Electronic Journal of Polish Agricultural Universities (EJPAU) founded by all Polish Agriculture Universities presents original papers and review articles relevant to all aspects of agricultural sciences. It is target for persons working both in science and industry,regulatory agencies or teaching in agricultural sector. Covered by IFIS Publishing (Food Science and Technology Abstracts), ELSEVIER Science - Food Science and Technology Program, CAS USA (Chemical Abstracts), CABI Publishing UK and ALPSP (Association of Learned and Professional Society Publisher - full membership). Presented in the Master List of Thomson ISI.
2014
Volume 17
Issue 3
Topic:
Agronomy
ELECTRONIC
JOURNAL OF
POLISH
AGRICULTURAL
UNIVERSITIES
Magnucka E. , Oksińska M. , Lewicka T. 2014. RESORCINOLIC LIPIDS IN WINTER WHEAT GRAINS AND ROOTS, EJPAU 17(3), #12.
Available Online: http://www.ejpau.media.pl/volume17/issue3/art-12.html

RESORCINOLIC LIPIDS IN WINTER WHEAT GRAINS AND ROOTS

Elżbieta G. Magnucka, Małgorzata P. Oksińska, Teresa Lewicka
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland

 

ABSTRACT

This study was carried out to compare contents of resorcinolic lipids both in grains and roots of four winter wheat cultivars, harvested in different parts of Poland. Moreover, under in vitro conditions, the effects of various temperatures on accumulation of these phenolic compounds in roots of tested cereal cultivars were estimated.

Most tested cultivars (Mikula Turnia and Natula) contained similar amounts of alkylresorcinols in their grains, up to 463.6 mg kg-1. The only exception was Satyna, in which kernels the level of resorcinolic lipids was markedly lower than in cultivars from south-eastern Poland (Mikula and Turnia).

In turn, alkylresorcinol occurrence in roots of 10-day-old seedlings of cultivars tested was noticeably different between cultivars and was markedly dependent on temperature conditions. It was found that the lowest tested temperature (18°C) mainly decreased the alkylresorcinol accumulation in wheat roots. Moreover, it is the first report about the presence of these compounds in root system of cereals.

Key words: alkylresorcinols, kernels, root system, Triticum aestivum L..

INTRODUCTION

Wheat is cereal crop that is cultivated worldwide. World economy role of this cereal production is significant both in terms of cultivated land and food supply, feeding and commerce. The role of different secondary metabolites of wheat grains is a subject of numerous studies. Among bioactive compounds, plant-derived phenols have received the attention of researchers in varied fields due to their innumerable biological activities. Phenols are widely distributed in plants and are produced mainly to protect plants from various stress factors, such as UV light, free radicals, or wounds, pathogens and herbivores. One of the major groups of phenolic compounds is resorcinolic lipids. These substances, known also as 5-n-alkylresorcinols (ARs) are a group of amphiphillic 1,3-dihydroxy-5-methylbenzene derivatives, with an odd-numbered alkyl chain at position 5 of the benzene ring (Fig. 1) [11, 12]. They widely occur in various plants, including cereals belonging to the Poaceae family [2, 19, 21]. Cereal alkylresorcinols are mixtures mainly of saturated homologues with 13–29 carbon chains [10, 18]. These phenolic lipids are found in grains of rye, wheat, triticale and barley, in different amounts range from 0.36 to 3.2, from 0.32 to 1.0, from 0.6 to 1,6, and  from 0.04 to 0.2 g kg-1 respectively [19, 25, 27]. Resorcinolic lipids occur in outer parts of kernels. They are located mainly in an intermediate layer of the caryopsis, including the hyaline layer, testa and inner pericarp [13]. Moreover, these phenolic compounds were isolated from cereal seedlings, especially from rye coleoptiles [2, 14, 20].

Fig. 1. General formula of resorcinolic lipids (R: 13–29 carbons in the saturated or unsaturated side chain)

Alkylresorcinols are potent antimicrobials and therefore considered an effective defensive chemical barrier against numerous bacterial and fungal pathogens [22, 23]. In fact alkylresorcinols were found as a strong inhibitors of growth and development of several fungal species including Aspergillus niger, A. parasticus, A. versicolor, Penicillium chrysogenum, P. roqueforti [6, 17], Fusarium culmorum, Rhizoctonia solani, R. cerealis [23], Alternaria alternata, Cladosporium cucumerinum [3], Trichophyton mentagrophytes and Saccharomyces cerevisiae [1]. For this reason they are often called the natural biofungicides [22]. Moreover, resorcinolic lipids isolated from rice seedlings shown allelopathic activities  towards major weeds colonizing rice fields [4].

As phenol metabolism in plants is affected by a variety of stimuli, the accumulation of alkylresorcinols in cereal plants may result not only from a progressive plant infection, but could also be evoked by many diverse biotic (e.g. by the presence of plant growth promoting rhizobacteria [16]) and abiotic factors (e.g. in the absence of light [2, 20], or under variable environmental and agricultural conditions [27]). Therefore, it is very important to know all plant tissues and organs within which these antimicrobial compounds will be accumulated and external stimuli responsible for changes in their level. Such knowledge allows developing new strategies for control of plant pathogens.

This work aimed at study of the presence of alkylresorcinols in both root systems and kernels of various cultivars of winter wheat. In addition, the effect of different temperature conditions on accumulation of these compounds in wheat roots was studied.

MATERIALS AND METHODS

Chemicals
Seed treatment, Vitavax200 FS, was obtained from Chempura Corporation Inc. (USA). Solvents and reagents of the highest available purity were from Polskie Odczynniki Chemiczne POCH S.A. (Gliwice, Poland) and from Chempur (Piekary Śląskie, Poland). Diazonic dye Fast Blue B Zn salt and bacteriological agar were bought from Sigma-Aldrich (USA).

Grain samples
Four cultivars of winter wheat (Triticum aestivum L.), Satyna, Turnia, Mikula, and Natula, were studied. The resistance of the tested cultivars was noticeably different according to cultivar vouchers published by the Central Laboratory for Studies of Cultivable Plants (COBORU; Słupia Wielka, Poland). Seeds were harvested in 2009. Natula and Satyna were provided by Kobierzyce Seed Center and Mikula and Turnia by Plant Growing Station in Mikulice and Kraków of Małopolska Plant Growing Company – HBP LLC.

Treatments and growth conditions
Grains of winter wheat (50 g) were treated with Vitavax 200 FS (a.i. carboxin 200 g mL-1 + thiram 200 g mL-1) according to the label information provided by producer.

Afterwards seeds were transferred to Petri dishes (diameter of 150 mm) containing 50 mL of 0.3% water agar (autoclaved and nutrient free). Four dishes (12.5 g seeds per each plate) made one repetition/treatment. Closed plates were incubated in darkness at three different temperatures: (18 ± 2), (24 ± 2) and (30 ± 2)°C. Roots of wheat seedlings were collected after 10 days of culturing and dried at 55 ± 5°C for 24 h. Then, their dry (DW) weight was determined gravimetrically. Experiment was carried out in triplicate.

Isolation and purification of alkylresorcinols
Alkylresorcinols were extracted from both whole grains (10 g) and dried roots of wheat with equal volumes of acetone for three times by 24 hrs. In the case of root material an acidified acetone was used (acetone + 0.25% (v/v) acetic acid). Each acetone fraction was filtered through filter paper to remove any solid particles. All three acetone filtrates were combinated and the solvent was removed by vacuum evaporation on a rotavapor at 40°C. The oily residue was diluted in n-propranol and organic fraction was concentrated in vacuo. Obtained residiue was dissolved in 0.5 mL chloroform and then applied on a 20 × 20 cm preparative TLC plate covered with silica gel Si60 (Merck, Germany). Separation was carried out in gradually, first in chloroform/ethyl acetate (82:25, v/v), then in n-hexane/ethyl ether/formic acid (70:30:1, v/v). Afterwards, 1-cm-wide strip of gel of the plate was sprayed with aqueous 0.05% (w/v) Fast Blue B Zn salt. Part of the gel containing these compounds was scrapped off the plate and re-extracted overnight with a mixture of acetone/methanol (4:1, v/v). After centrifugation (7500 g, 10 min), the supernatant was concentrated in vacuo. The fraction of pure alkylresorcinols was redissolved in methanol and used for further analysis. Each of the isolations was made in triplicate.

Determination of alkylresorcinol contents
The microcolorimetric method described by Gajda and coworkers [5] was used for quantitative determination of resorcinolic lipids. Alkylresorcinols were quantified by measuring the absorbance at 520 nm against the reagent blank after reaction with 0.05% (w/v) Fast blue B Zn salt in 1% acetic acid freshly diluted 6-fold with methanol. Tested samples of ARs evaporated with a stream of nitrogen gas. After evaporation of solvent to dry residue a portion of 2 mL of the fresh diazonic reagent was added. Reaction mixture was thoroughly vortexed and placed in the darkness for an hour. The content of alkylresorcinols was estimated using a calibration curve based on diluted stock solution of pure 5-n-pentadecylresorcinol (Sigma-Aldrich, USA) in the range from 0.1 to 10 micrograms. All determinations were carried out at least in triplicate.

Chromatographic analyses
Additional identification of resorcinolic lipids was carried out using a set of thin layer chromatography techniques. Composition of the homologues according to the length of the side chain was determined on the basis of reversed-phase thin layer chromatography on RP18 HPTLC plates [10]. The presence and pattern of homologues according to their unsaturation were determined by argentation chromatography on silica gel impregnated with 5% (w/v) AgNO3 in methanol [8]. Isolated, pure, individual saturated homologues were used as standards. After development of chromatograms and evaporation of solvents, the plates were sprayed with aqueous diazonic dye and alkylresorcinol homologues were identified by their characteristic reddish-violet colour and Rf values.

Statistical analyses
The data obtained were processed using Statistica for Windows version 5.1 (StatSoft Ltd, UK). Dixon’s Q test with 95% reliability was applied to the replicate data. Average mean values from each study are presented with standard deviation (SD). Fisher's LSD test was used to assess the differences among the means at 95% level of significance (P = 0.05). Also, the Pearson correlation coefficient was used for the analysis.

RESULTS AND DISCUSSION

Four winter wheat cultivars were analyzed for alkylresorcinol content in their grains. It was demonstrated that all of the tested cultivars (Mikula, Satyna, Turnia, and Natula) contained these phenolic lipids. Results of quantitative analysis of alkylresorcinols in the wheat samples are presented in Table 1. Resorcinolic lipid concentrations found ranged from 419.4 to 463.6 mg kg-1 of dry weight of grains. All values were also within the literature range of that previously mentioned [19], but near the lower end of this range.

Table 1. Resorcinolic lipid concentrations in winter wheat grains
Cultivar
Field location
Content
[mg kg-1] a
Satyna
south-western Poland
419.4 ± 13.8 a
Turnia
south-estern Poland
463.6 ± 15.1 b
Mikula
south-estern Poland
462.3 ± 9.6 b
Natula
south-western Poland
441.0 ± 0.0 ab
a – Mean values expressing concentration of alkylresorcinols (± SD) obtained from three independent experiments.
Values followed by different letters are significantly different at P = 0.05 according to Fishers's LSD test

There is not significant variation between three of four cultivars tested. Alkylresorcinol content in Natula kernels, the cultivar planted in the south-western part of Poland, was similar to cultivars harvested in the south-east region (Mikula and Turnia). In turn, the amount of resorcinolic lipids in Satyna grains, the second cultivar obtained from south-western Poland, was noticeably lower than in cultivars from south-eastern Poland.

It is common knowledge that a biochemical profile of a plant is not exclusively dependent on information included in genome, but is also affected by many external factors, like agronomy or climatic conditions. For example, Zarnowski and coworkers [26, 27] showed that the amount of alkylresorcinols in kernels can be affected also by crop location.

Therefore, all these factors might influence the concentration of alkylresorcinols in grains of cultivars tested and decided about their little diversity.

Moreover, based on chromatographic analyses, saturated compounds with 17, 19 and 21 carbon chains were found in all tested cultivars. According to the literature, these homologues, i.e. 5-n-heptadecylresorcinol (AR C17:0), 5-n-nonadecylresorcinol (AR C19:0), and 5-n-heneicosylresorcinol (AR C21:0) predominated in grains of this cereal [19].

Alkylresorcinols as low-molecular-mass secondary metabolitesplay a fundamental role in the plant defence system against fungal pathogens. Also noteworthy is the fact that specific, previously described location of alkylresorcinols in cereal kernels [13] qualified them to being used as potential biomarkers of whole-grain cereals and cereal products. Determination of these lipids in seeds of cereals is also very important due to increasing consumption of whole–grain as a valuable nutritional components of foods.

We have found the detectable amounts of ARs in roots of wheat seedlings. Effect of temperature on their level in root system of tested wheat cultivars was presented in Table 2. Significant effect of temperature on alkylresorcinol accumulation in roots was observed only for two of four tested cultivars. In the case of cv. Turnia, as the growth temperature increased, the level of these lipids in its roots also rose (r = 0.993). At the lowest tested temperature, however, these compounds were not detected in roots of this cultivar.

Table 2. Effect of temperature on alkylresorcinol content in wheat root system
Cultivar
Alkylresorcinol concentrations [mg kg-1 DW] a
30 ± 2ºC
24 ± 2ºC
18 ± 2ºC
Satyna
8.5 ± 1.5 ab
5.9 ± 0.3 ad
n.d
Turnia
15.2 ± 3.7 c
6.0 ± 2.1 ad
n.d.
Mikula
6.7 ± 1.8 a
10.3 ± 1.3 b
0.9 ± 0.3 e
Natula
6.7 ± 2.1 ad
3.7 ± 0.4 def
6.0 ± 1.3 af
a – Mean values expressing concentration of alkylresorcinols (± SD) obtained from three independent experiments.
Values followed by different letters are significantly different at P = 0.05 according to Fisher's LSD test
n.d. – not detected

By contrast, the AR amount in Mikula seedling roots grown at 24°C was significantly higher than those observed at the others. Additionally, the alkylresorcinol level in roots of seedlings grown 30°C was noticeably higher than that observed at 18°C. No significant effect of temperature on these lipid accumulation was noted only in Natula roots; insignificant differences in AR levels in roots of plants grown at all tested temperatures were noted. In turn, two temperatures studied (30 and 24°C) did not noticeably altered the concentration of alkylresorcinols in Satyna roots. Furthermore, alkylresorcinols were not detected in roots of this wheat cultivar grown at the lowest tested temperature. Interestingly, unlike the shoots of rye seedlings, high temperatures did not decrease the accumulation of these compounds in root system [14, 15].

The low content of alkylresorcinols in root samples, did not allow us for determination of their homologue composition.

It should be emphasized here that it is the first report about the presence of alkylresorcinols in wheat roots. Perhaps the use of acidified acetone as an extraction solvent for this lipids from roots was responsible for this fact. Most of plant phenolic compounds are associated especially with monosaccharides [7]. Among allelochemicals of rice tissue, for example, were glucosides of resorcinols [9]. In turn, these forms could degrade immediately under acidic conditions [7]. In addition, acetone has been reported in the literature as the best solvent to alkylresorcinol extraction [24].

Also some pesticides markedly modified the concentrations of these compounds in plants [14, 15]. So, it is highly probable that treatment of kernels with Vitavax 200 FS also determined the increased accumulation and consequently detection of this compounds in this under-ground part of wheat seedlings. However, the application of this synthetic seed dressing was necessary because standard surface disinfection procedures were ineffective towards tested wheat cultivars exhibiting different level of microbial contamination (date not shown). Therefore, to eliminate the action of various fungi on plant metabolism, especially on alkylresorcinol metabolism, wheat kernels were treated with this chemical preparation. In this manner, the effect on resorcinolic lipid biosynthesis was reduced to only one abiotic factor.

The presence of alkylresorcinols in both kernels and root systems of winter wheat seedlings undoubtedly provide resistance mainly against soil phytopathogens during germination and early stages of this plant development. Furthermore, resorcinolic lipids in roots may play an important role in rhizobacterial – plant interactions. Because plant roots are colonized by different soil microorganisms which, in large degree, decided about growth and development of whole plant. Therefore, their interaction, especially with plant growth promoting rhizobacteria merits special attention. The finding of alkylresorcinol accumulation in root system of different cultivars of wheat, suggested that breeding strategy focus on this antifungal and antibacterial metabolites should be consider.

CONCLUSIONS

Four winter wheat cultivars were analyzed for alkylresorcinol content in their grains. It was demonstrated that all of the tested cultivars (Mikula, Satyna, Turnia, and Natula) contained these phenolic lipids. Results of quantitative analysis of alkylresorcinols in the wheat samples are presented in Table 1. Resorcinolic lipid concentrations found ranged from 419.4 to 463.6 mg kg-1 of dry weight of grains. All values were also within the literature range of that previously mentioned [19], but near the lower end of this range.

Acknowledgment

This work was carried out and financed by a research project of The National Science Centre N N310 729240 entitled The role of wheat alkylresorcinols in plant – microbe interactions [Rola pszenicznych lipidów rezorcynolowych w kształtowaniu zależności typu ryzobakterie – roślina].

REFERENCES

  1. Adawadkar P.D., ElSohly M.A., 1981. Isolation, purification and antimicrobial activity of anacardic acids from Ginkgo biloba fruits. Fitoterapia, 52, 129–135.
  2. Deszcz L., Kozubek A., 2000. Higher cardol homologs (5-alkylresorcinols) in rye seedlings. Biochim. Biophys. Acta, 1483, 241–250.
  3. Droby S., Prusky D., Jacoby B., Goldman A., 1987. Induction of antifungal resorcinols in flesh of unripe mango fruits and its relation to latent infection by Alternaria alternata. Physiol. Mol. Plant Pathol., 30, 285–292.
  4. Fujii Y., Hiradate S., 2007. Allelopathy. New concepts and methodology, Science Publishers, Inc. Enfield, NH, USA.
  5. Gajda A., Kulawinek M., Kozubek A., 2008. An improved colorimetric method for the determination of alkylresorcinols in cereale and whole-grain cereale products. J. Food Comp. Anal., 21, 428–434.
  6. Garcia S., Garcia C., Heinzen H., Moyna P., 1997. Chemical basis of the resistance of barley seeds to pathogenic fungi. Phytochemistry, 44, 415–418.
  7. Garcia-Salaz P., Morales-Soto A., Segura-Carretero A., Fernandez-Gutierrez A., 2010. Phenolic-compound-extraction systems for fruit and vegetable samples. Molecules, 15, 8813–8826.
  8. Kaczmarek J., Tluscik F., 1984. Variability of alkylresorcinol content in rye (Secale cereale L.) grains. A comparative analysis with several species of the genus Triticum. Genet. Polon., 25, 349–358.
  9. Kong C., Xu X., Hu F., Chen X., Ling B., Tan Z., 2002. Using specific secondary metabolites as markers to evaluate allelopathic potentials of rice varieties and individual plants. Chin. Sci. Bull., 47(10), 839–843.
  10. Kozubek A., 1985. Isolation of 5-n-alkyl-, 5-n-alkenyl- and 5-n-alkadienyl-resorcinol homologs from rye grains. Acta Alimen. Polon., 9, 185–198.
  11. Kozubek A., Tyman J.H.P., 1999. Resorcinolic lipids, the natural non-isoprenoid phenolic amphiphiles and their biological activity. Chem. Rev., 99, 1–26.
  12. Kozubek A., Tyman J.H.P., 2005. Bioactive phenolic lipids [in:] Studies in Natural Products Chemistry, 30, 111–190.
  13. Landberg R., Kamal-Eldin A., Salmenkallio-Marttila M., Rouau X., Amna P., 2008. Localization of alkylresorcinols in wheat, rye and barley kernels. J. Cereal Sci., 48, 401–406.
  14. Magnucka E., Suzuki Y., Pietr S.J., Kozubek A., Zarnowski R., 2007. Effect of norflurazon on resorcinolic lipid metabolism in rye seedlings. Z. Naturforsch.,62c, 239–245.
  15. Magnucka E., Suzuki Y., Pietr S.J., Kozubek A., Zarnowski R., 2009. Cycloate, an inhibitor of fatty acid elongase, modulates metabolism of very-long-side-chain alkylresorcinols in rye seedlings. Pest Manag. Sci., 65, 1065–1070.
  16. Pietr S.J., Kita W., Sowiński J., Nowak W., Biliński Z., Nadziak J., Zarnowski R., 2002. The influence of Cedomon on yield and fungal infection of spring barley in field conditions in Poland. IOBC wprs Bull., 25, 333–336.
  17. Reiss J., 1989. Influence of alkylresorcinols from rye and related compounds on the growth of food-borne molds. Cereal Chem., 66, 491–493.
  18. Ross A.B., Kamal-Eldin A., Jung C., Shepherd M.J., Äman P., 2001. Gas chromatographic analysis of alkylresorcinols in rye (Secale cereale L.) grains. J. Sci. Food Agric., 81, 1405–1411.
  19. Ross A., Shepherd M.J., Schupphaus M., Sinclair V., Alfaro B., Kamal-Eldin A., Aman P, 2003. Alkylresorcinols in cereals and cereal products. J. Agric. Food Chem., 51, 4111– 4118.
  20. Suzuki Y., Saitoh C., Hyakutake H., Kono Y., Samurai A., 1996. Specific accumulation of antifungal 5-alk(en)ylresorcinol homologs in etiolated rice seedlings. Biosci. Biotech. Biochem., 60, 1786–1789.
  21. Verdeal K., Lorenz K., 1977. Alkylresorcinols in wheat, rye and triticale. Cereal Chem., 54, 475–483.
  22. Zarnowski R., Kozubek A., 2002. Resorcinolic lipids as natural biofungicides [in:] Dehne H.W., Gisi U., Kuck K.H., Russel P.E., Lyr H. (Eds.), Modern fungicides and antifungal compounds III, AgroConcept GmbH, Th. Mann Verlag, Bonn, 337–347.
  23. Zarnowski R., Kozubek A., Pietr S.J., 1999. Effect of rye 5-n-alkylresorcinols on in vitro growth of phytopathogenic Fusarium and Rhizoctonia fungi. Bull. Pol. Acad. Sci.: Biol. Sci., 47, 231–235.
  24. Zarnowski R., Suzuki Y., 2004. Expedient Soxhlet extraction of resorcinolic lipids from wheat grains. J. Food Comp. Anal., 17, 649–663.
  25. Zarnowski R., Suzuki Y., 2004. 5-n-alkylresorcinols from grains of winter barley (Hordeum vulgare L.). Z. Naturforsch., 59c, 315–317.
  26. Zarnowski R., Suzuki Y., Pietr S.J., 2004. Alkyl- and alkenylresorcinols of wheat grains and their chemotaxonomic significance. Z. Naturforsch., 59c, 190–196.
  27. Zarnowski R., Suzuki Y., 2004. 5-n-alkylresorcinols from grains of winter barley (Hordeum vulgare L.). Z. Naturforsch., 59c, 315–317.

Accepted for print: 4.08.2014


Elżbieta G. Magnucka
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
Grunwaldzka 53
50-375 Wrocław
Poland
email: elzbieta.magnucka@up.wroc.pl

Małgorzata P. Oksińska
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland
phone/fax: +48 71 320 5621
Grunwaldzka 53
50-375 Wrocław
Poland
email: malgorzata.oksinska@up.wroc.pl

Teresa Lewicka
Agricultural Microbiology Lab, Department of Plant Protection, Wrocław University of Environmental and Life Sciences, Poland

email: teresa.lewicka@up.wroc.pl

Responses to this article, comments are invited and should be submitted within three months of the publication of the article. If accepted for publication, they will be published in the chapter headed 'Discussions' and hyperlinked to the article.