Introduction

Due to their sessile nature, plants are constantly subjected to various abiotic factors. Hence, recognizing these sources of stress and deploying mechanisms to counteract them is essential for their survival (Barkla et al. 2013).

Water deficit is the most critical abiotic factor limiting plant growth, their morphology, as well as physiological and biochemical processes (Bosabalidis and Kofidis 2002; Wu et al. 2008; Cairns et al. 2012; Ghotbi‐Ravandi et al. 2014; Maatallah et al. 2016; Allahverdiyev 2016; Kumar et al. 2017). Growth is particularly compromised as low water availability causes loss of turgidity, thus reducing cell division and elongation (Lawlor and Cornic 2002; Jaleel et al. 2008; Ge et al. 2012). Water scarcity promotes also the formation of reactive oxygen species, such as superoxide radicals, singlet oxygen, hydroxyl radicals, and hydrogen peroxide, which impair protein, nucleic acid, and lipid synthesis (Mittler et al. 2004; Ozkur et al. 2009; Xie et al. 2016). Furthermore, plants respond to water deficit by inducing stomatal closure, restricting water loss by transpiration, reducing CO2 diffusion from intercellular spaces to the chloroplast and, ultimately, decreasing photosynthetic rates (Flexas et al. 2004; Chaves et al. 2009; Li et al. 2017).

Cellular and physiological responses to water limitation allow plants to mitigate cell damage while adapting to this condition. Responses include biosynthesis and accumulation of organic solutes such as proline, which plays an important role in osmotic adjustment (Hasegawa et al. 2000; Merwad et al. 2018), and synthesis of free radical scavenging enzymes, such as peroxidase, catalase, peroxide dismutase, and ascorbate peroxidase (Sofo et al. 2015). In addition, hormonal regulation determines changes in phytohormones, such as abscisic acid, cytokinins, gibberellic acid, auxins, and ethylene (Sharp 2002; Du et al. 2010; Wilkinson and Davies 2010; Peleg et al. 2011; Xuemei et al. 2011; Wilkinson et al. 2012).

Secondary plant metabolism is also modulated by water deficit, as manifested by variations in the quality and quantity of essential oils produced by aromatic plants (Petropoulos et al. 2008; Yadav et al. 2014; Mandoulakani et al. 2017; Morshedloo et al. 2017).

The biosynthesis of essential oil components occurs via two distinct pathways: the mevalonate pathway (MEV) and the methylerythritol phosphate pathway (MEP). The MEV pathway occurs in the cytosol, where sesquiterpenes are synthesized from the precursors pyruvate and acetylcoenzyme-A. The MEP pathway occurs in the plastids, where pyruvate and glyceraldehyde-3-phosphate are used as the precursors for the synthesis of monoterpenes (Chemat et al. 2013; Pérez Zamora et al. 2018). Examples of sesqui- and monoterpene synthesis genes are, respectively, nerolidol synthase and geraniol synthase (Castro et al. 2019).

The genus Lippia is distributed throughout Latin America and Africa, where it has adapted to various environments. Lippia alba (Verbenaceae) has great economic and social importance due to its essential oil rich in bioactive compounds (Vieira et al. 2016). The oil can prevent and treat cardiovascular disease, bronchitis, cough, asthma, stomach, and intestinal disorders (Pascual et al. 2001; Lorenzi and Matos 2008; Raut and Karuppayil 2014; Amin and Hosseinzadeh 2016; Saljoughian et al. 2018); it can be used as a food preservative (Peng and Li 2014; Szczepanski and Lipski 2014; Otoni et al. 2016; Pola et al. 2016); and it can serve as a pest control product in the agrochemical industry (Pavela and Govindarajan 2017; Benelli et al. 2018). Thus, the species is of interest for various applications, from traditional medicine to modern pharmacological and agrochemical industries.

The tissue culture techniques is an important tool to access the effects of abiotic stress on plants, as it creates conditions to better isolate these effects, allowing to develop resistant or tolerant lines for abiotic stresses (Pérez-Clemente and Gómez-Cadenas 2012). Given the wide applicability of essential oils, it is important to understand how different environments alter their production and biosynthesis routes. In spite of its relevance as a medicinal species, there is limited knowledge of how abiotic factors such as water deficit modulate primary and secondary metabolism in L. alba. The objective of this study was to evaluate the effect of water deficit on in vitro growth, photosynthesis, essential oil profile, and the expression of genes related to the biosynthesis of these compounds in L. alba.

Materials and methods

Plant material

Lippia alba plants (chemotype BGEN-04) were obtained from the Germplasm Bank of the Department of Biology, Federal University of Juiz de Fora (UFJF, Juiz de Fora, MG, Brazil), they were micropropagated from original plants granted by Embrapa Genetic Resources and Biotechnology (Cenargen, Brasília, DF, Brazil). The specimens were deposited at the Leopold Krieger Herbarium (Herbarium CESJ 48,372, UFJF). The plants were propagated in vitro in MS medium (Murashige and Skoog 1962) supplemented with 30 g L−1 sucrose, 100 mg L−1 myo-inositol, and 6.5 g L−1 Merck agar (Merck Millipore Corp., Darmstadt, Germany). The pH of the medium was adjusted to 5.7 ± 0.01. The medium was autoclaved at 120 °C and 108 kPa for 20 min.

Effect of water deficit

Eight nodal segments (~ 2 cm in length) were inoculated into glass flasks (600 mL capacity) containing 80 mL MS medium supplemented with 30 g L−1 sucrose, 100 mg L−1 myo-inositol, and 6.5 g L−1 Merck agar. The pH of the medium was adjusted to 5.7 ± 0.01. The medium was autoclaved at 120 °C and 108 kPa for 20 min. To induce an increasingly severe water deficit, polyethylene glycol (PEG) was added to the culture medium at three concentrations: 0, 1, 2, and 3% (w/v). Cultures were maintained in a growth room at 25 ± 1 °C, at a 16-h light photoperiod (from 6 to 22 h), and constant irradiance of 41 μmol m−2 s−1 provided by two white LED bulbs (SMD 100, 18 W; Vilux®, Vitória, ES, Brazil). The flasks were sealed with rigid polypropylene lids with two vents (10 mm diameter) covered with 0.45-μm membranes (MilliSeal® AVS-045 Air Vent, Millipore Merck, Billerica, MA, USA), allowing a CO2 exchange rate of 25 µL L−1 s−1 (Batista et al. 2017b).

After 40 days of culture (Fig. 1), the following parameters were analyzed: primary metabolites, growth, profile of essential oils, and expression of the genes nerolidol/linalool synthase and geraniol synthase. All samples were collected at 17 h.

Fig. 1
figure 1

Representative images of Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). Bar = 2 cm

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Biometric analysis

The following biometric analysis were performed: fresh mass (g), dry mass (g) by oven-dried at 60 ºC until constant weight, total shoot length (cm), total length of the largest root (cm), and leaf area (cm2). The latter was measured using ImageJ software (Schneider et al. 2012).

Quantification of photosynthetic pigments, carbohydrates, proteins, and amino acids

Samples from the shoots were collected, frozen in liquid nitrogen, ground, and lyophilized. To determine the concentration of photosynthetic pigments, approximately 10 mg of lyophilized tissues were used for extraction with acetone as described by Welburn (1994). For the determination of carbohydrates (sucrose, glucose, and fructose), 25 mg of lyophilized tissues were used in the extraction with methanol and evaluated as described by Fernie et al. (2001). Quantification of protein and total amino acids was conducted as suggested by Cross et al. (2006).

In vitro photosynthetic rate

Gas exchange and quantification of in vitro photosynthetic rate were performed as proposed by Costa et al. (2014) with modifications. An AQ-S151 infrared CO2 analyzer (Qubit Systems, Kingston, ON, Canada) was used for measurements and data collection was performed using LoggerLite 1.8.1 software (Vernier Software & Technology, Beaverton, OR, USA). Reference CO2 was calculated by inflowing air into an empty flask, pumped from the external environment at a constant air flow rate of 300 mL min−1, located within an illuminated chamber (white LED bulbs). The plants were maintained in the dark for a period of 8 h prior to analysis. Soon after measuring the reference CO2, the flasks containing the plants were coupled to the system and the CO2 was calculated at the stabilization point. Gas exchanges were calculated by computing the difference between the reference CO2 and the CO2 of the plants exposed to atmospheric air. Air temperature and humidity in the flask were measured by a Spec sensor (Thermo Recorder RS-11, Takai Spec Corp., Aichi, Japan).

The in vitro photosynthetic rate (A) was calculated by the following formula:

$${\text{A}} \left( {\upmu {\text{mol m}}^{ - 2} {\text{s}}^{ - 1} } \right) = \frac{{\Delta {\text{CO}}_{2} }}{{\text{Mol Flow}}}$$

where, \(\Delta {\text{CO}}_{2} \left( {{\text{ppm}}} \right) = {\text{Reference CO}}_{2} - {\text{Analysis CO}}_{2}\)

$${\text{Mol Flow }} = \frac{{{\text{Air flow rate }}\left( {{\text{L min}}^{ - 1} { }} \right){ }}}{{\left( {\frac{{{ }({\text{ Constant for perfect gases}}\left( {22.4} \right){ } \times {\text{ Temperature }}\left( {\text{K}} \right){ }}}{{\frac{6,00,000}{{\left( {{\text{Leaf dry weight per plant }}\left( {\text{g}} \right)} \right)}}}}} \right)}}.$$

Microextraction of essential oils

For microextraction of essential oils, 300 mg of leaves were collected and stored at − 18 °C in test tubes with screw caps. After freezing, 1 mL hexane and 0.5 mL methanol were added to each sample. The samples were kept in an ultrasonic bath (Thornton-INPEC, Vinhedo, SP, Brazil) at 70 kHz and room temperature for 1 h. Subsequently, the supernatant was filtered through a sterile cotton wick. The resulting samples consisting of a 1-μL clear solution containing the extracted oils were analyzed by gas chromatography.

Qualitative analysis of essential oils

Qualitative analysis of essential oils was carried out using a gas chromatographer coupled to a mass spectrometer (GCMS-QP2010 Plus; Shimadzu, Suzhou, China) and Rtx-5MS® column (Restek, Bellefonte, PA, USA) of 30 m × 0.25 mm. The oven temperature was set to 70 °C for 3 min and then increased by 6 °C min−1 to 300 °C. The injector was operated in split mode (1:10) at 240 °C. The interface and mass detector were operated at 300 °C. Helium was used as the carrier gas, with a flow of 1.53 mL min−1. A standard mixture of linear hydrocarbons (C9H20, C10H22…, C25H52, and C26H54) was injected under the same conditions as the samples. Identification of the constituents was performed by comparing the obtained mass spectra with those in the NIST 9.0 database (correlation > 97%) and confirmed by their retention indices (Kováts Index), which were calculated for each constituent and compared to published data (Adams 1997).

Extraction of mRNA, cDNA synthesis, and analysis by real-time PCR (RT-qPCR)

Total RNA was isolated from the leaves with TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and treated with DNAse I (Invitrogen) following the manufacturer’s recommendations. The quality and quantity of the RNA and cDNA were determined by a NanoDrop ND-2000 (NanoDrop Technologies, Wilmington, DE, USA). cDNA was synthesized using reverse transcriptase (Ludwig Biotec, Bela Vista-Alvorada, RS, Brazil) and 800 ng of total RNA. RT-qPCR was performed on a CFX96 Touch™ cycler (Bio-Rad, Hercules, CA, USA). The total reaction volume was 10 μL:1 μL cDNA, 3 μL diethyl pyrocarbonate water, 2 μL of 4 μM forward and reverse primers, and 4 μL SYBR-Green mix/Rox (Ludwig Biotec). Primers for nerolidol/linalool synthase (LaNES/LIS), geraniol synthase (LaGES), and the reference gene alcohol dehydrogenase (LaADH) were designed based on the de novo transcriptome sequence of L. alba.

Statistical analysis

Experiments were conducted following a completely randomized design. The experimental unit consisted of a culture flask containing eight plants. Statistical analyses were performed using Genes software version Windows/2004.2.1 (Cruz 2016). Data were submitted to analysis of variance (ANOVA) by the F test and the means were compared by the Scott and Knott test (P ≤ 0.05). RT-qPCR expression levels were calculated by the 2−ΔΔCt method (Livak and Schmittgen 2001) with three biological replicates and at least three technical replicates. Means were compared by Dunnett’s test (P ≤ 0.05).

Results

Water deficit slows the growth of Lippia alba plants in vitro

Water deficit affected growth and development of Lippia alba grown in vitro (Fig. 1). Shoot fresh and dry mass were higher in the control than in other treatments (Fig. 2a, b); whereas root fresh/dry mass and length (Fig. 2c, d, and f), as well as stem length (Fig. 2e) exhibited no significant difference. Leaf area was higher in the control and 1% PEG condition than in the 2 and 3% PEG treatments, in with water deficit induction was more severe (Fig. 2g). Finally, total chlorophyll content (Fig. 2h) showed no significant difference.

Fig. 2
figure 2

Growth variables of Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). a Shoot fresh weight, b shoot dry weight, c root fresh weight, d root dry weight, e shoot length, f root length, g leaf area, and h total chlorophyll. Data are presented as means (n = 4) and vertical bars denote the standard error. Equal letters indicate no difference according to the Scott & Knott test at 5% probability

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Water deficit augments total protein and amino acid contents, but not sugar levels in Lippia alba grown in vitro

Glucose, fructose, and sucrose levels were not modulated by water deficit treatments (Fig. 3a–c). In contrast, total protein levels increased in the treatments with 2 and 3% PEG (Fig. 3d). Amino acid levels were higher in plants exposed to 2% PEG (Fig. 3e).

Fig. 3
figure 3

Sugar, protein, and amino acid contents of Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). a Glucose, b fructose, c sucrose, d protein, and e amino acids. Data are presented as means (n = 4) and vertical bars denote the standard error. Equal letters indicate no difference according to the Scott and Knott test at 5% probability

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Severe water deficit reduces Lippia alba photosynthesis in vitro

In vitro photosynthesis of L. alba was reduced in plants grown in the presence of 3% PEG, and manifested as a significant decrease in photosynthetic rates compared to the other treatments (Fig. 4).

Fig. 4
figure 4

Photosynthetic rate (a) of Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). Data are presented as means (n = 4) and vertical bars denote the standard error. Equal letters indicate no difference according to the Scott and Knott test at 5% probability

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Linalool content increases in Lippia alba plants grown in vitro under water deficit

Water deficit changed the profile of essential oils in L. alba. Specifically, linalool content was significantly higher in plants subjected to water deficit induced by different levels of PEG than in control plants (Fig. 5); whereas germacrene content exhibited the opposite trend and was significantly higher in the control. Eucalyptol content did not differ statistically among the treatments.

Fig. 5
figure 5

Profile of essential oils in Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). Data are presented as means (n = 4) and vertical bars denote the standard error. Equal letters indicate no difference according to the Scott and Knott test at 5% probability

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Water deficit does not alter the expression of nerolidol/linalool synthase and geraniol synthase in Lippia alba

The relative expression of LaNES/LIS and LaGES was not altered by different water deficit levels (Fig. 6).

Fig. 6
figure 6

Geraniol synthase (LaGES) and neralidol/linalool synthase (LaNES/LIS) expression levels in Lippia alba after 40 days of in vitro culture under different concentrations of PEG (0, 1, 2, and 3%). Expression is normalized to that of alcohol dehydrogenase (LaADH). Data are presented as means (n = 3) and are compared by Dunnett's test at 5% probability, vertical bars denote the standard error

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Discussion

Abiotic factors are constantly modulating the primary and secondary metabolism of plants, affecting yield and/or composition of essential oils (Bahreininejad et al. 2014; Batista et al. 2016, 2017a; Mahmoud et al. 2018). Several studies have found a correlation between water deficit and essential oils profile (Yadav et al. 2014; Mandoulakani et al. 2017; Morshedloo et al. 2017). However, the present study is the first to provide a comprehensive picture of the effect of water deficit on primary metabolism, growth, photosynthesis, the profile of essential oils, and the expression of genes related to their biosynthesis in L. alba grown in vitro.

Regarding primary metabolism, this work evidences how the more severe water deficit treatments, using 2 and 3% PEG, promoted a decrease in shoot fresh/dry mass and reduced the leaf area. These results confirmed earlier observations by Ghotbi-Ravandi et al. (2014) on the negative effect of water deficit on the dry matter of Hordeum vulgare L. genotypes, and by Maatallah et al. (2016) on the decrease in relative growth rate, leaf area, and chlorophyll content following water deficit in Laurus nobilis L. Similarly, Allahverdiyev (2016) reported a reduction of leaf area in wheat cultivars subjected to water deficit. In general, the reduction in plant biomass caused by water deficit is related to a suppression of cell expansion. Specifically, the decrease in turgor pressure caused by lower cell water content results in reduced growth (Jaleel et al. 2008). Moreover, reducing leaf expansion is one of the strategies employed by plants to mitigate low water availability, resulting in decreased leaf area, stomatal closure, lower transpiration, and limited photosynthesis (Ge et al. 2012).

In L. alba plants grown in vitro, the water deficit elicited by treatment with 3% PEG promoted a reduction of photosynthetic rate. This response could be caused by limited CO2 diffusion from intercellular spaces to the chloroplast, which may constrain CO2 fixation (Flexas et al. 2004; Chaves et al. 2009). Water restriction often culminates in stomatal closure, an effective mechanism against water loss (Cornic 2000; Loreto et al. 2003; Molnar et al. 2005; Chaves et al. 2009), this effect was already observed in Lippia alba plants in vivo under water stress, where there was a decrease in stomatal conductance (Oliveira and Leite 2017) Li et al. (2017). observed a decreased photosynthetic rate in potato seedlings under severe water deficit, suggesting that this reduction was due to stomatal limitation and the damage to photosystem II and antioxidant enzymes. Ghotbi-Ravandi et al. (2014) reported a reduction in both CO2 assimilation rate and stomatal conductance in barley genotypes under severe water stress.

Here, water deficit did not affect the levels of glucose, fructose, and sucrose, but the 2% PEG treatment increased total amino acids content. In dry conditions, many metabolites accumulate to act as regulatory osmolytes. This is one of the mechanisms that plants develop to counteract environmental stresses such as drought (Seki et al. 2007). Sugars (raffinose family oligosaccharides, sucrose, trehalose, and sorbitol), amino acids (proline), sugar alcohols (mannitol), and amines (glycine, betaine, and polyamines) are considered compatible solutes that can be concentrated in the cell cytoplasm to promote water balance between vacuole and cytoplasm (Per et al. 2017; Merwad et al. 2018; Naeem et al. 2018).

Total protein content increased in plants subjected to moderate and severe water deficit conditions. Xie et al. (2016) observed that, compared to controls, water deficit increased and decreased the content of 260 and 206 proteins, respectively. Most of these proteins were involved in photosynthesis, metabolism, stress, and defense, indicating that water stress could modulate protein synthesis and influence plant physiology, promoting different stress responses.

Treatments in which water deficit was induced by 1, 2, and 3% PEG influenced the profile of essential oils, increasing linalool content but decreasing germacrene content. Similar effects under severe water stress have also been reported earlier, with increased content of methyl chavicol, methyl eugenol, β-myrcene, and α-bergamotene in Ocimum basilicum L. (Mandoulakani et al. 2017). Morshedloo et al. (2017) also reported that water deficit up- and down-regulated specific essential oil components in subspecies of Origanum vulgare L. resulting in increased content of essential oil. Yadav et al. (2014) observed a reduction in the density of glandular trichomes in leaves of Artemisia annua L., with a consequent decrease in the content of essential oils. On the other hand, Cruz et al. (2014), in a study with Lippia gracilis plants in the field, found no variation among plants subjected to different irrigation conditions, which highlights the importance of in vitro studies that offer more controlled conditions to detect subtle variations in secondary metabolism.

The relative expression of genes involved in the biosynthesis of L. alba essential oil components was not significant in the present study. This finding contrasts with that by Mandoulakani et al. (2017), who observed that essential oil biosynthesis genes in Ocimum basilicum L. were differentially expressed under the most severe water stress condition (50% field capacity). Specifically, O-methyl transferase and eugenol O-methyl transferase expression were increased by approximately 6.0 and 46.0 fold, respectively, which correlated strongly with the content of the essential oil compounds methyl chavicol and methyleugenol. At the same time, this increase was accompanied by a reduction in the expression of 4-coumarate:CoA ligase and cinnamate 4-hydroxylase, two enzymes involved in the metabolism of hydroxycinnamic acid.

In conclusion, our results show that water deficit is capable of modulating the primary metabolism of Lippia alba, as indicated by a reduction in growth, development, and physiological functions. Secondary metabolism was also slightly altered, with linalool and germacrene levels modified in plants under water deficit. Thus, the medicinal species L. alba can adapt to low water availability environments, while still being able to grow and produce essential oils.