Introduction

The photoperiod is established by the rotation of the Earth around its own axis and around the sun, and impacts significantly the metabolism of most living beings (Serrano-Bueno et al. 2017). In plants, several process are controlled by photoperiod, like growth, flowering induction, cold tolerance and regulation of the circadian clock (Bordage et al. 2016; Barros et al. 2017; Shin et al. 2017; Maurya and Bhalerao 2017; Mahmud et al. 2018; Triozzi et al. 2018).

Interactions between the circadian clock, primary metabolism, and signaling are thought to explain the importance of rhythmic behavior for plant growth and survival (Harmer et al. 2000; Dodd et al. 2005; Shin et al. 2017). Despite a variety of reports involving the circadian regulation of primary metabolism pathways in plants, it remains to be determined how rhythmic oscillators are influenced by metabolism (Dodd et al. 2005).

Several rhythmic genes are involved in regulating the endogenous clock in plants. In Arabidopsis thaliana, CCA1, late elongated hypocotyl (LHY), pseudo response regulator (PRR), and timing of cab expression 1 (TOC1) can be up- or down-regulated throughout the day and night (Ito et al. 2009; Huang et al. 2012; Haydon et al. 2013). CCA1 and LHY (Alabadí et al. 2001) are expressed together with PRR5, 7, and 9 (Farré et al. 2005) in the morning only. Similarly, TOC1 (Ito et al. 2009), gigantea (GI) (Park et al. 1999), early flowering 3 and 4 (ELF3/4), and luxar rhythmo (LUX) (Lu et al. 2009; Nusinow et al. 2011) transcripts become more numerous during the night period.

Some authors have sought a correlation between the plant endogenous clock and secondary metabolism. Barbosa et al. (2012) analyzed the influence of circadian rhythm on the composition of Piper divaricatum essential oils, but found no change in safrole content among leaves collected at different times of the day. Thus, the regulation of genes involved in the biosynthesis of essential oils remains unknown. Some transcription factors were predicted to regulate the expression of genes or groups of genes in different plant species. For example, the synthesis of isoprenoids in Populus is regulated by the transcription factors LHY and CCA1 (Loivamäki et al. 2007).

Medicinal and aromatic plants have been domesticated for thousands of years as a source of natural compounds with a broad range of pharmacological applications (Böhme et al. 2014; Wagas et al. 2016). The increasing interest in these compounds associated with technological advances has enabled large-scale production of secondary metabolites for pharmaceutical use (Wei et al. 2016).

Essential oils are bioactive compounds used by the pharmaceutical, cosmetic, food, and agrochemical industries. They are used in the treatment of cardiovascular diseases, bronchitis, cough, asthma, as well as stomach and intestinal disorders (Pascual et al. 2001; Lorenzi and Matos 2008; Saljoughian et al. 2018), whereby they provide analgesic, anti-inflammatory, anti-ulcerogenic, and anti-helmintic relief (Raut and Karuppayil 2014; Amin and Hosseinzadeh 2016). Furthermore, these compounds may also be used in controlling pests (Pavela and Govindarajan 2017; Benelli et al. 2018), neutralizing oxidizing agents (Proestos et al. 2014), and promoting food preservation (Peng and Li 2014; Szczepanski and Lipski 2014; Otoni et al. 2016; Pola et al. 2016).

Lippia alba has great cultural and economic importance, due to the medicinal properties of its essential oil extract (Pascual et al. 2001; Lorenzi and Matos 2008). A peculiarity of this genus is the chemical variability among its genotypes (chemotypes), with respect to the composition of essential oils and non-volatile compounds (Hennebelle et al. 2008). Furthermore, L. alba is characterized by high phenotypic and genomic plasticity (Reis et al. 2014; Viccini et al. 2006, 2014).

The physiological plasticity of this genus may help respond to different abiotic factors, which may affect secondary metabolism, and particularly the profile of essential oils (Ragagnin et al. 2014; Batista et al. 2016, 2017a; Lazzarini et al. 2018). Among these factors, spectral variation of light (quality), irradiance (intensity), CO2 concentration, and temperature seem to be particularly important (Morais 2009; Batista et al. 2016, 2017a; Lazzarini et al. 2018).

With the growing demand for essential oils in the pharmaceutical industry, L. alba is a relevant medicinal species, with emphasis on its capacity to modulate the profile of essential oils due to abiotic factors (Batista et al. 2016, 2017a). Such an insight will help establish strategies to optimize the production of essential oils in L. alba. Moreover, there have been no studies to date examining the influence of photoperiod on the metabolism and production of essential oils in this species, and new findings may be interesting even to extrapolate this relationship between photoperiod and secondary metabolism to other species. Hence, the objective of this work was to evaluate the effect of photoperiod on anatomy, growth, profile of essential oils, and expression of genes related to the synthesis of essential oils and circadian clock in L. alba cultured in vitro.

Materials and methods

Plant material

Lippia alba (BGEN-04) was obtained from the Germplasm Bank of the Department of Biology, Federal University of Juiz de Fora (UFJF, Juiz de Fora, MG, Brazil), and the propagules were granted by Embrapa Genetic Resources and Biotechnology (Cenargen, Brasília, DF, Brazil). The specimens were deposited in the Leopold Krieger Herbarium (Herbarium CESJ 48372, 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 at the Plant Tissue Culture Laboratory (LCT II), located at the Institute of Biotechnology Applied to Agriculture—BIOAGRO, Federal University of Viçosa (UFV).

Effect of photoperiod

Eight nodal segments (~ 2 cm in length) were inoculated into glass flasks (600 mL capacity) containing 80 mL of 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. Cultures were maintained in a growth room at 25 ± 2 °C and exposed to different photoperiods (4, 8, 16, and 24 h light) 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 PTFE membranes (MilliSeal® AVS-045 Air Vent, Milipore Merck) allowing for 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, anatomy, photosynthesis, profile of essential oils, as well as expression of genes involved in mono- and sesquiterpenes synthesis and the endogenous clock. All samples were collected at 17 h.

Fig. 1
figure 1

Lippia alba plants grown in vitro under different photoperiods (4, 8, 16, and 24 h) after 40 days of culture. Bar = 2 cm

Full size image

Development analysis

The following developmental characteristics were analyzed: fresh mass (g), dry mass (g), total shoot length (cm), total length of the largest root (cm), and leaf area (cm2). The latter was measured with ImageJ software (Schneider et al. 2012).

Anatomical analysis

For an anatomical structural characterization, samples of the second pair of expanded leaves, stems of the median region of the second internode, and main roots of the median region were fixed in Karnovsky solution (Karnovsky 1965). After fixation, the samples were dehydrated in increasing ethanolic series (10, 20, 30, 40, 50, 60, 70, 80, 85, 90, and 95%) and included in acrylic resin (Historesin; Leica Instruments, Jena, Germany). Cross-sections of 5-μm thickness were obtained on a rotary microtome (RM2155, Leica Microsystems Inc., Buffalo Grove, IL, USA) and stained with toluidine blue (pH 3.2) (O’Brien and McCully 1981). Slides were mounted on Permount® SP15-500 synthetic resin (Fisher Chemicals-Fisher Scientific, Springfield, NJ, USA). A light microscope (AX70 TRF; Olympus Optical, Tokyo, Japan) with the U-photo system, coupled to a digital photographic camera (Spot Insightcolour 3.2.0; Diagnostic Instruments Inc., Merrick, NY, USA) and microcomputer with Spot Basic image capture software were used to capture the images.

In vitro photosynthetic rate

Gas exchange and quantification of in vitro photosynthetic rate were performed as proposed by Costa et al. (2014) with some 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 absence of light 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:

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

where

$$\Delta {\text{CO}}_{2} \left( {\text{ppm}} \right) = {\text{Reference}}\,{\text{CO}}_{2} - {\text{Analysis}}\,{\text{CO}}_{2}$$
$${\text{Mol}}\,{\text{Flow}} = \frac{{{\text{Air}}\,{\text{flow}}\,{\text{rate}}\,\left( {{\text{L}}\,{ \hbox{min} }^{ - 1} } \right)}}{{\left( {\frac{{\left( {{\text{Constant}}\,{\text{for}}\,{\text{perfect}}\,{\text{gases}}\,\left( {22.4} \right)\, \times \,{\text{Temperature}}\,\left( {\text{K}} \right)} \right)}}{{\frac{600000}{{{\text{Leaf}}\,{\text{area}}\,\left( {{\text{cm}}^{2} } \right)}}}}} \right)}}$$

Photosynthetic pigments and quantification of carbohydrates, proteins, amino acids, and starch

Samples from the shoots were collected, frozen in liquid nitrogen, ground, and lyophilized. To determine the amount 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 (starch, sucrose, glucose, and fructose) content, 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).

Fluorescence of chlorophyll

Electron transport rate (ETR), non-photochemical quenching (NPQ), effective PSII quantum yield (Fv/Fm), and photochemical quenching (qP) were quantified in fully expanded L. alba leaves submitted to a light–dark cycle and using a MINI-PAM chlorophyll fluorometer (Walz, Effeltrich, Germany). The data for minimum fluorescence (Fo), maximum fluorescence (Fm), and variable fluorescence (Fv) were calculated as proposed by Kooten and Snel (1990). ETR, NPQ, and qP were calculated according to Maxwell and Johnson (2000).

Microextraction of essential oils

For microextraction of essential oils, ~ 300 mg of leaves were collected and stored at −18 °C in a test tube with a screw cap. 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. Resulting samples of 1 μL of clear solution containing the extracted oils were analyzed by gas chromatography.

Qualitative analysis of essential oils

The 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 started at 70 °C, maintained for 3 min, followed by an increase of 6 °C min−1 to 300 °C. The injector was operated in split mode (1:10) at 240 °C, and 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. Quantification was performed with 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), timing of cab expression 1 (LaTOC1), pseudo response regulator 5 (LaPRR5), late elongated hypocotyl (LaLHY), and the reference gene alcohol dehydrogenase (LaADH) were designed based on the de novo transcriptome sequence of L. alba (Souza et al. unpublished data).

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 & Knott test (P ≤ 0.05). RT-qPCR expression levels were calculated by the 2−ΔΔCt method proposed by Livak and Schmittgen (2001) with three biological replicates and at least three technical replicates. Means were compared by Dunnett’s test (P ≤ 0.05). Pearson correlation analysis was performed for the LaLHY, LaPRR5, and LaTOC1 gene expression data.

Results

Photoperiod interferes with growth of L. alba in vitro

Treatments with 16 and 24 h of light increased the values of all growth variables analyzed (Fig. 2a–g), with the 24-h light treatment resulting in the greatest shoot fresh weight (Fig. 2a), root length (Fig. 2f), and leaf area (Fig. 2g). Therefore, the longer was the period of light exposure, the greater was the overall growth response.

Fig. 2
figure 2

Growth variables of Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h). Means marked by the same letters do not differ according to the Scott & Knott test at a 5% probability. Data are presented as means (n = 4). Vertical bars represent the standard error

Full size image

The morphoanatomy of vegetative organs in L. alba cultivated in vitro is influenced by photoperiod

In vitro culture of L. alba over different photoperiods promoted variations in the anatomy of stems, leaves, and roots (Fig. 3). In the stem, it was possible to observe a higher cellular organization with increasing photoperiod. Specifically, plants grown under daily 16- and 24-h light regimens had a more differentiated vascular tissue, with larger and more organized bundles (Fig. 3a, d, g, j).

Fig. 3
figure 3

Cross-sections of stems (a, d, g, j), leaf blades (b, e, h, k), and roots (c, f, i, l) of Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h) and stained with toluidine blue. Bars = 20 μm

Full size image

The central rib region of plants grown over photoperiods of 16 and 24 h presented greater mesophyll cell organization and vascular bundles in comparison to those exposed to 4- and 8-h light regimens (Fig. 3b, e, h, k).

The cellular organization and thickness of the roots increased along with longer photoperiods, with plants exposed to light for 24 h having more spaced cells in the cortex (Fig. 3c, f, i, l). Furthermore, the vascular bundle was more organized and of greater caliber in relation to other treatments.

Photoperiod influences primary metabolism in L. alba cultured in vitro

Metabolites involved in osmoregulation, such as glucose and fructose, increased during the 24-h treatment, although the glucose level did not differ from 4 h (Fig. 4a, b); whereas sucrose levels decreased as the photoperiod increased (Fig. 4c).

Fig. 4
figure 4

Sugar, protein, and starch contents in leaves of Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h). a Glucose; b fructose; c sucrose; d protein; and e starch. Equal letters denote no difference according to the Scott & Knott test at a 5% probability. Data are presented as means (n = 3). Vertical bars represent the standard error

Full size image

Protein content increased with an increasing photoperiod (Fig. 4d). However, there was no difference in starch accumulation among the treatments (Fig. 4e).

The profile of essential oils in L. alba cultured in vitro is altered by photoperiod

Photoperiod altered the composition profile of essential oils in L. alba. The 4-h treatment was limited by the low amount of plant material in the samples and, thus, no essential oil components could be detected. However, plants submitted to uninterrupted light conditions (24-h photoperiod) showed a significant increase in linalool content when compared to the 8- and 16-h photoperiods. The levels of eucalyptol and germacrene did not differ significantly among treatments (Fig. 5).

Fig. 5
figure 5

Effect of photoperiod on the composition profile of essential oils in Lippia alba after 45 days of culture in vitro. Means were compared by the Scott & Knott test at a 5% probability. Data are presented as means (n = 3). Vertical bars represent the standard error

Full size image

A shorter photoperiod reduces photosynthetic capacity and alters chlorophyll a fluorescence in L. alba grown in vitro

In vitro photosynthesis of L. alba was reduced in plants cultivated for the shortest photoperiod; they exhibited a significant decrease in photosynthetic rates compared to those cultivated under 8, 16, and 24 h of light (Fig. 6).

Fig. 6
figure 6

Photosynthetic variables of Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h). a NPQ; b ETR; cFv/Fm; d qP; and e photosynthetic rate. Means were compared by the Scott & Knott test at a 5% probability. Data are presented as means (n = 4). Vertical bars represent the mean standard error. NPQ, non-photochemical quenching; ETR, electron transport rate; Fv/Fm, effective PSII quantum yield; qP, photochemical quenching

Full size image

Fluorescence measurements revealed that photoperiod was able to influence NPQ and ETR in the electron transport chain of L. alba plants. Plants grown over a 4-h photoperiod exhibited a three-fold NPQ induction compared to plants grown over a 24-h photoperiod. None of the treatments, however, showed significant differences in relation to Fv/Fm and qP (Fig. 6).

Photoperiod alters photosynthetic pigment levels in L. alba

Chlorophyll a content was highest with the 24-h photoperiod (Fig. 7a); whereas chlorophyll b was highest with the 8-h light treatment, followed in equal measure by 16- and 24-h photoperiods, and finally the 4-h treatment (Fig. 7b). The chlorophyll a/b ratio was highest for the 4-h photoperiod, followed by 16- and 24-h light treatments, and eventually the 8-h photoperiod (Fig. 7c). Overall, the highest levels of total chlorophyll and carotenoids were observed with the 8-, 16-, and 24-h light treatments (Fig. 7d, e).

Fig. 7
figure 7

Chlorophyll and carotenoid contents in Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h). Means were compared by the Scott & Knott test at 5% probability. Data are presented as means (n = 6). Vertical bars represent the mean error. Chl a, chlorophyll a; Chl b, chlorophyll b

Full size image

Photoperiod reduces geranial synthase expression, but does not alter expression levels of LaNES/LIS and circadian clock genes in L. alba

Expression of LaGES was significantly down-regulated following a 24-h photoperiod compared to control and other treatments; whereas relative expression of LaNES/LIS showed no significant difference between treatments (Fig. 8a, b).

Fig. 8
figure 8

Gene expression levels in Lippia alba after 45 days of in vitro culture under different photoperiods (4, 8, 16, and 24 h) and Pearson correlation analysis. LaGES, geraniol synthase; LaNES/LIS, neralidol/linalool synthase; LaTOC1, timing of cab expression 1; LaLHY, late elongated hypocotyl; LaPRR5, pseudo response regulator 5. Expression is relative to alcohol dehydrogenase (LaADH). Data are presented as means (n = 3) and are compared using Dunnett’s test at 5% probability

Full size image

Regarding circadian clock genes, the relative expression of LaLHY, LaPRR5, and LaTOC1 was not affected by light treatments (Fig. 8c–e). As for LaLHY and LaPRR5, a positive and significant correlation was detected between their expression levels and length of photoperiod (Fig. 8f).

Discussion

Plant growth and development are influenced by several abiotic factors, which influence also the biosynthesis of essential oils (Ragagnin et al. 2014; Batista et al. 2016, 2017a; Lazzarini et al. 2018). Several studies have investigated the impact of photoperiod on different physiological processes in plants (Bordage et al. 2016; Barros et al. 2017; Maurya and Bhalerao 2017; Mahmud et al. 2018; Song et al. 2018; Wang et al. 2018). This is the first study to investigate the effect of photoperiod on the profile of essential oils and physiological responses in L. alba.

Exposure to 16- and 24-h photoperiods resulted in an increase in both shoot and root dry mass, as well as increased stem and root length. Accordingly, increased exposure to light may have a positive influence on L. alba root growth, as observed by Mahmud et al. (2018) when evaluating the effect of photoperiod on the growth and development of roots in vines. Baerenfaller et al. (2015) described also a larger average leaf areas in plants developed under long photoperiods (16 h).

In contrast, exposure to short photoperiods resulted in stunted leaf area development, which may explain the worse photosynthetic performance and lower concentration of photosynthetic pigments and photoprotectors (carotenoids) in plants subjected to a 4-h photoperiod. Fréchette et al. (2016) observed a decline in the net assimilation of photosynthetic CO2 in Pinus strobus under short-day conditions, suggesting the importance of photoperiod in the regulation of photosynthesis.

Changes in the concentrations of photosynthetic pigments were dependent on the daily dose of light, as exemplified by reduced contents of a, b, and total chlorophylls but increased chlorophyll a/b ratio in plants subjected to a 4-h photoperiod. Similar results were obtained by Vogg et al. (1998) in Pinus sylvestris L., whose chlorophyll a, b, and total contents were found to be relatively lower on short days (9 h) compared to long days. Here, a short photoperiod (4 h) increased NPQ compared to plants exposed to light for 24 h. This result suggests that the input of photosynthetic energy may have been beyond what the plant could exploit and, therefore, the plants might have strategically acclimatized themselves by increasing the fraction of absorbed energy, dissipating it into heat (Bilger and Bjorkman 1990; Maxwell and Johnson 2000). Thus, there is likely a physiological plasticity at low light exposure, and neither Fv/Fm nor photochemical dissipation differed significantly relative to other treatments. Although ETR was lower in the uninterrupted photoperiod compared to other treatments, this did not have a negative impact on the physiological processes of the plants analyzed in this study. Shibaeva and Markovskaya (2013) reported a smaller ETR in cucumber (Cucumis sativus L.) exposed to a 24-h photoperiod, thus emphasizing the importance of photoperiod in agricultural production. Overall, in spite of decreased photosynthesis, leaf area, photosynthetic pigments, and carotenoids, our results indicate that L. alba possesses great adaptability to different photoperiods.

During the 4-h photoperiod, there was a reduction in protein content, indicating that photoperiod may be regulating protein synthesis of L. alba in vitro. Fréchette et al. (2016) reported that a reduced protein level during lower photoperiods might arise in response to photosynthetic limitation. This plasticity in protein levels under different photoperiods may allow for a faster growth during long photoperiods (Baerenfaller et al. 2015). Glucose and sucrose were generally higher during photoperiods of 4 h, but lower under 24-h light treatment. These findings are similar to those reported by Mengin et al. (2017), who noted that sucrose regulation during short photoperiods restricted growth and caused the export of carbon during the day. Here, the various treatments did not alter the levels of starch in L. alba in vitro, as opposed to other studies, whereby shorter photoperiod led to an increase in starch accumulation (Hädrich et al. 2012; Mengin et al. 2017). Hence, duration of the photoperiod in L. alba in vitro has a great influence on primary metabolism, with sugar and protein contents being important for controlling growth and development.

The in vitro culture of L. alba under different photoperiods affected morphogenesis, showing that longer photoperiods (16 and 24 h) improved structuring of the mesophyll, stem, and roots, and differentiation of vascular tissues, resulting in more organized and larger bundles. These results are consistent with those of Castro et al. (2005), who reported that vascular bundle size and arrangement were smaller under short-day photoperiods (8 h).

Shorter photoperiods did not influence the profile of essential oils as much as did uninterrupted light (24 h), demonstrating that photoperiod has a significant effect on the profile of essential oils of L. alba. Long days and elevated light intensity have been reported before to activate genes related to anthocyanin biosynthesis and promoting its accumulation in the leaves of Malus spp. (Lu et al. 2009), as well as flavonoids, anthocyanins, and catechins in Ipomoea batatas (L.) Lam. (Carvalho et al. 2010).

Here, linalool content was increased under the 24-h light photoperiod compared to the 8-, 16-, and 24-h periods. This increase was not followed by the expression of LaNES/LIS. However, expression levels of LaGES were reduced. Possibly, the increased synthesis of linalool was related to a lower expression of LaGES, which in turn hampered the synthesis of geraniol, another monoterpene. Thus, plant metabolism may be directing the precursor of the geranyl pyrophosphate (GPP) route to the synthesis of linalool, causing a significant increase in its levels. Viccini et al. (2014) reported that the main components of essential oils in L. alba were related to ploidy level; here, diploids, triploids, and tetraploids of all accessions were capable of producing linalool and citral. However, triploids are inefficient in catalyzing this additional biotransformation to citral, and thus predominantly generate linalool. Such inability may explain the observed reduction in LaGES expression as the accession used in this study was a triploid. In addition, it is probable that the 24-h photoperiod caused some oxidative damage in plant tissues, which resulted in the aforementioned variation in linalool levels. Furthermore, several studies have reported that monoterpenes are effective in protecting plants against stress (thermal or oxidative); their levels become increased under these circumstances to allow for reactive oxygen species removal and protection of photosynthetic systems (Chen et al. 2009; Zuo et al. 2017).

The relative expression of circadian clock genes (LaLHY, LaPRR5, and LaTOC1) was not affected by any of the treatments. Even though studies show that LHY expression is repressed by PRR5 and TOC1, photoperiod treatments did not alter the expression of clock genes in L. alba (Nakamichi et al. 2010; Gendron et al. 2012; Huang et al. 2012). Instead, we report here the co-expression of LaLHY and LaPRR5.

The present results indicate that photoperiod modulates primary and secondary metabolisms in L. alba plants cultured in vitro. The uninterrupted photoperiod treatment (24 h) provided greater growth; better anatomical organization of the mesophyll, stem, roots, and bundles; increased photosynthetic pigment levels, higher photosynthetic rate; and greater synthesis of total proteins. Batista et al. (2016) and Hsie et al. (2019) already demonstrated that Lippia alters its metabolism under different qualities of light, now we found that this physiological plasticity also is triggered by the photoperiod, with better performance during long days, which can be advantageous in the tropical regions. In addition, the biosynthesis of linalool, a major component of the L. alba chemotype used in this study, was characterized by reduced expression of LaGES. These results will allow a better understanding of how photoperiod affects the regulation of primary and secondary metabolism, which is of great importance given the species’ value for the pharmacological industry.