Nitrogen supply and shading affect morphology and composition of the essential oil in marigold (Tagetes erecta L.)

The experiment was carried out under greenhouse conditions (Table 1). Thirty-day-old marigold (T. erecta L.) cv. Inca plants were transplanted in 1 L black plastic pots with a substrate made up of tezontle (a porous volcanic rock) and perlite (60:40, v:v). After transplantation, plants were watered for 4 days; subsequently, irrigation was applied with a 5% Steiner nutrient solution (Steiner, 1984) supplemented with micronutrients (Tradecorp AZ™) at the concentrations described by Trejo-Téllez et al. (2013). The pH of the nutrient solution was adjusted to 5.5. Twice a day, a total volume of 150 mL of the nutrient solution per pot was applied through a drip irrigation system. The nutrient solution was stored in a 200-L tank connected to 1′ (2.54 cm) hydraulic polyvinyl chloride (PVC) pipe. Each dripper had two-way adapters with tubing and a peg, which were placed in each pot. The irrigation system was programmed with a controller and pumped using 1/2 HP pumps.

Two study factors at different levels were tested. The study factors were the N concentration in the nutrient solution at three levels (8.47, 12.71 and 16.94 mg · L⁻¹) and the shading percentage at two levels (0 and 70%). The N levels were established considering that the high level (16.94 mg · L⁻¹) is sufficient for this species in an open hydroponic system (free drainage) with daily nutrient supply. The shading percentage resulted from using or not a black 70% UV light duty screening shade cloth. The mean light intensities corresponding to the shading percentages of 0 and 70% were 680 and 207 μmol · m⁻² · s⁻¹, respectively (Table 1). Black shade cloth is commonly used in horticulture since it efficiently reduces photosynthetically active radiation (Stamps, 2009). Consequently, the experiment had a 3 × 2 factorial arrangement with a completely randomised distribution of treatments in the greenhouse. Treatments were applied during the flowering stage of marigold plants. One day after transplantation (31-day-old plants), the flower buds of plants were removed from all the plants since these were formed when the plants had not received the treatments yet. Each treatment had 40 replicates. The experimental unit was a single pot with a single plant.

The following morphological variables were measured 30 days after the beginning of treatments (dat). The height of side branches (HSB) was determined from the visible base of the aerial part of the plant to the apex of lateral branches, containing the flower buds, using a graduated flexometer. The number of primary branches (NPB) and number of secondary branches (NSB) of the plant were quantified. The number of opened flower buds (NOFB) per plant was also evaluated. The diameter of flower (DF) was measured with a digital vernier calliper.

Isolation of the essential oil was done 38 days after the beginning of the treatments. Four plants in the flowering stage were randomly sampled per treatment. The plants were separated into flowers, leaves and stems. Once separated, the samples were dried in a hot air stove (Felisa, FE291AD; Guadalajara, Mexico) at 40°C for 48 h according to Babatunde et al. (2017). The dried material was ground, and each plant organ was processed separately. Essential oil extraction was performed according to the method described by Bowman et al. (1997) with some modifications. From each sample, 0.125 g were weighed, and the material was placed in a vacuum flask with a glass stopper and 2.5 mL dichloromethane was added. The flask was connected to a vacuum system and allowed to stand for 24 h. The samples were filtered, and the obtained extracts were kept in amber-coloured bottles at −20°C. Subsequently, the extracts were concentrated at 0.5 mL with a gentle stream of chromatographic grade nitrogen gas.

To identify the compounds, a gas chromatography/mass spectrometry (GC-MS) instrument was used, with a Hewlett Packard HP-6890 Series^® (Agilent, Santa Clara, CA, USA) GC coupled to an HP-5973 mass detector. The gasified extract was run through an HP-5MS column (length 30 m, ID 0.250 mm, film 0.25 μm), with a mean speed of 36 cm · s⁻¹. The operation conditions of the chromatograph were initial temperature 40°C for 5 min, first ramp-up to 150°C, with an increase of 9°C · min⁻¹. This was maintained for 3 min; second ramp-up to 220°C with an increase of 9°C · min⁻¹; the ion trap at 230°C, with a quadrupole at 150ºC; splitless injector mode, the temperature at 220°C, 6.97 psi. The gas carrier at 99.9% purity was helium and 1 μL of the concentrated samples was injected manually. Identification of terpenoids was performed by retention times and comparing mass spectra using the NIST database of the US National Institute of Standards and Technology (NIST/EPA/NIH, 2002) according to Adams (2007).

A one-way analysis of variance (ANOVA) was carried out to analyse the morphological data and components of the essential oil. When statistical differences were found, the mean separation was done by using the Tukey test with α = 0.05. The SAS 9.0 software (SAS, 2011) was used for all statistical analyses carried out.

Nitrogen had significant effects on the variables NSB and DF, while shading influenced HSB, NPB and number of opened flower buds. The interaction of the study factors was significant on all morphological variables evaluated 30 dat (Table 2).

The N level in the nutrient solution was positively associated with the NSB (Figure 1A), which increased to 65.9 and 123.9% with the concentrations of 12.71 and 16.94 mg · L⁻¹ N, respectively, in comparison with the lowest level of N (8.47 mg · L⁻¹ N). Likewise, the DF increased to 1.2 and 5.9% with medium and high levels of N, as compared with the low level of N (Figure 1B).

Figure 1

Quality and quantity of flowers are greatly influenced by climatic, geographical and nutritional factors. Proper fertilisation is of paramount importance for growth and development of plants as well as for a good quality of flower production in marigold (Priyadarshini et al., 2018). In this research, higher N levels stimulated the growth of secondary branches and flower diameter. These responses may result from a more efficient nutrients flow into the plants, which consequently may enhance plant growth and emergence of auxiliary buds. The easy uptake of nutrient and simultaneous transport of growth-promoting substances to the auxiliary buds may stimulate a breakage of apical dominance and thus a faster mobilisation of photosynthates and early transition of the plant from vegetative to reproductive phase (Priyadarshini et al., 2018). In China aster (Callistephus chinensis), the balanced application of fertilisers resulted in increased carbohydrate assimilation leading to enhanced vegetative growth (Chaitra and Patil, 2007). These carbohydrates, when translocated to reproductive organs, underwent hydrolysis and got converted into the reducing sugars that ultimately increased flower size. Indeed, mineral contents of low transpiring plant organs, such as fruits and flowers, might differ from those of rapidly transpiring leaves. Therefore, one can expect changes in the contents of specific nutritional elements to be induced in flowers and thus improve quality and quantity parameters (Bernstein et al., 2005).

Shading reduced the HSB by 4.1% (Figure 2A), the NPB by 5.4% (Figure 2B) and the NOFB by 23.3% (Figure 2C), as compared with plants without shading. Shading is a common agricultural practice aimed to reduce heat stress in the summer. However, inappropriate shading decreases photosynthesis and consequently reduces plant growth (Qiu et al., 2018). Branching results from several interrelated processes: axillary bud formation, dormancy induction and release, bud outgrowth (involving the growth of preformed leaves), internode extension and initiation of new leaf primordia by the shoot apical meristem and then shoot extension (Leduc et al., 2014). Both endogenous (i.e. autonomous) and environmental (i.e. non-autonomous) pathways regulate the transition from vegetative to reproductive stages. Endogenous pathways function independently of environmental pathways and their inputs to flowering induction vary among, and even within, species (Amasino and Michaels, 2010). Importantly, environmental signals leading to floral transition include light, sucrose, cytokinins, gibberellins and reduced N compounds that are translocated in the phloem sap from leaves to the shoot apical meristem. Endogenous signals controlling flowering time function in cascades within four promotive pathways, all of which converge on the integrator genes Suppressor of Overexpression of Constans 1 (SOC1) and Flowering locus T (FT) (Corbesier and Coupland, 2006). The protein Constans (CO) is stabilised by light and rapidly degraded in darkness (Valverde et al., 2004). Consequently, CO accumulates during inductive long days, and its role in flowering is to activate the expression of the florigen FT (Amasino and Michaels, 2010).

Figure 2

Shading not only reduces radiation but also increases the fraction of diffuse light and alters the spectral quality. Diffuse light is utilised more efficiently by plants and can produce small decreases in direct radiation and enhance leaf CO₂ uptake, photosynthesis and growth. With increasing shading, the fraction of blue light (400–500 nm) increases while that of red light (600–700 nm) decreases, which may affect both physiological parameters (e.g. photosynthesis and chlorophyll biosynthesis) as well as plant morphology (Li et al., 2010). Plant species differ in their responses to light quality, but red and blue lights generally have the strongest effects on plant growth. Stomata regulate gas exchange and water loss in plants. Their opening and closure are influenced by many environmental factors, including light, CO₂, and temperature (Ye et al., 2017). Among all these factors, light is the main environmental signal that controls stomatal movement. Usually, stomata are open in the light and closed in the dark. Occurring in all green tissues, chlorophyll is one of the most important pigments in higher plants, responsible for capturing light for photosynthesis. Therefore, chlorophyll is a key player in the interaction with light during the entire life cycle of plants (Ye et al., 2017), and photoreceptors such as phytochromes may mediate the regulation of chlorophyll biosynthesis (Inagaki et al., 2015). Based on the wide distribution of light-harvesting pigments and photoreceptors in various plant organs, buds will contain all photoreceptor types, thus affecting plant responses to light (Leduc et al., 2014). Furthermore, phytohormones are also affected by phytochromes, and thus they participate in the regulation of stem elongation and shoot branching (Krishna-Reddy and Finlayson, 2014; Lymperopoulos et al., 2018).

The highest HSB was recorded in plants treated with the medium dose of N and no shading; it was 7.1 and 6.6% higher than those found in plants exposed to shading receiving medium and low N doses (Figure 3A). The NPB in the treatments with medium dose of N was influenced by the light intensity; a 12% reduction in this variable in shaded plants was observed, compared with unshaded plants (Figure 3B). The NSB was positively related to the N dose in the nutrient solution (Figure 3C).

Figure 3

Nitrogen concentration in plant tissues often decreases in both elevated concentrations of CO₂ and low nutrient availability, whereas it increases in low light environments (Bernacchi et al., 2007). The adverse effects of over-illumination can be aggravated by N limitation since the shortage of this nutrient can disrupt protein synthesis and impair the capacity of chloroplast proteins involved in the photosynthetic electron transfer chain and carbon assimilation. Lower photosynthetic capacity results in an excess of electrons in the thylakoid membrane and limitation of electron acceptors on the PSI side, which in turn stimulates the formation of reactive oxygen species (Cohen et al., 2019). Under N-deficient conditions, a limited sink development might induce downregulation feedback of the photosynthetic machinery and, ultimately, growth inhibition. Nitrogen uptake may be regulated by shoot-borne light signals, resulting in the upregulation of root N transporters and, ultimately, in an additional increase in N uptake (Cohen et al., 2019). Excessive N supply under high light intensity has adverse effects on plant growth (Fu et al., 2017).

On average, the NOFB was 32.3% higher in plants without shading than in plants with shading, independent of the N level in the nutrient solution. Opened flower buds of plants with low N level were more affected by shading, with 39% reductions as compared with the same N level without shading (Figure 4A). Flowers of plants without shading and high N level exhibited the higher diameter, surpassing in 9.5 and 6.1% DF with shade and medium level of N, and plants without shading and with low levels of N, respectively (Figure 4B).

Figure 4

Apart from N supply and light intensity, stress conditions such as heat, salt and UV light can promote flowering (Lin and Tsay, 2017). Moreover, the capacity of plants to develop new sink organs also depends on photosynthesis and N availability.

In calendula (Calendula officinalis), the application of 160 kg · ha⁻¹ N significantly increased the number of flowers, but the diameter was not affected (Król, 2011). In different grass species (S. secundatum, A. compressus, P. clandestinum), shading increased the growth and nutritious quality of the forage when the N supply was increased (Samarakoon et al., 1990). These results partially coincide with those obtained herein, since only in the case of plants with shade an increase in the NOFB was observed (Figure 4A).

In this study, a slight increase of 1.7% in the DF in shaded plants was observed, with respect to plants without shade (Figure 4B).

Fifteen different compounds were identified in the plant organs of T. erecta cv. Inca analysed (Table 3).

The compound content and amount in the essential oils are affected by factors such as the environment where the plant grows (Karousou et al., 2005), its phenological stage, the different parts from where the oils are extracted, the composition of the soil and mineral fertilisation (Fischer, 1991). Therefore, it is common to find differences even among plants of the same species. In some Mexican species of this genus, such as T. erecta (Sefidkon et al., 2004; Ogunwande and Olawore, 2006), T. filifolia (Serrato-Cruz et al., 2008), T. lucida (Cicció, 2004) and T. patula, (Sagar et al., 2005), secondary metabolites have been identified, although the chemical composition of the essential oils in most of these species is still unknown.

Despite variations in the relative concentrations, the main constituents of T. minuta, T. erecta and T. patula oils are monoterpenes (Krishna et al., 2002). In T. minuta, limonene, myrcene and spathulenol have been identified (Gil et al., 2000). These compounds coincide with those reported in the present study. Also, limonene has been reported in T. argentina (Vázquez et al., 2011).

In this research, the main effects of the study factors (nitrogen and shading) and their interactions were significant in almost all the components studied, as observed in Table 4.

With exception of carene, where the study factor shading had no significant influence, the main effects of nitrogen and shading and its interaction had significant influence in all components of essential oil in leaves (Table 4). In leaves, four compounds were consistently identified in all the treatments: d-limonene, ocimene, β-myrcene and α-pinene, with mean percentages of 13.0, 16.8, 2.5, and 1.2%, respectively (Table 5). These compounds have also been reported in T. erecta leaves, although in lower percentages as compared with those recorded herein, specifically limonene 7.6%, (E)-β-ocimene 0.6%, β-myrcene 0.4% and α-pinene 0.3% (Krishna et al., 2004).

The most abundant compounds found in this study were ocimene, d-limonene and piperitone (Table 5). The effects of nitrogen fertilisation on growth and composition of essential oils in different species have been previously reported in aromatic plants (Baranauskiené et al., 2003; Ashraf et al., 2005; Yang et al., 2005; Sifola and Barbieri, 2006; Martins et al., 2007). Nitrogen limitations increase secondary metabolite production in annual plants (Said-Al Ahl et al., 2009). In this study, the abundant components had the highest percentages with the low N dose (8.47 mg · L⁻¹) and no shading, with mean values of 36.78, 24.13 and 23.6%, respectively. Contrary to the present results, in basil (Ocimum basilicum L.), the presence of limonene was registered with high N doses (300 kg · ha⁻¹), with percentages of 13.2% in the cultivar Monstruoso mammouth, 17.3% in ‘Genovese profumatissimo’ and 16.8% in ‘Napoletano a foglia di lattuga’ (Sifola and Barbieri, 2006).

With the lowest N dose in the nutrient solution, there was an inhibition in the synthesis of caryophyllene, β-phellandrene, trans-pinene and γ-elemene in leaves. The percentages of β-myrcene (2.50%) and α-pinene (2.54%) in leaves are also outstanding with the lowest N dose with no shading, compared with the rest of the treatments. Contrary to the present observations, in Summer savory (Satureja hortensis), α-pinene contents increased from 0.774 to 0.785% when N applications were raised from 0 to 150 kg · ha⁻¹ (Mumivand et al., 2011).

Some components of the oil were found only in the leaves with the extreme doses of the tested treatments (either at the minimum or the maximum levels). Thus, γ-elemene was only identified in leaves of plants exposed to 70% shading and a high N dose. Conversely, sabinene and terpinolene were only identified in the treatment with no shading and a low N dose (Table 3). Sabinene has been identified as a derivate of the resin extract of Pinus oocarpa, useful in the chemical–pharmaceutical, cosmetic and textile industries (Iñiguez et al., 2014). Likewise, it has been identified in T. patula leaves in percentages of 0.6–0.9 of four varieties (Stojanova et al., 2000).

Also, trans-pinene was influenced positively by shading. Contrarily sabinene, α-pinene and terpinolene in shaded leaves were not identified (Table 3). In black sage (Varronia curassavica Jacq.) under different light intensities (20, 50, 70 and 100%), α-pinene has been identified; this compound showed the highest percentages in intermediate shading, 50 and 70%, while in 20 and 100% shadings it decreased. Thus, the synthesis of this component is influenced by light. The biosynthesis of isopropene, a basic component of α-pinene, is also affected by light (Feijó et al., 2014).

In general, ocimene, d-limonene and piperitone were the most abundant compounds in leaves. Importantly, the latter two are the most abundant commercial components in T. erecta essential oil (Krishna et al., 2004). Piperitone is used as a fragrance in home products (Meshkatalsadat et al., 2010), as a biodegradable industrial solvent, as a dispersant agent in paints and printer inks and as a raw material in the synthesis of carvone that is highly an important compound in the perfume industry (Trytek et al., 2007).

In flowers, the main effects of the study factors and their interactions were significative on compounds of flower essential oil, with exception of nitrogen on caryophyllene and shading on d-limonene, ocimene and piperitone (Table 4).

Interestingly, caryophyllene was constantly found in flowers of the six treatments evaluated. Even though the effect of N was not significant as already indicated, and it was negatively affected by shading. On average, this compound was present in percentages lower than 1% (Table 5). For T. minuta, the main compound in flowers is β-ocimene, with 45.4% (Chamorro et al., 2008). In the present research, this compound was identified in flowers only in two treatments: medium N and shading, and high N and no shading. Caryophyllene has been identified in other plant species such as clove (Syzygium aromaticum), basil, oregano (Origanum vulgare), black pepper (Piper nigrum) and true cinnamon (Cinnamomum zeylanicum), among others. It may act as a weed suppressor, and its synthesis increases with increments in light intensity (Stokłosa et al., 2012). On the other hand, trans-pinene was the most abundant compound (16.3%) in flowers of plants receiving the medium N level without shading (Table 6). The presence of verbenol only stood out in flowers of plants exposed to high N dose and shading. Verbenol is a natural compound with pharmaceutical applications, which is used as raw material to synthesise antitumour products. This compound is obtained from monoterpene geraniol and pinene, which is used in biotransformation processes of different microorganisms (Pescheck et al., 2009).

The main effects of the study factors and their interactions had a significant effect on the compounds of the essential oil from stems, as is observed in Table 4.

In the oil obtained from stems, only three compounds were identified. The positive effect of N on the synthesis of β-farnesene was observed in the medium and high doses under evaluation. In plants treated with low N doses (i.e. 8.47 mg · L⁻¹), this last compound and caryophyllene were not identified in the stems. Piperitone was the component that had the highest percentage with the low N level without shading (6.3%) (Table 7).