Effect of light intensity and water availability on plant growth, essential oil production and composition in Rosmarinus officinalis L.

Abstract

The effect of light intensity (LI) and water availability (WA) on rosemary (Rosmarinus officinalis L.) plant growth, essential oil (EO) production and composition was investigated by a two-factorial field experiment, where the first factor was LI (100%, 50% or 25% of natural sunlight) and the second factor was WA (irrigation set at 85%, 70% or 55% of field capacity during plant growing). The EO obtained by steam distillation of the dried aerial part of the plant was analysed by GC/MS. Reduction of LI from 100 to 25% of natural sunlight markedly lowered plant biomass production, whereas reduction of WA from 85 to 55% had a smaller lowering effect on plant growth. High shading (25% of LI) markedly reduced EO yield on a plant basis (− 43%), whereas intermediate shading (50% of LI) increased EO yield as % content of the fresh biomass (+ 29%) when compared to full solar radiation. WA markedly influenced EO yield, as expressed on a plant basis, but only in plants exposed to 100% LI. Moreover, changes in LI and WA seemed to have an opposite effect on the relative abundance of EO constituents that are formed through the activity of two groups of enzymes, pinene synthases (α- and β-pinene, camphene and myrcene) and, respectively, bornyl diphosphate synthases (borneol, camphor and bornyl acetate). Accurate management of light conditions and water availability, in greenhouse as well as open field conditions, may allow to optimize rosemary EO yield and modulate EO profile in view of different potential uses.

Introduction

Rosemary is a perennial aromatic herb and member of the Lamiaceae family, native and widely cultivated in the Mediterranean basin, where it is traditionally used as a spice herb for culinary purposes [1]. Rosemary essential oil (EO) is reported to be commercially used in quantities between 100 and 500 tons per year [2], namely as fragrance in cosmetic products [3], flavouring and active ingredient in products for aromatherapy and food preservation [1]. Innovative potential applications as ingredient of functional foods [4] or active packaging for food products [5] are focus of current intense research.

It is well established that, not only plant growth and morphology, but also EO production are strongly influenced in medicinal and aromatic plant (MAP) species by climatic conditions [6,7,8,9,10,11,12]. Indeed, even though the composition of EO is primarily under genetic control [13], its formation is also highly dependent on environmental conditions, such as day length, irradiance, temperature and water supply [14]. Light intensity (LI) is, in general, strongly associated with plant photosynthetic activity and, thus, carbon fixation, vegetative growth and dry matter accumulation in several species, while secondary metabolites, including EO constituents, are formed from photosynthetic carbon [15]. However, not all experimental studies on the effect of LI on EO production in MAP species showed univocal results: in some cases, a higher LI was associated to a higher EO yield, as observed on Thymus vulgaris [16], Origanum vulgare [17], Ocimum basilicum [18] and Mentha piperita [19]. On the contrary, in other experiments on Salvia officinalis [16], Mentha piperita [20], Eucalyptus citriodora [21], plants subjected to a reduced level of solar irradiance produced higher yields of EO, suggesting that effects on EO production may not simply parallel those on photosynthetic activity. Also water availability (WA) has been observed to markedly influence formation of EO constituents in several species [22]. Water shortage generally leads to an increase of EO concentration in the plant organs, even though due to the marked concurrent decline of biomass production, total EO content on a whole plant basis generally decreases, as observed in Salvia officinalis [22, 23], Lavandula angustifolia [23], and Mentha spicata [24]. As regards rosemary, while it has been thoroughly assessed that EO composition is mainly dependent on genetic background and origin [25], it has also been observed that severe water stress conditions significantly enhance EO concentration within plant biomass, even though due to the marked biomass reduction slight changes in EO yield result on a plant basis [24]. On the contrary, no information is available about the effect of more moderate water stress conditions, which would be compatible with crop cultivation management, nor about the influence of LI. In addition no information have been reported on rosemary about the interaction between the two factors, LI and WA, while it has been emphasized that interference of multiple factors may be relevant when analysing the influence of environmental conditions on EO production [22].

Cultivation of rosemary generally takes place in the open field, where solar irradiance may significantly differ depending on latitude, field slope and orientation, or geographical features, such as the shading of tall hedge. Thus an improved knowledge of the effects of light conditions on EO production could have useful implications on management of product quality and standardization, both in open field and in greenhouse conditions [17]. Moreover, WA may be deliberately modulated in the field to optimize cultivation strategies aimed at improving product quality and yield [22]. In fact, the importance of both light and drought-related stress conditions to be used in elicitation methods to enhance the synthesis of secondary metabolites in MAP species is generally recognized [26].

In the present study the effects of light intensity (LI) and water availability (WA), along with their interactions, on rosemary (Rosmarinus officinalis L.) plant growth and EO production and composition were investigated through a two-factorial field experiment, where the first factor was LI (100%, 50% and 25% of natural sunlight) and the second factor was WA (by setting irrigation regime at 85%, 70% and 55% of field capacity during plant pot growing). Aim of the study was to provide an in-depth knowledge of rosemary plant growth response to these key environmental factors along with their influence on EO yield and composition, with the purpose of optimizing cultivation strategies to improve crop productivity and EO quantity and quality.

Materials and methods

Plant materials and growing conditions

The trial was performed at Sanremo (latitude 43°49′05.23″N, longitude 7°45′26.12″E), Italy, in the experimental fields of CREA-Research Centre for Vegetable and Ornamental Crops, located about 80 m a.s.l., in a climatic area suitable for citrus cultivation in the open air. A rosemary (Rosmarinus officinalis L.) variety widely cultivated in Italian aromatic pot plant production, commonly named “erect rosemary” and characterized by an α-pinene/1,8-cineole chemotype, was used. Plants propagated by a local grower were potted in 7.5-L plastic pots on 3 April 2017 in a commercial substrate (Terflor Hochmoor® Vulcan Invernale pumex) suitable for nursery crops. A controlled release fertilizer (Geogreen Horti-Cote®Plus, 6 months release period, N–P–K–Mg 16–6–11–2 plus microelements) was added in the amount of 5 g L−1 of substrate. Two fertigations were accomplished after 7 and 12 days with Ferty 1 (Planta Düngemittel GmbH, N–P–K–Mg 20–7–10–2, 1.5 g L−1). Application of different levels of LI and WA started on 15 April. Three light environments in the open air were arranged, where reduction of full sunlight was obtained by shading the plants with black plastic nets of suitable cutoff. The three LI treatments corresponded to (a) 100%, or full, sunlight; (b) 50% sunlight and (c) 25% sunlight. The plants subjected to each light treatment were grown under an arched metallic structure 2.3 m tall; each structure was completely covered (except for 100% sunlight) by the appropriate shading net and, only on the top, by a polythene transparent film to avoid rainfall into the pot substrate (all treatments). In each light environment the plants were subjected to three different WA regimes: (a) 85%, (b) 70%, (c) 55% of field capacity. To accomplish these regimes during plant growth, water was not supplied until the scheduled field capacity percentage was reached, restoring the 100% field capacity level by irrigation after the threshold was overcome. The water amount in each pot and the corresponding percentage of field capacity was calculated daily by weighing the pots, then subtracting the weight of the pot (fixed), of the dried substrate (a representative sample was measured before experiment) and of the plant (on sample plants a destructive relief was carried out monthly). In that way a two-factorial scheme was obtained, with 9 treatments (3 LI levels × 3 WA levels), and a completely randomized experimental design was arranged; 15 plants for each treatment were used. Measurements on biomass growth and plant structures were performed. Data on temperature of leaves and pot external surface were also collected by an infrared thermometer (Minolta Cyclops Compac 3, Osaka). The trial lasted 154 days (15 April–15 September 2017).

Essential oil extraction

For each treatment, the aerial part of 3 plants (3 replicates), without the woody base (about 7 cm tall) carrying the branch insertions, was harvested, reduced to small pieces and air dried at room temperature (20–28 °C) in a shaded environment for 15 days after the trial end. Weight loss was determined for each plant replicate. The resulting biomass from each plant replicate was accurately mixed and transferred into a 2-L round-bottomed flask, 1 L of water was added and the mixture was steam distilled for 2 h using a Clevenger-type apparatus, according to the method recommended by the Italian Pharmacopoeia [27]. At the end of distillation the volume and weight of the obtained EO was determined.

GC–MS analysis of essential oil profile

Extracted EO samples were dried over Na2SO4, then 1 mL of oil was diluted to total 100 mL with tert-butyl methyl ether, containing butyl benzene as internal standard (2 μL mL−1). GC/MS analyses were performed on an Agilent 6890 GC 5973 N MS system equipped with a quadrupole mass filter for mass spectrometric detection (Agilent Technologies, Palo Alto, CA) and a DB1-MS column (0.25 mm i.d. × 60 m, 0.5 μm film thickness; J&W, Agilent Technologies, Palo Alto, CA) for GC separation. Chromatographic conditions were as follows: split (50:1 ratio) injection, injector temperature at 250 °C, oven temperature programme from 70 to 200 °C at 4 °C min−1, and then to 270 °C (5 min) at 50 °C min−1, constant flow of He carrier gas was 1.5 mL min−1 corresponding to a linear velocity of 32 cm s−1. The MS detector operated in the electronic impact ionization mode at 70 eV; transfer line, source, and quadrupole temperatures were set, respectively, at 300, 230, and 150 °C. Detection was performed in the full scan mode, over the mass range 30–250 amu. Identification of EO constituents was accomplished by comparison of linear retention indexes (LRI) and mass spectra of chromatographic peaks with those obtained on standards solution of pure reference compounds (purchased from Sigma-Aldrich, Milan, Italy). Linear retention indexes were determined by analysing, in the same conditions used for EO samples, a standard solution of C7–C30 saturated alkanes, and by applying the equation proposed by van den Dool and Kratz. When a pure compound was not available tentative identification was based on the comparison of determined linear retention indexes with those reported in the NIST Chemistry WebBook database [28], and on the comparison of mass spectra with those reported in the NIST/EPA/NIH Mass Spectra Library 2005. To evaluate the relative abundance of each EO constituent (expressed as percentage content) peak area normalization measurement was carried out, based on average data obtained by a duplicate GC run on the same EO diluted sample.

Statistical analysis

A 2-way ANOVA was performed on plant growth data and on EO (yield and composition) data to highlight significant effects associated to each of the experimental factors and to their interactions. A 1-way ANOVA was conducted on plants temperature data. When significant effects were observed, the Tukey’s multiple comparison test was performed to highlight significant differences between the levels of the two factors. ANOVA and Tukey’s test were performed by using the XLStat software (ver. 2016.05; Addinsoft, New York, NY).

Dataset on EO composition was formed by average (n = 2) data on 28 constituents determined on the 27 EO samples (9 treatments × 3 plant replicates): percentage data on relative content of EO constituents were transformed by the arcsin function before statistical analysis. A principal components analysis (PCA) was carried out on EO constituents data by using the PAST software [29].

Results and discussion

Effects on plant growth

Both whole plant fresh weight and dry weight were negatively affected by LI reduction (Table 1). In plants exposed to a 25% LI level, a 55% and a 59% decrease of whole plant fresh and dry weight was observed with respect to 100% LI. A similar effect of LI reduction was found on fresh and dry weight of the plant aerial part (49% and 57% decrease) and an even more strong effect on roots fresh and dry weight (77% and 72% decrease). Reduction of LI also decreased stem diameter, the number of total shoots, even though plants exposed to the highest LI showed a decrease of the nodes developed on the trunk during the last 2 months of cultivation. As regards the effects on leaf characteristics, reduction of LI induced an increase of the leaf area and a decrease of specific leaf weight, both fresh and dry.

Table 1 Data on rosemary plant growth

Differently from the effect of LI, only plant fresh weight, but not dry weight, was affected by WA, with a significant reduction at the lowest WA level (55% of field capacity) (Table 1). This effect was much smaller than that due to reduction of LI, amounted approximately to a 11% plant fresh weight decrease and could be ascribed to a reduced plant water content. The effect of WA reduction on the aerial part fresh weight (17% decrease) was similar to that one observed on the whole plant. On the contrary, reduction of WA promoted roots biomass accumulation, the lowest values being recorded at the highest WA level (85% of field capacity). Consequently, while reduction of LI increased the aerial part/roots weight ratios (fresh and dry weight ratios), the opposite effect was associated to WA reduction. Reduction of WA, similarly to LI, was associated to lowered stem diameter and number of total shoots, whereas leaf characteristics seemed to be scarcely affected by WA changes.

ANOVA results also showed the occurrence of significant interactions between the two factors for some plant growth parameters (Table 1), in particular for the aerial part weight. Evaluation of data related to individual treatments (Fig. 1) highlighted that the effect of WA reduction on formation of the aerial part biomass occurred only in plants subjected to 100% LI, whereas the influence of WA was negligible in plants exposed to lower LI levels. This observation was even more clear when evaluating the plant foliage biomass weight (Fig. 1S), instead of the global weight of the aerial part. In a similar way, the effect of WA reduction on formation of roots biomass occurred only in plants exposed to 100% LI, whereas no effect was observed at lower LI levels (Fig. 2).

Fig. 1
figure1

Aerial part fresh (a) and dry (b) weight (g) of rosemary plants grown under different light intensity (LI) and water availability (WA) conditions

Fig. 2
figure2

Roots fresh (a) and dry (b) weight (g) of rosemary plants grown under different light intensity (LI) and water availability (WA) conditions

As reported in the literature for many species, a lower LI generally reduces plant growth (in terms of biomass production), extent of branching, stem diameter, leaf thickness and specific leaf weight, and these effects may be linked to reduced photosynthetic activity, and resulting lowered carbon fixation [6,7,8, 10, 11]. In addition, a reduction of LI is associated to an increase of the aerial part/roots weight ratio, mainly due to a remarkable reduction of the root biomass, and of leaf size. This adaptation can be interpreted as a strategy to maximize the capture rate of radiant energy [30]. Similar changes in plant growth have also been found in MAP species, such as Salvia sclarea [31], Salvia officinalis [32] and Ocimum basilicum [18]. Results reported in this paper show that rosemary plant responds in a similar way to the other species reported above when subjected to increasing levels of shading. Moreover, reduction of biomass of the aerial part at the 25% LI level was higher than that observed on Salvia officinalis, another aromatic species typically cultivated in the open air [32], confirming the sun-loving nature of rosemary plant, which, accordingly, in a wild environment tends to behave like a pioneer species.

In general, plant growth is adversely affected by water stress due to a decrease in stomatal opening, which restricts CO2 diffusion into leaves and, thus, reduces photosynthetic activity [12]. Minor effects on plant growth have been obtained in this study by applying decreasing levels of WA, mainly consisting of a decrease of whole plant and aerial part biomass, stem diameter and number of total shoots, and of an increase of root biomass. In analogy to LI effects, changes associated to variation in WA can be interpreted as a plant strategy for growth optimization: in this case, under conditions of reduced WA, plant response involves enhanced investment of dry matter into structures that absorb water, while structures that consume water are downsized. This is shown also by the reduction of the aerial part/root weight ratio, similarly to that previously reported in the literature on various species, including rosemary, under conditions of water stress [9, 33]. This kind of effects occurred in the present study in spite of the relatively moderate water stress imposed: it must be remarked that this study was designed to evaluate plant growth in normal cultivation conditions, where water stresses can sometimes occur but are not usually severe. Interestingly, in the present study the combined application of two factors, LI and WA, allowed to highlight also that the above effects due to WA occurred only in plants exposed to the full solar radiation. In essence the moderate water stress condition caused a moderate effect on plant growth and this effect was observable only under conditions of maximum solar radiation and photosynthetic activity. This was in accordance with previous observations of water stress effects on rosemary plant growth [24], where it was observed that decreasing levels of WA reduced biomass formation through declining photosynthetic activity and that the effect of reduction of photosynthetic activity was higher at higher levels of photosynthetic active radiation.

Effects on EO yield

Essential oil yield, expressed as percentage of fresh biomass weight, ranged in the 9 treatments between 0.29 and 0.43% (Fig. 3), being relatively low when compared to Italian national production standard, 0.5–0.6% [34], or with data reported in the international literature, 0.5–0.9% [1]. This relatively low yield could be due to the harvest season in the present study, in mid September, whereas the highest yields are known to be obtained during late spring and early summer. Secondly, dried material used for EO extraction included almost all the upper aerial parts of the plant, comprising a higher fraction of ligneous parts than in common practice. This procedure for plant material sampling was preferred to obtain enough material for each sample to be used for EO distillation.

Fig. 3
figure3

Essential oil (EO) yield of rosemary plants grown under different light intensity (LI) and water availability (WA) conditions. EO yield expressed as % of fresh biomass (a) and as amount per plant (b)

Results from the two-factorial experiment showed that both LI and WA had a significant effect on EO yield when expressed as amount per plant, whereas only LI significantly affected EO yield when expressed as % of fresh biomass weight (Table 2). The interaction between the two factors was significant only in the case of EO yield expressed as amount per plant. Globally, the extent of the effect due to changes in LI was higher than the one related to variations in irrigation conditions. As regards the effect of LI, plants exposed to 50% sunlight produced, on average, the highest EO level when expressed as % content on fresh biomass. This situation partly changed when considering EO yield on a plant basis, due to the negative effect of decreasing levels of LI on fresh biomass production (Table 1): for this reason, EO yields on a plant basis were not significantly affected by LI reduction from 100 to 50%, whereas they were markedly reduced in plants grown under 25% LI. WA significantly affected EO yield on a plant basis, by reducing it at the lowest irrigation level (55%). All these effects may be observed in detail in Fig. 3, where mean values for each treatment are reported. In particular, the interaction between the two investigated factors in affecting EO yield on a plant basis (Fig. 3b) is also clearly highlighted. EO yield on a plant basis became lower and lower as WA decreased, but this effect only occurred on plants exposed to 100% LI; under this light condition, EO yield on a plant basis was reduced by approximately one-third when reducing WA from 85 to 55% of field capacity (Fig. 3b).

Table 2 Data on rosemary EO yield and composition (% level of each EO constituent)

Terpenoids, which are the constituents of rosemary EO, are secondary metabolites synthesized in leaf protuberances termed peltate glandular trichomes from freshly fixed carbon and their accumulation is thought to depend on CO2 acquisition from air and formation of photosynthesis intermediates [15]. Differently from what observed in the present study, a decreased % EO content of the fresh biomass in plants exposed to reduced solar radiation has been observed in Thymus vulgaris [16], Origanum vulgare [17], Ocimum basilicum [18], Mentha piperita [19], and has been generally attributed to reduced photosynthetic activity and corresponding level of basic metabolites. Results from other studies also suggested that more intense solar radiation can lead to higher accumulation of secondary metabolites as part of a plant defence mechanism against exceeding level of radiation [21]. On the contrary, in other cases, shading of solar radiation was associated to increased % EO content of the fresh biomass as observed in Salvia officinalis [16], in Eucalyptus citriodora [21] and Mentha piperita [20]. In these two last cases, this was explained based on the protection conferred by shading against the severe radiation stress imposed on the plants by exposure to very intense and harmful irradiance level. This explanation, however, did not seem to apply to the enhanced EO yield observed at the 50% sunlight level in the present study, because in this case plants exposed to the highest LI also produced the highest level of biomass, thus not denoting a condition of severe radiation stress. In accordance with this observation, leaf temperatures recorded throughout a day in August (Table 3) suggests that the aerial part of plants grown at 100% sunlight level did not experience a heat stress condition more than the other plants.

Table 3 Temperature (°C) of leaves and pot external surface recorded throughout a day during the last phase of growth on plants subjected to the different levels of light intensity

Data from the present study suggest that at a low level of LI (25% of natural sunlight) the reduced amount of radiation is the dominant factor in determining a lowered production of biomass and EO, according to most results reported in the above cited literature. On the contrary, at the highest level of LI, production of EO seems to be decoupled from biomass production. For this unexpected result one possible explanation may be proposed. Results reported in Table 1 show that the number of nodes developed on the trunk in the last 2 months of growth was much lower in plants exposed to full sunlight than under shading. This means that in the final time of cultivation leaves on plants under full sunlight were, on the whole, older than the ones on plants exposed to the other LI treatments, and so volatiles produced on these leaves could have been already partially lost at the harvest time. The growth rate reduction of these plants during the last phase of growth (mid and late summer) may be associated to a heat stress occurred at the roots level, only during this phase, as the temperature of pot external surface during the day tended to be higher at full sunlight than under shading, rising up to more than 48 °C in the afternoon (Table 3). When considering the effects of WA, differently from what observed in the present study, reduction of WA strongly increased EO % content of fresh biomass in a previous study on rosemary [24]. However, in that experiment [24], the extent of WA reduction was much higher (50 to 100% reduction of estimated plant water use was compared to 0% reduction) than in the present study, where the purpose was to explore irrigation conditions within ranges compatible with crop cultivation management rather than evaluation of extreme environmental stress conditions. As a consequence, the smaller extent of WA reduction may explain the absence of an effect on EO % content of fresh biomass in the present study. In the previous study, the strong increases in EO % content of fresh biomass corresponded to slight increases or reduction of EO yield when expressed on a plant basis, due to the parallel reduction of produced biomass [24]. For the same reason, in the present study the absence of an effect of water reduction on EO % content of fresh biomass corresponded to a reduction of EO yield per plant. This effect occurred only in plants exposed to 100% LI, because only these plants experienced a significant decrease of biomass formation due to WA reduction (Fig. 2S), for the reason discussed above. In any case, results from plants exposed to full sunlight highlighted a marked negative effect of reduction of WA on productivity, in terms of EO yield, and this would suggest taking into account this effect when applying water shortage strategies in the cultivation of this crop.

Effects on EO composition

GC–MS analysis allowed to determine 28 major constituents of the EO of the “erect rosemary” variety used for the experiment, whose profile resulted to be similar to that one of the ecotype Cevoli from a not distant geographical area [34]. Determined compounds belonged to the chemical classes of monoterpene hydrocarbons (n. 12), oxygenated monoterpenes (n. 10), sesquiterpenes (n. 2) and unidentified terpenoid compounds (n. 4), whereas major constituents were α-pinene (28.9–39.4%), 1,8-cineole (12.3–13.8%), camphene (6.3–8.2%), camphor (5.1–9.0%) and borneol (4.3–9.8%) (Table 2). Global effects of the investigated factors on the EO profile were shown by results of the PCA reported in Fig. 4. A substantial percentage of the variance within the dataset was explained by the first two PCs, 91.6% and 4.8%, respectively. Similarly to what observed on EO yield data, the effect of LI was more pronounced than that associated to WA, as highlighted by the fact that treatments corresponding to the same LI level can be grouped, as shown in Fig. 4, whereas this was not possible for treatments belonging to the same WA level. PC1 tended to discriminate plants subjected to 50% LI from plants exposed to the other light levels, whereas PC2 discriminated plants subjected to 100% and 25% LI. Data on PC loadings (Table 1S) highlighted a high contribution of α-pinene (positive loadings), borneol, verbenone and camphor (negative loadings) to the PC1, and a high contribution of borneol, 1,8-cineole (positive loadings), verbenone and bornyl acetate (negative loadings) to the PC2. A higher impact of the evaluated light conditions than irrigation regimes was also confirmed by results of ANOVA of data on EO composition (Table 2). LI conditions affected the percent level of 27 out of the determined 28 EO constituents, whereas WA affected the level of only 6 compounds. Interactions between the two factors were not significant. Again, as regards the effect of LI, major differences were observed between plants subjected to 50% LI and the other two groups of plants. A group of compounds, such α-pinene, 1,8-cineole, camphene, β-pinene, myrcene, α-phellandrene, α-terpinene and β-caryophillene showed enhanced relative abundances in plants exposed to 50% sunlight, whereas a second group of compounds, such as 3-carene, camphor, borneol, 1-terpinen-4-ol, α-terpineol, verbenone, humulene and three unknown compounds (unknown 1, 2, 3), showed lowered abundances in these plants. Major changes due to reduction of light intensity from 100 to 50% were observed for α-pinene (on average + 24%), camphor (− 24%), borneol (− 44%) and verbenone (− 45%). ANOVA results also allowed to highlight effects of WA level on EO composition. Reducing water availability significantly lowered the relative content of α-pinene (on average − 14%), myrcene and p-cymene, while increased the relative abundance of borneol (+ 38%), bornyl acetate (+ 41%) and compound unknown 4 (Table 2).

Fig. 4
figure4

PCA of data of composition of rosemary EOs obtained from plants subjected to different levels of light intensity and water availability. Bi-plot (score and loadings plot) of the first two PCAs. Labels in black character correspond to data points of the score plot (EO samples). The first number of the label denotes the level of LI (100%, 50% or 25% of natural sunlight), the second number denotes the level of WA (85%, 70% or 55% of field capacity), the third number denotes the treatment replicate (1, 2 or 3). Labels in blue character correspond to data points of the loadings plot (EO constituents). Coloured area are delimited by convex hulls, which are the smallest convex polygon enclosing all the data points (EO samples) corresponding to each level of LI (green: 25% LI; red: 50% LI; blue: 100 LI)

Biosynthesis of terpenoids occurs through diverse pathways, by which precursors of both monoterpenoids (geranyl diphosphate) and sesquiterpenoids (farnesyl diphosphate) are formed. Then, from these precursors all the other members of the groups are formed by complex series of reactions, which are all under enzymatic control [35]. Some of the key enzymes involved in monoterpene biosynthesis (monoterpene synthases) have been studied in detail in Salvia officinalis, where some pinene synthases have been recognized as responsible for the synthesis of α- and β-pinene and camphene, and to contribute to the synthesis of myrcene, whereas bornyl diphosphate synthase has been recognized as responsible for the synthesis of borneol and camphor, and 1,8-cineole synthase for the synthesis of 1,8-cineole [36]. Results from several studies have shown that changes in LI and WA can have a significant effect on EO composition [17,18,19, 22, 37], whereas different mechanism have been postulated to mediate these effects [19, 22, 37]. In the present study, major changes in EO composition where observed in plants subjected to 50% sunlight. Formation of compounds catalysed by pinene synthase enzymes, as for the major constituent α-pinene, but also β-pinene, camphene and myrcene, seemed to be significantly promoted by the 50% LI condition, when compared to the other LI levels, whereas the contrary was observed for compounds formed through the activity of bornyl diphosphate synthase, such as borneol and camphor. Also the relative abundance of 1,8-cineole, which is formed by 1,8-cineole synthase, is enhanced in plants exposed to 50% LI, even though the extent of this effect was smaller than that observed on α-pinene. An effect of the considered LI conditions on these enzymatic activities may be involved in the observed changes in the EO profile and could be confirmed by future investigations on other rosemary cultivars. Interestingly, verbenone, which may be produced by conversion of α-pinene, was markedly influenced by light conditions in an opposite way when compared to its plausible precursor α-pinene. A similar opposite effect on pinene synthase derived compounds and bornyl diphosphate synthase derived compounds was also observed in relation to irrigation regimes, where reduction of WA promoted the accumulation of borneol and bornyl acetate (bornyl diphosphate synthase related compounds), while inhibiting the formation of α-pinene and myrcene (pinene synthase related compounds). Interestingly, the effects observed on borneol and bornyl acetate, and also a similar though not significant effect on camphor, seemed to confirm recent results by Radwan et al. [37], who observed that a moderate drought stress strongly up-regulates bornyl diphosphate activity in Salvia officinalis, thus promoting the accumulation of camphor in that species. On the contrary, in a previous study on rosemary, no changes in EO composition were observed in plants subjected to severe drought stress [24]. In any case, in the present experiment, it seemed that changes in both light and water conditions affected in opposite ways the formation of compounds derived from the activity of pinene synthase and bornyl diphosphate synthase.

Conclusions

In the pot growing conditions considered in the present experiment changes in LI markedly affected rosemary plant growth and architecture as well as EO yield. High shading of natural sunlight markedly hindered plant biomass production and EO accumulation, whereas intermediate shading conditions significantly promoted EO yield when expressed as % content of fresh biomass, but not significantly when expressed as amount per plant, with respect to 100% natural sunlight. Reduction of WA, within ranges compatible with crop cultivation conditions, may markedly reduce crop productivity in terms of EO yield on a plant basis, but only when combined with exposure to full sunlight radiation. This highlights the importance of taking into account the possible occurrence of interactions between multiple factors affecting EO yield when evaluating the influence of environmental factors in crop management. In addition both LI and WA levels significantly affected EO composition. Changes in the level of the two factors seemed to have an opposite effect on the relative abundance of volatiles that are formed through the activity of two groups of enzymes, pinene synthases and, respectively, bornyl diphosphate synthases. This may have implications on EO sensory quality, given that volatiles derived from the two enzyme groups, such as α-pinene, camphene, borneol, camphor, are known to be key odorants of rosemary EO and are characterized by different aroma notes [38]. Moreover, variation in rosemary EO composition is associated to changes in its biological properties, such as antimicrobial and antioxidant activities [39]. Thus results from the present study also suggest the importance of a careful management of light and irrigation conditions to standardize the composition of rosemary EO or to modulate it in view of different potential uses.

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Acknowledgements

The study was carried out with the financial support of the Ministry of Agriculture, Food and Forestry Policies and Tourism, Italy, within the project “Implementation of the FAO International Treaty on Plant Genetic Resources for Food and Agriculture”.

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Raffo, A., Mozzanini, E., Ferrari Nicoli, S. et al. Effect of light intensity and water availability on plant growth, essential oil production and composition in Rosmarinus officinalis L.. Eur Food Res Technol 246, 167–177 (2020). https://doi.org/10.1007/s00217-019-03396-9

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Keywords

  • Rosemary
  • Terpenoids
  • Monoterpene synthases
  • Solar radiation
  • Aroma
  • Irrigation