysis buffer is undesirable as it degrades buffer properties and interferes with PI staining¹⁶. This might be related to polyphenol oxidase (PPO), which catalyzes the oxidation of several phenols into o-quinones²¹. Sodium metabisulfite is a reducing agent, it can react with o-quinone and form diphenols, so the polymerization of o-quinone into a coloured pigment-or browning-can be prevented^22-23. Thus, it is essential to complement the staining dye with DNA and PI-staining inhibitor.

Fig. 2(a-d):

Histogram of three-year-old E. guineensis cv. Tenera Suratthani 7 analyzed by using LB01 and modified LB01 buffer with gating (M1 represents G0/G1 phase, applied to calculate DNA content, whereas M2 represents G0/G1, S and G2/M phases, applied to draw region of intact nuclei, (a) LB01, (b) LB01+10 mM sodium metabisulfite, (c) LB01+20 mM sodium metabisulfite and (d) LB01+30 mM sodium metabisulfite

DNA content analysis of a three-year-old oil palm tree: We found that the effects of sodium metabisulfite concentration in tannic acid suppression can be attributed to the remaining quantity of tannic acid (decreased by 50% in FOL). Since sodium metabisulfite caused an increase in FL and a decrease in CV, while LB01 based buffer responded with a decreased FL and an increased CV, the nuclear DNA content was increased with the dose level of sodium metabisulfite. We hypothesized that the expected DNA content of a three-year-old oil palm tree might be similar to the DNA content of oil palm embryos (3.7 pg)¹⁰ that are free from phenolic compounds. The mean 2C DNA content of three-years old E. guneensis cv was shown in Table 5. Tenera Suratthani 7 from the Suratthani Oil Palm Research Center ranging from 3.7 pg (10 and 20 mM sodium metabisulfite) to 3.9 pg (30 mM sodium metabisulfite). Thus, genome size of oil palm had values ranging from 1800 to 1900 bp.

There are many factors affecting FCM estimation, such as components of lysis buffer, type of dyes, chopping procedure and DNA PI-staining inhibitor²⁴. Although the modified LB01 buffers result in high values of FL and give consistent PI-histograms, high YF and low DF were found for the LB01 based buffer. Interestingly, after modifying with 10 mM sodium metabisulfite, the buffer gave the lowest CV at 5%. In general, the CV of the peaks generated in the G0/G1 phase when below 3% is considered acceptable²⁵. The FOL from three-year-old oil palm trees having higher levels of tannic acid than in young or UOL was collected. It is known that the tannic acid²⁶ in FOL could affect FL2-histograms giving elevated CV. The range 3.7-3.8 pg of DNA contents in oil palm cv. Tenera has been reported^2,3,27 and the expected DNA content in three-year-old oil palm cv. Tenera is expected to be similar to the previous results.

Although modifying with sodium metabisulfite showed a negligible effect on DF and YF, higher FL and lower CV influenced the consistency of histograms enabling improved accuracy in DNA content estimates. Only two levels tested (10 and 20 mM sodium metabisulfite) exhibited acceptable YF (0.27 and 0.35 nuclei s^–1 mg^–1) compared to 0.45 nuclei s^–1 mg^–1 of LB01 based buffer, suggesting that very high level of sodium metabisulfite was not a good choice for FCM buffer of oil palm. Similar to current findings in oil palm, the DNA content estimated as 3.7-3.9 pg also falls into the short range as described by previous authors^2-4,27.

CONCLUSION

This study demonstrated the presence of tannic acid in oil palm leaves, which could interfere with FCM resulting in histogram errors affecting DNA content and genome size estimates. The optimization of the FCM protocol was supported by modifying the most suitable/popular lysis buffer with 10-20 mM sodium metabisulfite for plants rich in phenolic compounds, to obtain improved accuracy in DNA content and genome size estimates by FCM.

SIGNIFICANCE STATEMENT

This research documents information on significant modified reducing agent for oil palm FCM analysis, which was analyzed from the exhibited phenolic compounds after suppressed by the reducing agent. Here, we explore an existing of the optimum FCM criteria based on the hypothesis of “if phenolic compounds released from oil palm tissue and appeared in the lysis buffer solution, verifying and suppressing the phenolic compounds could help an effort for development of suitable nuclear lysis buffer for producing FCM peak/2c-DNA content of oil palm”. Our method of analysis provides the fact on the present of tannin while information of tannin inhibition can effectively be used for application in the specific lysis buffer for oil palm FCM analysis.

ACKNOWLEDGMENTS

This study was funded by Prince of Songkla University, project ref. S&T550410S. We are thankful to Associate Professor Dr. Seppo J. Karrila for editing the manuscript and grateful to the Suratthani Oil Palm Research Center for providing samples of Elaeis guineensis cv. Suratthani 1, 2 and 7.