Abstract
The cultivation of aromatic species to obtain essential oils has great economic importance, presenting an increasing demand from different industrial sectors, especially to menthol mint (Mentha arvensis L.) essential oil, rich in menthol (70-80%). Consortium cultivation has been an important practice in agricultural systems whose land use is necessary, consequently promoting strong competition for light in reduced space. Thus, this study aimed verifying if different light intensities might promote chemical, metabolical and anatomical alterations in menthol mint. Plants were grown in greenhouse at different average of light intensities (137, 254, 406 and 543 µmol photons m2 s1). Samples were collected 43 days after germination and submitted to following analyses: Gravimetric test, photosynthetic pigments, soluble fractions, enzymatic activity, N-total, trichome density and histochemistry and chemometric test based on essential oil chemical profile. Fresh mass gain, trichome density, essential oil content and soluble sugars were positively influenced by light intensity increase. On the other hand, total-N, NO3−-N and pigments content have decreased influenced by light intensity increase. In the secretion from the trichomes, phenolic substances were reported, as well as lipophilic ones in the peltate ones. The increase of oxygenated monoterpenes was favored by light intensity decrease.
Key words
Mentha arvensis; glandular trichomes; histochemistry; essential oil; nitrate reductase
INTRODUCTION
Among various essential oils types, the ones from Mentha genus have been the most requested, mainly from M. arvensis L. (menthol mint, japanese mint, vique) with menthol as its principal component (Croteau et al. 2005CROTEAU RB, DAVIS EM, RINGER KL & WILDUNG MR. 2005. (−)-Menthol biosynthesis and molecular genetics. Naturwissenschaften 92: 562-577., Shasany et al. 2010SHASANY AK, GUPTA S, GUPTA MK, NAQVI A, BAHL JR & KHANUJA PS. 2010. Assessment of menthol mint collection for genetic variability and monoterpene biosynthetic potential. Flavour Fragr J 25: 41-47., Kamatou et al. 2013KAMATOU GPP, VERMAAK I, VILJOEN AM & LAWRENCE BM. 2013. Menthol: A simple monoterpene with remarkable biological properties. Phytochemistry 96: 15-25.). Menthol is an oxygenated monoterpene with a crystalline appearance, with aromatic and medicinal properties, which has a soothing and refreshing effect on the skin and mucous membranes and is, therefore, an ingredient widely exploited by the pharmaceutical and cosmetic industry among others (Mishra et al. 2018MISHRA A, JAIN P, LAL RK & DHAWAN SS. 2018. Trichomes and Yield Traits in Mentha arvensis: Genotype Performance and Stability Evaluation. J Herbs, Spices Med Plants 24: 1-14., Zhao et al. 2018ZHAO Y, DU L-D & DU G. 2018. Menthol, in: Natural Small Molecule Drugs from Plants. Springer Singapore, Singapore, p. 289-294.). In the reason of its high demand, menthol mint essential oil production has been the second greatest worldwide, and second one only to citrus fruits considered as juice industry sub-products (Baser & Buchbauer 2010BASER KHC & BUCHBAUER G. 2010. Handbook of essential oils: Science, technology, and applications. CRC Press, Boca Raton, FL., Bizzo et al. 2009BIZZO HR, HOVELL AMC & REZENDE CM. 2009. Óleos essenciais no Brasil: aspectos gerais, desenvolvimento e perspectivas. Quim Nova 32: 588-594., Kothari 2005KOTHARI R. 2005. The Indian Essential Oil Industry. Perfum Flavorist 30: 46-50.). According to Jain (2017)JAIN MK. 2017. Mentha oil: Global demand lifts mentha oil prices; outlook bullish - The Economic Times [WWW Document]. Bennett, Coleman Co. Ltd. URL https://economictimes.indiatimes.com/markets/commodities/views/global-demand-lifts-mentha-oil-prices-outlook-bullish/articleshow/60062122.cms (accessed 2.2.19).
https://economictimes.indiatimes.com/mar...
total global production of mint essential oil was 48,000 tons between 2016-2017, of which India contributed 80%, followed by China and Brazil with 9% and 7% respectively.
Menthol mint volatiles biosynthesis exclusively occurs in the secretory cells from the capitate and peltate secretory trichomes, as well as genes involved with menthol route synthesis are expressed in these cells, making this a single site where all biochemical reactions in regarding to volatiles synthesis have occurred (McConkey et al. 2000MCCONKEY ME, GERSHENZON J & CROTEAU RB. 2000. Developmental Regulation of Monoterpene Biosynthesis in the Glandular Trichomes of Peppermint. Plant Physiol 122: 215-224., Sharma et al. 2003SHARMA S, SANGWAN NS & SANGWAN RS. 2003. Developmental process of essential oil glandular trichome collapsing in menthol mint. Curr Sci 84: 544-550., Croteau et al. 2005CROTEAU RB, DAVIS EM, RINGER KL & WILDUNG MR. 2005. (−)-Menthol biosynthesis and molecular genetics. Naturwissenschaften 92: 562-577., Tiwari 2016TIWARI P. 2016. Recent advances and challenges in trichome research and essential oil biosynthesis in Mentha arvensis L. Ind Crops Prod 82: 141-148.). Some works have pointed to a positive correlation between the glandular trichomes density and the essential oil yield and content (Gupta et al. 2017GUPTA AK, MISHRA R, SINGH AK, SRIVASTAVA A & LAL RK. 2017. Genetic variability and correlations of essential oil yield with agro-economic traits in Mentha species and identification of promising cultivars. Ind Crops Prod 95: 726-732., Mishra et al. 2018MISHRA A, JAIN P, LAL RK & DHAWAN SS. 2018. Trichomes and Yield Traits in Mentha arvensis: Genotype Performance and Stability Evaluation. J Herbs, Spices Med Plants 24: 1-14., Souza et al. 2016SOUZA MAA DE, SANTOS LA DOS, BRITO DMC DE, ROCHA JF, CASTRO RN, FERNANDES MS & SOUZA SR DE. 2016. Influence of light intensity on glandular trichome density, gene expression and essential oil of menthol mint (Mentha arvensis L.). J Essent Oil Res 28: 138-145.). Thus, the extensive research on the cultivation and elucidation of metabolic pathway responsible for essential oil biosynthesis has made substantial contribution in research focusing on trichomes and production of essential oil (Tiwari 2016TIWARI P. 2016. Recent advances and challenges in trichome research and essential oil biosynthesis in Mentha arvensis L. Ind Crops Prod 82: 141-148.).
Availability of natural light to crop suffers little or no management in agricultural systems, however, inclusion of intensive soil practices promoted an intense dispute over this resource in intercropping systems. Light restriction compromises plant development, as well as may directly affect metabolism and relevant quantitative aspects such as volatiles production (Fernandes et al. 2013FERNANDES VF, DE ALMEIDA LB, FEIJÓ EVR DA S, SILVA D DA C, OLIVEIRA RA DE, MIELKE MS & COSTA LC DO B. 2013. Light intensity on growth, leaf micromorphology and essential oil production of Ocimum gratissimum. Rev Bras Farmacogn 23: 419-424., Shafiee-Hajiabad et al. 2016SHAFIEE-HAJIABAD M, NOVAK J & HONERMEIER B. 2016. Content and composition of essential oil of four Origanum vulgare L. accessions under reduced and normal light intensity conditions. J Appl Bot Food Qual 89: 126-134., Fadil et al. 2016FADIL M, FARAH A, IHSSANE B, HALOUI T, LEBRAZI S, ZGHARI B & RACHIQ S. 2016. Chemometric investigation of light-shade effects on essential oil yield and morphology of Moroccan Myrtus communis L. SpringerPlus 5: 1062.). In addition, there are works showing the effect of light intensity on the biomass and essential oil yield, several metabolites and glandular trichomes density, as well as the relation between them (Fernandes et al. 2013FERNANDES VF, DE ALMEIDA LB, FEIJÓ EVR DA S, SILVA D DA C, OLIVEIRA RA DE, MIELKE MS & COSTA LC DO B. 2013. Light intensity on growth, leaf micromorphology and essential oil production of Ocimum gratissimum. Rev Bras Farmacogn 23: 419-424., Gupta et al. 2017GUPTA AK, MISHRA R, SINGH AK, SRIVASTAVA A & LAL RK. 2017. Genetic variability and correlations of essential oil yield with agro-economic traits in Mentha species and identification of promising cultivars. Ind Crops Prod 95: 726-732., Souza et al. 2016SOUZA MAA DE, SANTOS LA DOS, BRITO DMC DE, ROCHA JF, CASTRO RN, FERNANDES MS & SOUZA SR DE. 2016. Influence of light intensity on glandular trichome density, gene expression and essential oil of menthol mint (Mentha arvensis L.). J Essent Oil Res 28: 138-145.).
In this context, this study aimed verifying if light intensities variations might promote alterations in biomass production, anatomical characteristics, nutritional aspects, influencing volatiles production and quality in menthol mint.
MATERIALS AND METHODS
General aspects and seedlings preparation
Menthol mint (M. arvensis L.) seedlings from IAC 701 variety provided by Linax® (Votuporanga, SP) in a greenhouse at Department of Chemistry from Universidade Federal Rural do Rio de Janeiro (UFRRJ) were cultivated. Plants were produced from shoots middle-third cuttings treated with 2% sodium hypochlorite (10 min), washed with distilled water and fixed on polystyrene support. Part from cuttings was immersed in a Hoagland and Arnon nutrient solution modified by 15 mM NO3 --N at one-fourth strength under constant aeration. After 2 weeks, seedlings by leaves number and root size were standardized. Furthermore, a sample from plants used in this experiment to herborization was subjected and in the herbarium (RBR, Botany Department, UFRRJ) under RBR32886 code was registered.
Hydroponic system and nutrient solution
Seedlings were placed in 2 cm thick Styrofoam plates with the help of synthetic foam, and then allocated on 1.8 L pots connected by flexible caliber tubes to an electromagnetic air compressor at 65 L min-1 flow rate, adjusted at 45 min activity per hour. Hydroponic system was composed by twenty-four pots and in each one two plants were placed. During the experiment a Hoagland and Arnon nutrient modified solution with 2 mM NO3−-N, 2 mM NH4 + -N, 5 mM Ca2 + and 9.5 mM S at one-fourth strength were used (Hoagland & Arnon 1950HOAGLAND DR & ARNON DI. 1950. The water-culture method for growing plants without soil. Calif Agric Exp Stn, p. 1-32.).
Experiment description and treatment
The experiment was carried out in a greenhouse at Chemistry Institute (UFRRJ). The mean temperature ranged from 18°C to 31°C and the mean radiation was 543 µmol photons m-2 s-1 inside, as well as 797 µmol m-2 s-1 outside greenhouse. Three types of share cloths aiming 25, 50 and 75% radiation reduction were used, and the mean radiation was 137, 254 and 406 µmol photons m-2 s-1, respectively. Every day nutrient solution volume and pH value were adjusted to 1.6 L and 5.8, respectively, and radiation (from 400 to 700 nm) was measured by a Basic Quantum meter QMSW from Apogee Instruments® Inc. (Logan, USA) at 9, 12 and 15 hours (local time). Nutrient solution was weekly replaced.
Fresh mass and photosynthetic pigments analyses
After 43 days, harvest was performed and roots, stems and leaves were separated and used for fresh mass analysis. Leaf samples discs with 9 mm diameter (4th node) were immersed in dimethyl sulfoxide and placed in a steam bath at 64°C for 3 hours pigment extraction (Hiscox & Israelstam 1979HISCOX JD & ISRAELSTAM GF. 1979. A method for the extraction of chlorophyll from leaf tissue without maceration. Can J Bot 57: 1332-1334.). The extract was used for a and b chlorophyll, and carotenoids analyses (Wellburn 1994WELLBURN AR. 1994. The Spectral Determination of Chlorophylls a and b, as well as Total Carotenoids, Using Various Solvents with Specttrophotometers of Different Resolution. J Plant Physiol 144: 307-313.).
Soluble fraction analysis and dry matter content
One gram from roots, stems and 4th node from leaves in 80% ethanol was separately homogenized. After chloroform partitioning, soluble fraction for determining NH4+-N, NO3−-N, free amino-N and free sugar levels (3rd and 4th nodes) were weighed and dried in a forced-air oven at 65°C for 72h (Felker 1977FELKER P. 1977. Microdetermination of Nitrogen in Seed Protein Extracts with the Salicylate-Dichloroisocyanurate Color Reaction. Anal Chem 49: 1080., Cataldo et al. 1975CATALDO DA, MAROON M, SCHRADER LE & YOUNGS VL. 1975. Rapid colorimetric determination of nitrate in plant tissue by nitration of salicylic acid. Commun Soil Sci Plant Anal 6: 71-80., Yemm & Cocking 1955YEMM EW & COCKING EC. 1955. The determination of amino-acid with ninhydrin. Anal Biochem 80: 209-213., Yemm & Willis 1954YEMM EW & WILLIS AJ. 1954. The estimation of carbohydrates in plant extracts by anthrone. Biochem J 57: 508-514., respectively), then grounded and weighed for evaluating total-N and phosphorus according to Kjeldahl method (Tedesco et al. 1995TEDESCO JM, GIANELLO C, BISSANI CA, BOHNEN H & VOLKWEISS SJ. 1995. Análise de solo, plantas e outros materiais, 2a ed., Porto Alegre.).
Nitrate reductase activity (NRA)
Nitrate reductase (NR, EC1.7.1.1) activity (NRA) in 0.2 g fresh plant tissue samples (roots, stems, 3rd and 4th nodes from leaves) from each plant according to methodology cited by literature was determined (Jaworski 1971JAWORSKI EG. 1971. Nitrate reductase assay in intact plant tissues. Biochem Biophys Res Commun 43: 1274-1279.). Samples were placed in 5 mL phosphate buffer solution (100 mM KH2PO4 pH 7.5, 3% n-propanol and 200 mM KNO3) and kept in bath at 30°C for 60 min. Samples aliquots (0.4 mL) were combined with 0.3 mL sulfanilamide 1% in 3 M HCL and 0.3 mL n-naphthyl-ethylenediamine 0.02%. After 20 min, 4 mL water was added, and absorbance (540 nm) against NaNO2 standard by spectrophotometry equipment was measured.
Essential oil extraction and analysis
Menthol mint essential oil was extracted by hydrodistillation in a Clevenger apparatus from 100 g fresh plant material for 30 min. Essential oil content (% w/w) was evaluated and composition by gas chromatography (GC) was analyzed. A Hewlett Packard 5890 Series II (Palo Alto, USA) with flame ionization detection and a splity/splitless injection in a 1:20 split ratio was used for separating and detecting essential oil constituents. Substances were separated by a CP-Sil-8CB (30 m x 0,25mm x 0,25µm) fused silica capillary column. Equipment operating conditions were the same ones previously published (Souza et al. 2016SOUZA MAA DE, SANTOS LA DOS, BRITO DMC DE, ROCHA JF, CASTRO RN, FERNANDES MS & SOUZA SR DE. 2016. Influence of light intensity on glandular trichome density, gene expression and essential oil of menthol mint (Mentha arvensis L.). J Essent Oil Res 28: 138-145.). Gas chromatography coupled to mass spectrometry (GC-MS) for essential oil analysis by a Varian Saturn 2000 (Palo Alto Ca) was used. Helium gas carrier flow capillary column and temperature conditions for the GC-MS analysis were the same ones described for the GC, and previously published (Souza et al. 2016SOUZA MAA DE, SANTOS LA DOS, BRITO DMC DE, ROCHA JF, CASTRO RN, FERNANDES MS & SOUZA SR DE. 2016. Influence of light intensity on glandular trichome density, gene expression and essential oil of menthol mint (Mentha arvensis L.). J Essent Oil Res 28: 138-145.). Essential oils constituent identification was based comparing their GC linear retention indexes (LRI) and their mass spectra with those ones from authentic standards [(-)- pulegone, (-)- menthone, (+)- neomenthol and (-)- menthol from Sigma – Aldrich (USA)], NIST – 2008 database and literature retention indexes (Adams 2007ADAMS RP. 2007. Identification of essential oil components by gas chromatography/mass spectroscopy, 4th ed., Allured Publishing Corporation, Carol Stream.). LRI was obtained from a C8-C40 (Fluka, Louis, USA) alkane standard solution co-injection and calculated according to literature (van Den Dool & Kratz 1963VAN DEN DOOL H & KRATZ PD. 1963. A generalization of the retention index system including linear temperature programmed gas—liquid partition chromatography. J Chromatogr A 11: 463-471.).
Density and glandular trichome histochemistry
For anatomical study, fresh material and 70% ethyl alcohol stored samples (Johansen 1940JOHANSEN DA. 1940. Plant microtechnique. MacGraw-Hill, New York.) were selected by the Ranvier microtome in the middle-third region of the leaf blade. Section (10-12 µm) were clarified by 20% sodium hypochlorite, neutralized in 1% acetic water, washed in distilled water, stained by blue astra-safranin (Bukatsch 1972BUKATSCH F. 1972. Bemerkungem zur doppel far burng Astrablau-Safranin. Mikrokosmos 61: 255.) and confectioned between lamina and laminula with 50% glycerin (Strasburger 1924STRASBURGER E. 1924. Handbook of practical botany. 8th ed., George Allen e Nonviirn, Ltd., London.). Photomicrographies were confectioned by Olympus BX-51 microscopy with Q color 5 camera capture system and Image-Pro Express software. When necessary images were edited in Corel Photo-Paint® 15 and boards in Corel DRAW® 15.
To verify different chemical compounds in trichomes and nature of fresh material from all walls, these ones were treated with Sudan IV (Johansen 1940JOHANSEN DA. 1940. Plant microtechnique. MacGraw-Hill, New York.) and Sudan Black B (Pearse 1980PEARSE AGE. 1980. Histochemistry: Theoretical and Applied, 4th ed., Longman Press, London.) for lipid groups identification; 10% Potassium Dichromate for phenolic compounds detection (Gabe 1968GABE M. 1968. Techiniques Histologiques. Masson & Cie, Paris.); 0.02% Ruthenium Red for pectic compounds (Jensen JENSEN WA. 1962. Botanical histochemistry: principles and pratice. San Francisco.1962); Xylidine Ponceau for protein detection (Amaral et al. 2001AMARAL LIV DO, PEREIRA M DE FDA & CORTELAZZO ÂL. 2001. Formação das substâncias de reserva durante o desenvolvimento de sementes de Urucum (Bixa orellana L. - Bixaceae). Acta Bot Brasilica 15: 125-132., Cortelazzo & Vidal 1991CORTELAZZO A & VIDAL B. 1991. Soybean seed proteins: Detection in situ and mobilization during germination. Rev Bras Bot 14: 27-33.); Periodic Acid/Schiff Reagent (PAS) for neutral polysaccharides (Amaral et al. 2001AMARAL LIV DO, PEREIRA M DE FDA & CORTELAZZO ÂL. 2001. Formação das substâncias de reserva durante o desenvolvimento de sementes de Urucum (Bixa orellana L. - Bixaceae). Acta Bot Brasilica 15: 125-132., Cortelazzo 1992CORTELAZZO A. 1992. Detecção e quantificação de amido em cotilédones de Canavalia ensiformis e C. gladiata durante o desenvolvimento inicial da planta. Rev Bras Bot 15: 157-162., Taboga & Vilamaior 2001TABOGA S & VILAMAIOR PSL. 2001. Citoquímica. In: A Célula. Manoli Ltda, Barueri, p. 19-27.). Control treatment was applied to histochemical tests according to authors mentioned above.
Experimental design and statistical
Each treatment was composed by three replicates (six plants) arranged in a completely randomized experimental design and in each pot, two plants (one replicate) were placed. Average, standard deviation (SD), standard error mean (SEM) and graphics were calculated and created in GraphPad Prism, version 6.01 (Graph Pad Software® Inc., San Diego, USA). Principal Component Analysis (PCA) and its graphics were made by PAST Program, version 3.13 (Hammer et al. 2001HAMMER Ø, HARPER DAT & RYAN PD. 2001. PAST: Paleontological statistics software package for education and data analysis. Palaeontol Electron 4: 1-9.).
RESULTS AND DISCUSSIONS
Biomass and photosynthetic pigments
Positive linear correlation between light intensity increase and fresh mass gain of stems, leaves and root/shoot ratio was observed (Figure 1a-c), highlighting the increase in roots mass was proportionally higher than increases in aerial part mass (Figure 1c). On the other hand, negative linear correlation in regarding to light intensity and pigments concentration was observed (Figure 1d-f).
Stems (a) and fresh mass (b), roots/shoot ratio (c), chlorophyll a (d), chlorophyll b (e) and carotenoids (c+x) (f) contents in menthol mint for 43 days and submitted to different light intensities (25, 50, 75 and 100%). Lines correspond to linear correlations and bars to average standard deviation (n = 3).
Results previously obtained have shown that fresh mass production in genus Mentha is modulated by environmental and nutritional factors (Souza et al. 2007SOUZA MAA, ARAÚJO OJL, FERREIRA MA, STARK EMLM, FERNANDES MS & DE SOUZA SR. 2007. Produção de biomassa e óleo essencial de hortelã em hidroponia em função de nitrogênio e fósforo. Hortic Bras 25: 41-48., 2014SOUZA MAA, ARAÚJO OJL, BRITO DMC, FERNANDES MS, CASTRO RN & DE SOUZA SR. 2014. Chemical Composition of the Essential Oil and Nitrogen Metabolism of Menthol Mint under Different Phosphorus Levels. Am J Plant Sci 5: 2312-2322., 2016, Fernandes et al. 2014FERNANDES VF, BEZERRA L DE A, MIELKE MS, SILVA D DA C & COSTA, LC do B. 2014. Leaf anatomy and ultrastructure of Ocimum gratissimum under different light radiation levels. Ciência Rural 44: 1037-1042.), mainly luminosity and day and night temperatures with strong influence (Burbott & Loomis 1967BURBOTT AJ & LOOMIS WD. 1967. Effects of Light and Temperature on the Monoterpenes of Peppermint. Plant Physiol 42: 20-28., Clark & Menary 1980aCLARK R & MENARY R. 1980a. The effect of irrigation and nitrogen on the yield and composition of peppermint oil (Mentha piperita L.). Aust J Agric Res 31: 489.). Availability of light has also influenced photosynthetic pigments concentration, since light intensity increase has usually decreased chlorophyll content, and light reduction has induced to an increase of these pigments concentration in order to enlarge light capture area (Enríquez & Sand-Jensen 2003ENRÍQUEZ S & SAND-Jensen K. 2003. Variation in Light Absorption Properties of Mentha aquatica L. as a Function of Leaf Form: Implications for Plant Growth. Int J Plant Sci 164: 125-136., Zervoudakis et al. 2012ZERVOUDAKIS G, SALAHAS G, KASPIRIS G & KONSTANTOPOULOU E. 2012. Influence of light intensity on growth and physiological characteristics of common sage (Salvia officinalis L.). Brazilian Arch Biol Technol 55: 89-95.).
Carotenoids are photoprotective pigments cooperating with light harvest complexes (Lichtenthaler & Buschmann 2001LICHTENTHALER HK & BUSCHMANN C. 2001. Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. In: Current Protocols in Food Analytical Chemistry. J Wiley & Sons, Inc., Hoboken, NJ, USA, p. F431-F438.). However, carotenoids concentration increase was not observed with light intensity increase, in contrast in higher light intensity treatment drastic decrease in pigments concentration (Figure 1f), below values usually found in plants with C3 type metabolism was not reported.
Soluble fractions, total N and nitrate reductase activity (NRA)
Increased soluble sugars values were observed, mainly in stems due to light intensity increase (Figure 2a). On the other hand, light intensity increase resulted in NO3 --N contents decrease in menthol mint plant parts (Figure 2b). Marked decrease in N-amino contents in stems due to light intensity was observed. Roots presented the lowest N-amino content (Figure 2c).
Soluble sugar (a), NO3 --N (b), amino-N (c), total-N (d) contents and nitrate reductase activity (e). Correlation among NO3 --N (f), NRA (g) and total-N (h) with soluble sugar variable. Menthol mint cultivated in hydroponic system for 43 days and submitted to different light intensities (25, 50, 75 and 100%). Lines correspond to linear correlations and bars to average standard deviation (n = 3).
As shown in Figure 2, stems presented the highest soluble fractions contents. The nitrate accumulation in the stems has been observed as a reserve N strategy, likewise stem high soluble sugars content indicated that stem might be a storage place or a photoassimilates intense traffic between source and drain tissues (Hirel et al. 2007HIREL B, LE GOUIS J, NEY B & GALLAIS A. 2007. The challenge of improving nitrogen use efficiency in crop plants: towards a more central role for genetic variability and quantitative genetics within integrated approaches. J Exp Bot 58: 2369-2387., Fageria et al. 2008FAGERIA NK, BALIGAR VC & LI YC. 2008. The Role of Nutrient Efficient Plants in Improving Crop Yields in the Twenty First Century. J Plant Nutr 31: 1121-1157.).
Nitrate reductase activity (NRA) in roots was positively influenced in regarding to light intensity increase, however, NRA decreased in leaves (Figure 2e). NRA in the leaf was higher than in the roots, except at 100% light treatment, presenting higher NRA in the roots. Reduction in light availability also reduces photoassimilates one in menthol mint (Figure 2a). Thus, menthol mint more intensively reduced leaf nitrate as a result of photoassimilates supply, however, when photoassimilates levels are higher, nitrate reduction may also occur in the roots.
Most of the nitrogen in plants is assimilated into amino acids, manly composing proteins structure. However, until proteins can be synthetized, much energy is necessary for the maintenance of process involving absorption, assimilation and their own synthesis. For this reason, sugars flow to anabolic processes is essential, and it has been expected that light intensity increasing, higher total-N contents will be found. However, this result was not confirmed in this study in the reason that higher leaves total-N contents followed by roots and stems have decreased due to light intensity increase, mainly in leaves (Figure 2d).
Plants with low light intensity have probably invested most energy resources in proteins synthesis related to leaves light energy capture and processing (light harvest complex and photosystems), as well as absorption and roots and stems nutrients transport to the detriment of the vegetative growth (biomass) which is stimulated in higher light intensity conditions (Enríquez & Sand-Jensen 2003ENRÍQUEZ S & SAND-Jensen K. 2003. Variation in Light Absorption Properties of Mentha aquatica L. as a Function of Leaf Form: Implications for Plant Growth. Int J Plant Sci 164: 125-136., Zervoudakis et al. 2012ZERVOUDAKIS G, SALAHAS G, KASPIRIS G & KONSTANTOPOULOU E. 2012. Influence of light intensity on growth and physiological characteristics of common sage (Salvia officinalis L.). Brazilian Arch Biol Technol 55: 89-95.). Linear negative correlation among soluble sugars contents, NO3−-N, NRA and total-N in menthol mint leaves was observed (Figure 2f-h). These results corroborated the thesis in regarding tissues where endergonic processes have been intense, the soluble sugar contents are lower (Fernandes 1990FERNANDES MS. 1990. Efeitos de fontes e níveis de nitrogênio sobre a absorção e assimilação de N em arroz. Rev Bras Fisiol Veg 2: 1-6.).
Density and glandular trichome histochemistry
Secretory trichomes found on both sides of leaves lamina were classified as two types: peltate and capitate, formed by 10 and 3 cells, respectively (Figure 3). Peltate secretory trichomes are composed by a basal cell, an unicellular peduncle, and an eight secretory cells head, covered by thin cuticle. Presence of conspicuous subcutilar space filled with secretion was observed. Thicker sidewalls from the peduncle cells and impregnated by lipid substances were reported (Figure 3a, b).
Peltate secretory trichome (a, b) and capitate secretory trichomes (c, d) in menthol mint leaves, IAC 701 cultivar.
Capitate secretory trichomes consist of a basal cell, an unicellular peduncle and a globular or oval secretory head, covered by thin cuticle. Sidewalls from peduncle cells are impregnated by lipids substances and also thicker. Subcuticular space is undeveloped and filled with secretion (Figure 3c, d).
The anatomical description of the secretory trichomes from IAC 701 cultivar is in line with results presented by other authors (Shanker et al. 1999SHANKER S, AJAYAKUMAR PV, SANGWAN NS, KUMAR S & SANGWAN RS. 1999. Essential oil gland number and ultrastructure during Mentha arvensis leaf ontogeny. Biol Plantarum1 42: 379-387., Sharma et al. 2003SHARMA S, SANGWAN NS & SANGWAN RS. 2003. Developmental process of essential oil glandular trichome collapsing in menthol mint. Curr Sci 84: 544-550., Deschamps et al. 2006DESCHAMPS C, ZANATTA JL, ROSWALKA L, OLIVEIRA M DE C, BIZZO HR & ALQUINI Y. 2006. Densidade de tricomas glandulares e produção de óleo essencial em Mentha arvensis L., Mentha x piperita L. e Mentha cf. aquatica L. Cienc e Nat 28: 23-34., Mishra et al. 2017MISHRA A, LAL RK, CHANOTIYA CS & DHAWAN SS. 2017. Genetic elaborations of glandular and non-glandular trichomes in Mentha arvensis genotypes: assessing genotypic and phenotypic correlations along with gene expressions. Protoplasma 254: 1045-1061.).
Histochemical tests showed lipid substances, proteins, phenolic substances, acid and neutral polysaccharides. Reactions were equally positive in the peltate and capitate trichomes cells protoplast (Table I). Great amount of phenolic and lipophilic substances deposition in the endoplasmatic reticulum and in peppermint vesicles justifying positive tests for these substances in cytoplasm were reported (Turner et al. 2000TURNER GW, GERSHENZON J & CROTEAU RB. 2000. Distribution of Peltate Glandular Trichomes on Developing Leaves of Peppermint. Plant Physiol 124: 655-664.).
In subcuticular space from both trichomes it might be observed lipophilic material drops composing the secretion. Reaction for phenolic substances is more intense in protoplasm of capitate trichomes cells, on the other hand, no proteins, pectic substances and neutral polysaccharides in peltate and capitate trichomes secretion were detected (Table I).
Results presented in Table I were also observed in Mentha genus (Martins 2002MARTINS MBG. 2002. Estudos de microscopia óptica e de microscopia eletrônica de varredura em folhas de Mentha spicata e de Mentha spicata x suaveolens (Lamiaceae). Bragantia 61: 205-218.), as well as were divergent in regarding to pectin presence in peltate and capitate trichomes of M. pulegium (Rodrigues et al. 2013RODRIGUES L, PÓVOA O, TEIXEIRA G, FIGUEIREDO AC, MOLDÃO M & MONTEIRO A. 2013. Trichomes micromorphology and essential oil variation at different developmental stages of cultivated and wild growing Mentha pulegium L. populations from Portugal. Ind Crops Prod 43: 692-700.). However, according to some authors capitate trichomes from peppermint plants did not present any monoterpenes secretion (Brun et al. 1991BRUN N, COLSON M, PERRIN A & VOIRIN B. 1991. Chemical and morphological studies of the effects of ageing on monoterpene composition in Mentha × piperita leaves. Can J Bot 69: 2271-2278.), and its secretion is mainly composed by proteins, lipids and polysaccharides mixture (Turner et al. 2000TURNER GW, GERSHENZON J & CROTEAU RB. 2000. Distribution of Peltate Glandular Trichomes on Developing Leaves of Peppermint. Plant Physiol 124: 655-664.).
In regarding to secretory trichomes density, 4th and 5th nodes leaves were evaluated and was verified that from 25-50% to 75-100% light intensity increase promoted significant increase in trichomes density, mainly the capitate ones, being decreased density from proximal to distal region in both foliar faces (Figure 4). No studies in the literature were observed in relation to light intensity effect over secretory trichomes density or distribution in menthol mint leaves, showing this result reported in this study as pioneer. However, Fernandes et al. (2013)FERNANDES VF, DE ALMEIDA LB, FEIJÓ EVR DA S, SILVA D DA C, OLIVEIRA RA DE, MIELKE MS & COSTA LC DO B. 2013. Light intensity on growth, leaf micromorphology and essential oil production of Ocimum gratissimum. Rev Bras Farmacogn 23: 419-424. verified that light intensity variations did not affect peltate and capitate secretory trichomes density in Ocimum gratissimum leaves.
Secretory trichome density according to type (peltate and capitate), face (adaxial an abaxial) and region (proximal, medial and distal) in menthol mint leaves cultivated in hydroponic system for 43 days and submitted to different light intensities (25, 50, 75 and 100%). Bars represent averages standard deviation (n = 3). Density of secreted trichomes pelted on the adaxial face (a), capitate (b), total (P+C = peltate + capitate) (c) and average (P+C = peltate + capitate) (d) in menthol mint leaves. Density of secreted trichomes pelted on the abaxial face (e), capitate (f), total (P+C = peltate + capitate) (g) and average (P+C = peltate + capitate) (h) in menthol mint leaves.
It was also observed that secretory trichomes (peltate + capitate) density average is twice as large in the abaxial face, and capitate secretory trichome density average is three times higher than secretory peltate one in foliar surface (Figure 4). In menthol mint plant, relation of four peltate trichomes for each capitate one was described in literature (Shanker et al. 1999SHANKER S, AJAYAKUMAR PV, SANGWAN NS, KUMAR S & SANGWAN RS. 1999. Essential oil gland number and ultrastructure during Mentha arvensis leaf ontogeny. Biol Plantarum1 42: 379-387., Sharma et al. 2003SHARMA S, SANGWAN NS & SANGWAN RS. 2003. Developmental process of essential oil glandular trichome collapsing in menthol mint. Curr Sci 84: 544-550.).
For menthol mint, spearmint and water mint, higher concentration of peltate and capitate trichomes in abaxial face were reported by previously published results, however, peppermint demonstrated higher trichomes density in adaxial face (Turner et al. 2000TURNER GW, GERSHENZON J & CROTEAU RB. 2000. Distribution of Peltate Glandular Trichomes on Developing Leaves of Peppermint. Plant Physiol 124: 655-664., Martins 2002MARTINS MBG. 2002. Estudos de microscopia óptica e de microscopia eletrônica de varredura em folhas de Mentha spicata e de Mentha spicata x suaveolens (Lamiaceae). Bragantia 61: 205-218., Deschamps et al. 2006DESCHAMPS C, ZANATTA JL, ROSWALKA L, OLIVEIRA M DE C, BIZZO HR & ALQUINI Y. 2006. Densidade de tricomas glandulares e produção de óleo essencial em Mentha arvensis L., Mentha x piperita L. e Mentha cf. aquatica L. Cienc e Nat 28: 23-34.).
Presence of phenolic compounds has been cited as a group of substances of great importance in regarding to protection against herbivores, microorganisms, ultraviolet radiation excess, and also protecting cellular protoplast maintaining its integrity when submitted to water stress (Carmelo et al. 1995CARMELO SM, MACHADO SR & GREGÓRIO EA. 1995. Ultrastructural aspects of the secretory duct development in Lithraea molleoides (Vell.) Engl. (Anacardiaceae). Rev Bras Bot 18: 95-103., Paiva & Machado 2008PAIVA EAS & MACHADO SR. 2008. The Floral Nectary of Hymenaea stigonocarpa (Fabaceae, Caesalpinioideae): Structural Aspects During Floral Development. Ann Bot 101: 125-133., Swain 1979SWAIN T. 1979. Tannins and lignins. In: Jansen GA and Rosenthal DH (Eds), Their Interactions with Secondary Plant Metabolites. Academic Press, New York, p. 657-682., Taiz & Zeiger 2006TAIZ L & ZEIGER E. 2006. Plant physiology, 4th ed., Sinauer Associates, Sunderland.). Pharmacologically, phenolic substances present astringent, healing, antiseptic, antioxidant, vasoconstricting, hemostatic and anti-inflamatory properties (Cunha & Batista 1999CUNHA DE AP & BATISTA MT. 1999. Taninos In: Cunha de AP (Ed), Farmacognosia e Fitoquímica. Fundação Calouste Guilbenkian, Lisboa, p. 291-316., Kuklinski 2000KUKLINSKI C. 2000. Farmacognosia: Estudio de las drogas y sustancias medicamentosas de origen natural. Barcelona., Rocha et al. 2002ROCHA JF, NEVES L DE J & PACE LB. 2002. Estruturas secretoras em folhas de Hibiscus tiliaceus L. e Hibiscus pernambucensis Arruda. Rev Univ Rural Série Ciências Vida 22: 43-55., Osadebe & Okoye 2003OSADEBE PO & OKOYE FBC. 2003. Anti-inflammatory effects of crude methanolic extract and fractions of Alchornea cordifolia leaves. J Ethnopharmacol 89: 19-24., Raphael & Kuttan 2003RAPHAEL KR & KUTTAN R. 2003. Inhibition of experimental gastric lesion and inflammation by Phyllanthus amarus extract. J Ethnopharmacol 87: 193-197.). According to Rocha et al. (2002)ROCHA JF, NEVES L DE J & PACE LB. 2002. Estruturas secretoras em folhas de Hibiscus tiliaceus L. e Hibiscus pernambucensis Arruda. Rev Univ Rural Série Ciências Vida 22: 43-55. other functions related to phenolic substances may be present although some doubts in relation to the totality of their roles were reported. In addition to secretory structures, production and secretion sites of secondary metabolites as phenolic compounds in non-specialized cells, as the parenchyma one, were also reported. According to Barros & Teixeira (2008)BARROS GMCC & TEIXEIRA SDP. 2008. Estudo farmacobotânico de duas espécies de Anileira (Indigofera suffruticosa e Indigofera truxillensis, Leguminosae) com propriedades farmacológicas. Rev Bras Farmacogn 18: 287-294. secondary metabolites production in Indigofera (Leguminosae) species non-specialized cells were observed.
Mucilage, described as a mixed nature secretion, mainly consisting by acid and/or neutral heteropolysaccharides, proteins and phenolic substances present wide distribution in plants constituting colloidal solutions becoming viscous in contact with water (Gregory & Baas 1989GREGORY M & BAAS P. 1989. A survey of mucilage cells in vegetative organs of the dicotyledons. Isr J Bot 38: 125-174., Priolo de Lufrano & Caffini 1981PRIOLO DE LUFRANO NS & CAFFINI NO. 1981. Mucílagos foliares de Chorisia H.B.K. (Bombacaceae): Analisis fitoquimico y enfoque quimiotaxonomico. Phyton (Argentina) 40: 13-20., Roshchina & Roshchina 1993ROSHCHINA VV & ROSHCHINA VD. 1993. The excretory function of higher plants. Springer-Verlag, Berlin, p. 56-57). These substances may play different roles in plants, including protection of developing structures or organs water retention, carbohydrates reserves, perspiration reduction, radiation protection by dispersing or reflecting incident light, protection against herbivores, roots apex lubricant, insects capture in insectivorous plants as an adhesive in seed dispersion and in seed germination regulation (Gregory & Baas 1989GREGORY M & BAAS P. 1989. A survey of mucilage cells in vegetative organs of the dicotyledons. Isr J Bot 38: 125-174., Clifford 2002CLIFFORD SC. 2002. Mucilages and polysaccharides in Ziziphus species (Rhamnaceae): localization, composition and physiological roles during drought-stress. J Exp Bot 53: 131-138., Pimentel et al. 2011PIMENTEL RR, MACHADO SR & ROCHA JF. 2011. Estruturas secretoras de Pavonia alnifloia (Malvaceae), uma espécie ameaçada de extinção. Rodriguésia 62: 253-262., Fahn 1979FAHN A. 1979. Secretory tissues in plants. Academic Press, London., Martini et al. 2003MARTINI MH, LENCI CG & TAVARES DQ. 2003. Mucilage pockets in cotiledon tissue of Theobroma speciosum. Acta Microsc 12: 65-69., Roshchina & Roshchina 1993ROSHCHINA VV & ROSHCHINA VD. 1993. The excretory function of higher plants. Springer-Verlag, Berlin, p. 56-57).
Mucilage in secretory trichomes from the species studied in this survey is composed by acid and neutral polysaccharides and phenolic substances. Gregory & Baas (1989)GREGORY M & BAAS P. 1989. A survey of mucilage cells in vegetative organs of the dicotyledons. Isr J Bot 38: 125-174. suggested that in Althaea officinalis different mucilage fractions presented different functions. Water reserve is made by the acid fraction reaching its production peaks in the summer, as well as carbohydrates reserve is made by the neutral one showing maximum production in the winter. Presence of phenolic compounds in mucilage, mainly tannins, has antimicrobial importance and protection against herbivores, constituting a relevant chemical protection barrier, as well (Carmelo et al. 1995CARMELO SM, MACHADO SR & GREGÓRIO EA. 1995. Ultrastructural aspects of the secretory duct development in Lithraea molleoides (Vell.) Engl. (Anacardiaceae). Rev Bras Bot 18: 95-103., Swain 1979SWAIN T. 1979. Tannins and lignins. In: Jansen GA and Rosenthal DH (Eds), Their Interactions with Secondary Plant Metabolites. Academic Press, New York, p. 657-682.). Pimentel et al. (2011)PIMENTEL RR, MACHADO SR & ROCHA JF. 2011. Estruturas secretoras de Pavonia alnifloia (Malvaceae), uma espécie ameaçada de extinção. Rodriguésia 62: 253-262. identified phenolic substances presence in different morphological types of secretory structures and reported these substances might perform functions attributed by authors above cited.
Mucilage produced by menthol mint trichomes probably presented as main function desiccation as reported by Meyberg (1988)MEYBERG M. 1988. Cytochemistry and Ultrastructure of the Mucilage Secreting Trichomes of Nymphoides peltata (Menyanthaceae). Ann Bot 62: 537-547. for Nymphoides peltata (S. G. Gmel) Kuntze secretory trichomes. In different morphological types from menthol mint secretory trichomes, diversity of chemical substances classes was reported. These substances protect in plant surface against desiccation and may perform as water and carbohydrates reserves inside the plant, in water balance, drought resistance and due to phenolic compounds presence protecting against herbivores and pathogens, as well. It was considered that reports of the use from other genus species with medicinal purposes and different classes of chemical substances evidenced in menthol mint secretory trichomes have been sufficiently strong indications for further varieties investigations, from pharmacological point of view.
Essential oil
Essential oils may present insect repellent function or be toxic to animals, operating against hervivores (Rosenthal & Janzen 1979ROSENTHAL G & JANZEN D. 1979. Herbivores, their interaction with secondary plant metabolites. Academic Press, New York.), lipophilic secretions, mainly terpenes have generally been reported as plants chemical defensors, protecting against herbivores and pathogens (Werker & Fahn 1981WERKER E & FAHN A. 1981. Secretory Hairs of Inula viscosa (L.) Ait.-Development, Ultrastructure, and Secretion. Bot Gaz 142: 461-476., Ascensão et al. 1995ASCENSÃO L, MARQUES N & PAIS MS. 1995. Glandular Trichomes on Vegetative and Reproductive Organs of Leonotis leonurus (Lamiaceae). Ann Bot 75: 619-626., Corsi & Bottega 1999CORSI G & BOTTEGA S. 1999. Glandular Hairs of Salvia officinalis: New Data on Morphology, Localization and Histochemistry in Relation to Function. Ann Bot 84: 657-664.), as well as might also perform in attracting or repelling animals (Rodriguez et al. 1984RODRIGUEZ E, HEALEY P & MEHTA I. 1984. Biology and chemistry of plant trichomes. Plenum, New York.).
Essential oils main substances is presented in Figure 5a. Profile related in all treatments is according to a menthol mint essential oil presenting around 70% menthol, and other p-mentans such as: mentone, iso-mentone, neomenthol. Significant difference among treatments was observed from menthol (68.9% minimum and 72.5% maximum) and alcohols/esters monoterpenes number (71.5% minimum and 76.1% maximum) (Figure 5a).
Total ions chromatogram, GC-MS (a) and principal component analysis, scores (b), based on the menthol mint essential oil composition cultivated at different light intensities (triangle diamond, square and circle: 25, 50, 75 and 100, respectively). Components 1 and 2 represents 96.3 and 2.4% variance, respectively.
Main components analysis based on essential oil chemical profile allowed to only differentiate plants group submitted to the lowest light intensity, monoterpenes, alcohols and esters positively contributed for group separation (25%), as well as mentone and Ketone monoterpenes negatively contributed (Figure 5b).
Previously published results pointed to shading effect in the anticipation of essential oil maturation in peppermint plants, characterized by Ketone monoterpenes contents decrease and monoterpenes alcohols and esters contents increase (Burbott & Loomis 1967BURBOTT AJ & LOOMIS WD. 1967. Effects of Light and Temperature on the Monoterpenes of Peppermint. Plant Physiol 42: 20-28., Costa & Chagas 2014COSTA AG & CHAGAS JH. 2014. Níveis de sombreamento e tipos de malha no crescimento e produção de óleo essencial de hortelã-pimenta. Hortic Bras 32: 194-199., Clark & Menary 1980bCLARK RJ & MENARY RC. 1980b. Environmental Effects on Peppermint (Mentha pipevita L.). II. Effects of Temperature on Photosynthesis, Photorespiration and Dark Respiration in Peppermint with Reference to Oil Composition. Aust J Plant Physiol 7: 693-697.).
Essential oil content reported in this study presented moderate correlation with soluble sugars and leaves production, on the other hand, strong one with secretory trichomes density. Secretory trichomes density presented strong correlation with leaves fresh mass and soluble sugar levels (Table II).
Pearson correlation coefficient among essential oil contents values and secretory trichomes total density and other associated variables to menthol mint leaves.
Previously published studies pointed to the existence of a positive correlation between quantitative aspects, such as fresh leaves mass and essential oil content (Enríquez & Sand-Jensen 2003ENRÍQUEZ S & SAND-Jensen K. 2003. Variation in Light Absorption Properties of Mentha aquatica L. as a Function of Leaf Form: Implications for Plant Growth. Int J Plant Sci 164: 125-136.). Correlation analysis among descriptions from different menthol mint accesses showed no correlation between secretory trichomes density and essential oil, however, moderate negative correlation between secretory trichomes density and menthol contents was observed (Mishra et al. 2017MISHRA A, LAL RK, CHANOTIYA CS & DHAWAN SS. 2017. Genetic elaborations of glandular and non-glandular trichomes in Mentha arvensis genotypes: assessing genotypic and phenotypic correlations along with gene expressions. Protoplasma 254: 1045-1061.). Studies relating secretory trichome density versus fresh leaves mass and soluble sugars were not found in literature.
Previously published results related secretory trichomes density increase with foliar maturation and with essential oil production menthol mint plants, as well (Shanker et al. 1999SHANKER S, AJAYAKUMAR PV, SANGWAN NS, KUMAR S & SANGWAN RS. 1999. Essential oil gland number and ultrastructure during Mentha arvensis leaf ontogeny. Biol Plantarum1 42: 379-387.). On the other hand, Deschamps et al. (2006)DESCHAMPS C, ZANATTA JL, ROSWALKA L, OLIVEIRA M DE C, BIZZO HR & ALQUINI Y. 2006. Densidade de tricomas glandulares e produção de óleo essencial em Mentha arvensis L., Mentha x piperita L. e Mentha cf. aquatica L. Cienc e Nat 28: 23-34. reported lower essential oil production with higher secretory trichomes density in menthol mint leaves.
Results corroborated strong influence of light availability in menthol mint plants cultivation, since increase in light intensity positively influenced fresh mass gain, which was strongly correlated to essential oil content and secretory trichomes density. Therefore, it might be suggested further studies to establish the best conditions for menthol mint cultivation, indicating crop spacing and/or ideal intercropping condition with other crops, as well.
ACKNOWLEGMENTS
The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for the scholarship and grants that supported this work. This work is part of the D.Sc. thesis of the first author in Programa de Pós-Graduação em Química (PPGQ) at Universidade Federal Rural do Rio de Janeiro (UFRRJ).
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Publication Dates
-
Publication in this collection
26 June 2020 -
Date of issue
2020
History
-
Received
9 Apr 2018 -
Accepted
20 June 2019