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Growth and production of secondary compounds in leaves of matico (piper aduncum L.) grown under altered ambient light.

Growth and production of secondary compounds in leaves of matico (piper aduncum L.) grown under altered ambient light.

Secondary Compounds in Matico Leaves Cultivated Under Altered Ambient Light

The species Piper aduncum L., native to the Americas, holds significant biological benefits for the production of secondary compounds such as phenolics and flavonoids. Among the factors influencing plant metabolism. Light is considered one of the most critical in promoting physiological changes that directly interfere with growth and secondary compound production.

This study aimed to evaluate the growth also production of different secondary compounds (total phenolic acids, flavonoids, lignin, and phenylalanine ammonia-lyase – PAL activity) of Piper aduncum L. The treatments included four shade netting greenhouses (modified ambient light) with 50%, 70% of natural irradiance, red also

blue nets. And full sun (100% natural irradiance), each with 20 replicates per treatment.

Growth and Production:

The growth of Piper aduncum was affected by the altered ambient light, showing increased growth under the blue net. However, the species exhibited responses enabling survival in full sun, such as increased root production. Secondary compound production is also affect by light, with the highest phenolic and lignin compound production (respectively 0.18 and 11.7 µg g DM-1) in the blue net greenhouse treatment. Additionally, PAL can be consider the key enzyme for phenolic compound production in this species.

The concentration of flavonoids in the 100% irradiance treatment was the lowest. Indicating the species has another protection mechanism against high irradiance.

Abbreviations: DAT – Days After Transplant; PAL – Phenylananine Ammonia Liase; LDM – Leaf Dry Mass; SDM – Steam Dry Mass; RDM- Roots Dry Mass; TDM – Total Dry Mass; SLA – Specific Leaf Area; LAR – Leaf Area Ratio; RWR – root weight ratio; RN – Red Net; BN – Blue Net.

matico plant
This type of plant (Piper aduncum) has a good metabolism with biological benefits such as phenolics and flavonoids.

Introduction:

Piper aduncum (Piperaceae) is a shrub native to the Americas, and has potential for commercial use (Rocha et al., 2008). It produces essential oils with a high yield (2.5 to 3.5%). Has low toxicity and is rich in dillapiole (31.5 to 91.1%) (Maia et al., 1998; Sousa et al., 2008).

Piper aduncum

This compound exhibits antimicrobial properties and acts as an insecticide and molluscicide (Orjala et al, 1994; Fazolin et al., 2005; Lara-Junior et al., 2012; Misni et al, 2011). However, according to Bernard et al. (1995) the genus Piper can still produce many types of secondary metabolites. Such as phenylpropanoids, flavonoids and lignoids, which can be exploit by the chemical and pharmaceutical industry. However, the production and quality of secondary compounds are modify by several environmental factors. The intensity and quality of light are important environmental factors that alter the synthesis of these compounds because they affect plant morphology and physiological processes. The biosynthesis of flavonoids and other phenolic acids, for example. Require larger amounts of irradiance, or are increase under these conditions (Ghasemzadeh and Ghasemzadeh, 2011).

Previous studies show that changes in light intensity also quality lead to increased production of total flavonoids and phenolics in medicinal plants (Ghasemzadeh et al., 2010; Karimi et al., 2013). Therefore, in medicinal plant cultivation. Microclimate management could be a promising alternative for the control of phytochemical levels and quality of plant material.

Light for Flavonoid Increase:

Light control and uniformity can result in increased growth. Better yield planning, and better control of contamination or insect attack (Karimi et al., 2013). However, the existence of interspecific differences in the microenvironment can influence plant accumulation and distribution of total phenolics and flavonoids (Jaafar and Rahmat, 2008). Flavonoid and phenolic compounds are important for plants against UV light also protect reactive oxygen species. A phenylalanine ammonia-lyase (PAL) is an enzyme involv in their biosynthesis. PAL activity is regulate by many factors, such as age, herbivory and mostly light conditions (KUMARI et al., 2009; NAWKAR et al., 2013). Studies conducted with Kalanchoe pinnata showed an increase in production.

 Piper aduncum
Piper aduncum is a shrub that produces essential oils with a high yield.

Results and Discussion:

Growth Relationships:

Piper aduncum grow is affect by different irradiance treatments (Fig. 1). Plants grown under red and blue meshes showed the highest growth in height also stem diameter throughout the experiment (Fig. 1A, 1B). Similar results were observ for Ocimum selloi, Mikania glomerata and Mikania laevigata, which showed the best height in the red also blue mesh treatments (Souza et al., 2007; Costa et al., 2010). Total leaf area was greater in plants grow under blue also red meshes (Fig. 1D).

The increase in leaf area under photoselective screens can be consider a means for the plant to increase photosynthetic surface area. With greater use of low light intensities and, therefore, compensating photosynthetic rates per unit area. Which is a characteristic of shade-adapted leaves (Jones and McLeod, 1991). Oliveira (2009) obtained similar results with the species Artemisia vulgaris. Which presented a larger leaf area under red and blue meshes, compared to irradiances of 50% and 100%. Plants grow under red and blue meshes. Where the proportions of the red and blue color range are altered also the radiation intensity is block, presented the highest leaf area averages.

Percentage of Piper aduncum:

Smaller leaf area is observ in the 50% and 70% irradiance treatments. Indicating light quality as a decisive factor for leaf blade expansion in matico. The number of leaves is higher in plants grow under red and blue nets at 70% and 100% irradiance. In the 100% and 70% treatments, leaf area reduced, but leaf numbers increased. Similar responses were observ in Ocimum gratissimum, where high irradiance levels led to decrease leaf area but increased leaf numbers. Leaf dry mass was higher in the 50% and 100% irradiance treatments, as well as in red and blue nets.

Radiation Control for Growth:

This result underscores the importance of radiation intensity and quality for plant growth. Not only for supplying energy for photosynthesis but also for generating signals that regulate its development. Leaf weight ratio was higher in the 100% and 50% irradiance treatments and lower under red nets. Root weight ratio was higher in plants grow with 100% and 70% irradiance and under red nets. The largest leaf area and total dry mass found in plants grown under blue nets indicate that increased leaf area allowed for greater light interception, favoring assimilate production. The root to shoot ratio was higher in plants grow with 100% and 50% irradiance and lower under red nets.

Furthermore, the reduction in leaf thickness in shade-grown plants results from differences in photoassimilate distribution and consumption for leaf expansion. Especially in plants grown under blue nets, which had larger leaf areas and larger leaf area ratios. Similarly, a reduction in leaf thickness is observ in Ocimum selloi when grow under blue nets.

These results allow us to implement medicinal plant cultivation with better production control. As there are few studies on crops in protected environments. Understanding growth responses as we direct the growth environment to increase secondary compound also biomass production.

piper aduncum
This plant has different treatments depending on the irradiance of the sun.

Phenolic Activity and PAL:

Irradiance also had an effect on the production of total phenolics (Fig. 3). The content of phenolic acids and lignin was higher in the treatment with blue mesh and lower in the treatment with red mesh and 100% irradiance (Figs. 4A, 4B). Such a result indicates that secondary compounds are influence by the quality and quantity of irradiance. Studies with Protea cynaroides L., grown in vitro, showed that it also presented a higher concentration of total phenolics. When grown under blue light (LED) and a lower amount under red light (Wu and Lin, 2012). However, ginger (Zingiber officinale Roscoe) grown under different natural light intensities presented different results. Where high irradiances caused an increase in these compounds (Ghasemzadeh et al., 2010).

Increased lignin content in plants grown (Piper aduncum) under blue screens may be a response to anatomical changes. Usually induced under adverse conditions that provide cell protection (Ghasemzadeh et al., 2010). Therefore, increased production of total phenolics and lignin may be related to increased resistance in plants (Martti et al., 2004). The increase in total phenolic compounds by treatment with blue mesh may be beneficial to plants in combating diseases or herbivore attack in Piper aduncum. In addition, the synthesis of these compounds is apparently influence by light quality. The flavonoid content is lower in the treatment with 100% irradiance and no significant differences were observed in the other treatments (Fig. 4C). This result indicates the high influence of irradiance intensities on flavonoid production.

Ghasemzadeh and Ghasemzadeh

Ghasemzadeh and Ghasemzadeh (2011) observed that leaves of Zingiber officinale Roscoe had higher flavonoid content when exposed to shade. Flavonoids are know as protective substances against damage induced by excess light and as antioxidants (Jaakola et al., 2004; Nascimento et al., 2013).

Benefit of Piper aduncum growth

The growth of Piper aduncum at 100% irradiance was not beneficial for the stimulation of these defenses. Or this species still presents other protection mechanisms against high irradiances. Pacheco et al. (2013) observed in full sun conditions, an increase of carotenoids. Therefore, these pigments may be associate with photoprotection mechanisms for this species. PAL activity increased in treatments with 50% and 70% irradiance and blue meshes (Fig. 4D). the production of photoassimilates from primary metabolism can cause an increase in the concentrations of phenolic compounds at high irradiances (Warren et al., 2003). However, when light, water and nutrients are sufficient, plant grow and adaptation are prioritize. Therefore, a high amount of phenylalanine is use for protein synthesis, and consequently the production of phenolic compounds decreases.

The lower synthesis of secondary compounds observed in plants grown at 100% irradiance is related to the induction of stress that forces their adaptation under these conditions. However, further studies are need to determine the increase in secondary metabolite production due to the decrease in primary metabolite production through photosynthesis or stress induce by irradiance conditions (Ghasemzadeh and Ghasemzadeh, 2011).

green shade net
Using blue shade netting brings more benefits than using red shade netting.

Materials and methods

Location and plant material.

The experiment is conduct at the Federal University of Lavras, Brazil. Piper aduncum seedlings were produce in the Biology Department, from seeds. The seeds were previously germinate in Petri dishes, on three sheets of filter paper and were keep in a Mangelsdorf germination chamber. At 25°C and during a 12-hour photoperiod, for 30 days. After this period, the seedlings were transfer to polypropylene trays containing the commercial substrate Tropstrato HA® (Vida Verde©, Brazil) and maintain in greenhouses with 50% shading until they reach 2.5 cm in height. Plants intended for cultivation in full sun (100% irradiance) were previously acclimatized for 7 days at 70% irradiance and then for 7 days in full sun before being transplanted to the final substrate.

location and climate

Irrigation is perform daily and the soil is maintain under field capacity condition. After acclimatization, the seedlings were transplant into plastic pots. With a capacity of 6 liters, containing a substrate composed of subsoil, sand and bovine manure, in a ratio of 2: 1: 1: 1. Which is arrange in the different irradiance treatments. The experiment is conduct between April and July 2012, at the Gota da Esperança farm. Belonging to the Department of Agriculture, with the following geographical coordinates: 21°14’07 “S and 44°58’22 “W, at 879 m altitude. The average climatic conditions observed during the experiment were provided by the Climatological Station of the Department of Agricultural Engineering, had a maximum temperature of 30.2°C and a minimum of 6.3°C, a rainfall of 1.28 mm and a relative humidity of 72.7%.

piper aduncum
Several experiments were conducted at the Federal University Lavras of Brazil with the seed of this species and obtained several results.

Altered environmental light

The treatments were characterize by growing plants for 120 days under four altered environmental lights, produced by shaded greenhouses with 70% and 50% of natural incident irradiance, two colored shading nets, in red (RN) and blue (BN), blocking 50% of the incident radiation and a full sun treatment. With the help of a portable spectroradiometer (USB650 Red Tide) coupled to a DT-MINI electromagnetic radiation source (200 to 2000 nm) and a R400-7-VIS-NIR reflective probe (US BioSolutions Ocean Optics®), the radiation spectrum of different environments is evaluate, with a spectral resolution of 1 nm.

The normalized irradiances observed in the 50% treatment were 6.54 W/m², 13.08 W/m² in the 70% treatment, 15.42 W/m² of 100%, 8.86 W/m² in BN and 9.07 W/m² in BN. Each environment presented the highest values in terms of amount and size of the spectrum for the atmosphere with 100% irradiance, followed by the NR atmosphere. Where the value found was 70%, and 50% irradiance for the environment with BN. It is also observ that the blue grid provided irradiances of approximately 450-550 nm, and the red grid between 490 and 690 nm.

Growth analysis

Grow is evaluate in twenty plants of each treatment and the following morphological variables were take as a basis: height, stem diameter, leaf area (LA), number of leaves, leaf dry mass (LDM), stem dry mass (SDM), root dry mass (RDM) and total dry mass (TDM).

The following ratios were also calculate: root/aerial parts (R/AP = RDM/ (LDM + TDM)), specific leaf area (SLA = LA/LDM), leaf area ratio (LAR = LA/TDM), leaf weight ratio (LWR = LDM/TDM) and root weight ratio (RWR = RDM/TDM), using equations according to Benincasa (2003). Height, stem diameter, number of leaves and leaf area were measure in 15 days. Height is measure with a graduate species, an accuracy of 1.0 mm. And distances from the surface to the ground were measure. Stem diameter is measure accurately with a 0.01 mm digital caliper at the soil surface.

The variables leaf dry mass, stem dry mass, root dry mass and total dry mass were obtain at the end of the period. (120 days). The dry mass is obtain by drying the leaves; the stem and roots. Previously separate, were dry in a forced air oven, at 70°C, at constant weight.

hoja de matico
It had excellent growth based on its development.

Secondary metabolites

Secondary compounds were analyze 150 days after transplanting and the contents of total phenolic compounds, lignin and flavonoids were quantified. In addition, phenylalanine ammonia-lyase (PAL) activity is also check. Two leaves were collect, fully expanded, located between the second and third node of five plants for each treatment;

Compositional analyses were perform in triplicate. Total phenolic acid and lignin contents are determine according to Zieslin and Ben-Zaken (1993) with modifications. For extraction, tissues were crush in liquid nitrogen and then freeze-dried for six hours. The lyophilized material is weigh (30 mg) and transfer to a 2-ml tube, 1.5 ml of 80% methanol is add and keep under agitation for 15 h at room temperature and in the dark. Also the extract is centrifuged at 12,000 x g for 15 min;

The supernatant is use for total phenolics quantification and the precipitate for lignin extraction. For quantification of total phenolic acids, 30 µL of Folin-Ciocalteu reagent is mix with methanol extract (0.25 N) in an ELISA plate and allowed to stand for 5 minutes. Then, 30 µL of 1M sodium carbonate is add and homogenized for 10 minutes.

Characterization of Piper aduncum

Completed 160 µL of distilled water, rested for 1 hour and centrifuged at 2,250 x g for 5 minutes. Then, 180 µL of this mixture is collect and deposit on another ELISA plate. The whole procedure is perform at room temperature. Readings are take at 725 nm. Phenolic acid content is calculate as a function of catechol standard curve and expressed as µg catechol per gram of dry matter. Lignin is extract from the resuspended precipitate in 1.5 ml of 80% methanol and centrifuged at 12,000 x g for 10 min. The supernatant is discard and the precipitate is dry at 65°C for four hours. The dried precipitate is resuspend in 1.5 ml of a 1:10 solution of thioglycolic acid and 2N hydrochloric acid and homogenize in a water bath.

Piper aduncum reading time

Again, the supernatant is discard and the precipitate is resuspend in 1.5 ml of distilled water and centrifuged at 12,000 x g for 10 min at 4°C. The dry precipitate is suspend in 1.5 ml of a 1.10 solution of thioglycolic acid and 2N hydrochloric acid and homogenized in a water bath. The supernatant is transfer to a new 2-ml tube and 200 µL of concentrated hydrochloric acid is add. It is then keep on ice for four hours and centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant is discard and the precipitate is resuspend in 2 mL of 0.5 M sodium hydroxide and homogenized. 200 µL of the extract is collect and deposit on an ELISA plate.

Reading results

Readings are take at 280 nm. Lignin content was calculated based on the lignin standard curve and the value was expressed in µg of lignin per gram of dry matter. Total flavonoids were extracted from leaves according to a method adapted from Santos and Blatt (1998). Approximately 250 mg of dry matter was kept in 70% (v/v) methanol for 24 hours. A 50 µl aliquot part of the supernatant was transferred to test tubes containing 1.8 ml of 70% methanol. A 50 µl aliquot part of the supernatant was transferred to test tubes containing 1.8 ml of 70% methanol. Then, 130 µL of an aluminum chloride solution (5 g of aluminum chloride in 100 mL of 70% methanol) and 6.7 ml of 70% methanol were added and shaken vigorously.

piper aduncum
The secondary metabolism of the plant was that its phenolic and flavonoid compounds were quantified.

Statistical analysis of Piper aduncum

The experimental design was completely randomized. In which 20 replicates per treatment were used for growth evaluations and 5 replicates for biochemical analyses. Data obtained over an extended time (quantitative) were analyzed by polynomial regression (p ≤0.05). While qualitative data were subjected to analysis of variance, and measurements were compared by Tukey’s test (p ≤0.05), using the SAEG program (SAEG-2007).

Table 1. Ratio of roots/aerial parts (R/AP), specific leaf area (SLA), leaf area ratio (LAR), leaf weight ratio (LWR) and root weight ratio (RWR) of Piper aduncum grown under different irradiance conditions.

tabla Tukey mallas rojas y mallas azules

Fig 1. Height (A), stem diameter (B), number of leaves (C) and leaf area (D) of Piper aduncum grown under different irradiances (50, 70, 100% irradiance and red netting – RN and blue netting – BN), at 120 DAT (days after transplanting). Lavras, Brazil.

Fig 2. Leaf dry mass (LDM), stem dry mass (SDM), root dry mass (RDM) and total dry mass (TDM) of Piper aduncum grown under different irradiance conditions (50, 70, 100% irradiance and red meshes – RN and blue meshes -BN). Means followed by the same letter in the columns do not differ according to Tukey’s test (p≤0.05). Lavras, Brazil.

Fig 3. Total phenolic acids (A), lignin (B), flavonoids (C) and PAL activity (D) in leaves of Piper aduncum L. grown under different irradiance conditions (50, 70, 100% irradiance and red meshes – RN and blue meshes -BN). *Means followed by the same letter in the columns do not differ according to Tukey’s test (p≤0.05) .Lavras, Brazil.

Conclusions

Growth and synthesis of secondary compounds (total phenolic acids, lignin and flavonoids) of Piper aduncum were influenced by different irradiance conditions. Plants grown under blue photoselective shading screens showed higher growth and dry mass production. The highest amount of secondary compounds was obtained in plants grown under blue shading screens. Therefore, given that this is the species that produces bioactive compounds with commercial potential, its cultivation in environments supplemented with blue light is recommended.

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Secondary Compounds in Matico Leaves Cultivated Under Altered Ambient Light

The species Piper aduncum L., native to the Americas, holds significant biological benefits for the production of secondary compounds such as phenolics and flavonoids. Among the factors influencing plant metabolism. Light is considered one of the most critical in promoting physiological changes that directly interfere with growth and secondary compound production.

This study aimed to evaluate the growth also production of different secondary compounds (total phenolic acids, flavonoids, lignin, and phenylalanine ammonia-lyase – PAL activity) of Piper aduncum L. The treatments included four shade netting greenhouses (modified ambient light) with 50%, 70% of natural irradiance, red also

blue nets. And full sun (100% natural irradiance), each with 20 replicates per treatment.

Growth and Production:

The growth of Piper aduncum was affected by the altered ambient light, showing increased growth under the blue net. However, the species exhibited responses enabling survival in full sun, such as increased root production. Secondary compound production is also affect by light, with the highest phenolic and lignin compound production (respectively 0.18 and 11.7 µg g DM-1) in the blue net greenhouse treatment. Additionally, PAL can be consider the key enzyme for phenolic compound production in this species.

The concentration of flavonoids in the 100% irradiance treatment was the lowest. Indicating the species has another protection mechanism against high irradiance.

Abbreviations: DAT – Days After Transplant; PAL – Phenylananine Ammonia Liase; LDM – Leaf Dry Mass; SDM – Steam Dry Mass; RDM- Roots Dry Mass; TDM – Total Dry Mass; SLA – Specific Leaf Area; LAR – Leaf Area Ratio; RWR – root weight ratio; RN – Red Net; BN – Blue Net.

matico plant
This type of plant (Piper aduncum) has a good metabolism with biological benefits such as phenolics and flavonoids.

Introduction:

Piper aduncum (Piperaceae) is a shrub native to the Americas, and has potential for commercial use (Rocha et al., 2008). It produces essential oils with a high yield (2.5 to 3.5%). Has low toxicity and is rich in dillapiole (31.5 to 91.1%) (Maia et al., 1998; Sousa et al., 2008).

Piper aduncum

This compound exhibits antimicrobial properties and acts as an insecticide and molluscicide (Orjala et al, 1994; Fazolin et al., 2005; Lara-Junior et al., 2012; Misni et al, 2011). However, according to Bernard et al. (1995) the genus Piper can still produce many types of secondary metabolites. Such as phenylpropanoids, flavonoids and lignoids, which can be exploit by the chemical and pharmaceutical industry. However, the production and quality of secondary compounds are modify by several environmental factors. The intensity and quality of light are important environmental factors that alter the synthesis of these compounds because they affect plant morphology and physiological processes. The biosynthesis of flavonoids and other phenolic acids, for example. Require larger amounts of irradiance, or are increase under these conditions (Ghasemzadeh and Ghasemzadeh, 2011).

Previous studies show that changes in light intensity also quality lead to increased production of total flavonoids and phenolics in medicinal plants (Ghasemzadeh et al., 2010; Karimi et al., 2013). Therefore, in medicinal plant cultivation. Microclimate management could be a promising alternative for the control of phytochemical levels and quality of plant material.

Light for Flavonoid Increase:

Light control and uniformity can result in increased growth. Better yield planning, and better control of contamination or insect attack (Karimi et al., 2013). However, the existence of interspecific differences in the microenvironment can influence plant accumulation and distribution of total phenolics and flavonoids (Jaafar and Rahmat, 2008). Flavonoid and phenolic compounds are important for plants against UV light also protect reactive oxygen species. A phenylalanine ammonia-lyase (PAL) is an enzyme involv in their biosynthesis. PAL activity is regulate by many factors, such as age, herbivory and mostly light conditions (KUMARI et al., 2009; NAWKAR et al., 2013). Studies conducted with Kalanchoe pinnata showed an increase in production.

 Piper aduncum
Piper aduncum is a shrub that produces essential oils with a high yield.

Results and Discussion:

Growth Relationships:

Piper aduncum grow is affect by different irradiance treatments (Fig. 1). Plants grown under red and blue meshes showed the highest growth in height also stem diameter throughout the experiment (Fig. 1A, 1B). Similar results were observ for Ocimum selloi, Mikania glomerata and Mikania laevigata, which showed the best height in the red also blue mesh treatments (Souza et al., 2007; Costa et al., 2010). Total leaf area was greater in plants grow under blue also red meshes (Fig. 1D).

The increase in leaf area under photoselective screens can be consider a means for the plant to increase photosynthetic surface area. With greater use of low light intensities and, therefore, compensating photosynthetic rates per unit area. Which is a characteristic of shade-adapted leaves (Jones and McLeod, 1991). Oliveira (2009) obtained similar results with the species Artemisia vulgaris. Which presented a larger leaf area under red and blue meshes, compared to irradiances of 50% and 100%. Plants grow under red and blue meshes. Where the proportions of the red and blue color range are altered also the radiation intensity is block, presented the highest leaf area averages.

Percentage of Piper aduncum:

Smaller leaf area is observ in the 50% and 70% irradiance treatments. Indicating light quality as a decisive factor for leaf blade expansion in matico. The number of leaves is higher in plants grow under red and blue nets at 70% and 100% irradiance. In the 100% and 70% treatments, leaf area reduced, but leaf numbers increased. Similar responses were observ in Ocimum gratissimum, where high irradiance levels led to decrease leaf area but increased leaf numbers. Leaf dry mass was higher in the 50% and 100% irradiance treatments, as well as in red and blue nets.

Radiation Control for Growth:

This result underscores the importance of radiation intensity and quality for plant growth. Not only for supplying energy for photosynthesis but also for generating signals that regulate its development. Leaf weight ratio was higher in the 100% and 50% irradiance treatments and lower under red nets. Root weight ratio was higher in plants grow with 100% and 70% irradiance and under red nets. The largest leaf area and total dry mass found in plants grown under blue nets indicate that increased leaf area allowed for greater light interception, favoring assimilate production. The root to shoot ratio was higher in plants grow with 100% and 50% irradiance and lower under red nets.

Furthermore, the reduction in leaf thickness in shade-grown plants results from differences in photoassimilate distribution and consumption for leaf expansion. Especially in plants grown under blue nets, which had larger leaf areas and larger leaf area ratios. Similarly, a reduction in leaf thickness is observ in Ocimum selloi when grow under blue nets.

These results allow us to implement medicinal plant cultivation with better production control. As there are few studies on crops in protected environments. Understanding growth responses as we direct the growth environment to increase secondary compound also biomass production.

piper aduncum
This plant has different treatments depending on the irradiance of the sun.

Phenolic Activity and PAL:

Irradiance also had an effect on the production of total phenolics (Fig. 3). The content of phenolic acids and lignin was higher in the treatment with blue mesh and lower in the treatment with red mesh and 100% irradiance (Figs. 4A, 4B). Such a result indicates that secondary compounds are influence by the quality and quantity of irradiance. Studies with Protea cynaroides L., grown in vitro, showed that it also presented a higher concentration of total phenolics. When grown under blue light (LED) and a lower amount under red light (Wu and Lin, 2012). However, ginger (Zingiber officinale Roscoe) grown under different natural light intensities presented different results. Where high irradiances caused an increase in these compounds (Ghasemzadeh et al., 2010).

Increased lignin content in plants grown (Piper aduncum) under blue screens may be a response to anatomical changes. Usually induced under adverse conditions that provide cell protection (Ghasemzadeh et al., 2010). Therefore, increased production of total phenolics and lignin may be related to increased resistance in plants (Martti et al., 2004). The increase in total phenolic compounds by treatment with blue mesh may be beneficial to plants in combating diseases or herbivore attack in Piper aduncum. In addition, the synthesis of these compounds is apparently influence by light quality. The flavonoid content is lower in the treatment with 100% irradiance and no significant differences were observed in the other treatments (Fig. 4C). This result indicates the high influence of irradiance intensities on flavonoid production.

Ghasemzadeh and Ghasemzadeh

Ghasemzadeh and Ghasemzadeh (2011) observed that leaves of Zingiber officinale Roscoe had higher flavonoid content when exposed to shade. Flavonoids are know as protective substances against damage induced by excess light and as antioxidants (Jaakola et al., 2004; Nascimento et al., 2013).

Benefit of Piper aduncum growth

The growth of Piper aduncum at 100% irradiance was not beneficial for the stimulation of these defenses. Or this species still presents other protection mechanisms against high irradiances. Pacheco et al. (2013) observed in full sun conditions, an increase of carotenoids. Therefore, these pigments may be associate with photoprotection mechanisms for this species. PAL activity increased in treatments with 50% and 70% irradiance and blue meshes (Fig. 4D). the production of photoassimilates from primary metabolism can cause an increase in the concentrations of phenolic compounds at high irradiances (Warren et al., 2003). However, when light, water and nutrients are sufficient, plant grow and adaptation are prioritize. Therefore, a high amount of phenylalanine is use for protein synthesis, and consequently the production of phenolic compounds decreases.

The lower synthesis of secondary compounds observed in plants grown at 100% irradiance is related to the induction of stress that forces their adaptation under these conditions. However, further studies are need to determine the increase in secondary metabolite production due to the decrease in primary metabolite production through photosynthesis or stress induce by irradiance conditions (Ghasemzadeh and Ghasemzadeh, 2011).

green shade net
Using blue shade netting brings more benefits than using red shade netting.

Materials and methods

Location and plant material.

The experiment is conduct at the Federal University of Lavras, Brazil. Piper aduncum seedlings were produce in the Biology Department, from seeds. The seeds were previously germinate in Petri dishes, on three sheets of filter paper and were keep in a Mangelsdorf germination chamber. At 25°C and during a 12-hour photoperiod, for 30 days. After this period, the seedlings were transfer to polypropylene trays containing the commercial substrate Tropstrato HA® (Vida Verde©, Brazil) and maintain in greenhouses with 50% shading until they reach 2.5 cm in height. Plants intended for cultivation in full sun (100% irradiance) were previously acclimatized for 7 days at 70% irradiance and then for 7 days in full sun before being transplanted to the final substrate.

location and climate

Irrigation is perform daily and the soil is maintain under field capacity condition. After acclimatization, the seedlings were transplant into plastic pots. With a capacity of 6 liters, containing a substrate composed of subsoil, sand and bovine manure, in a ratio of 2: 1: 1: 1. Which is arrange in the different irradiance treatments. The experiment is conduct between April and July 2012, at the Gota da Esperança farm. Belonging to the Department of Agriculture, with the following geographical coordinates: 21°14’07 “S and 44°58’22 “W, at 879 m altitude. The average climatic conditions observed during the experiment were provided by the Climatological Station of the Department of Agricultural Engineering, had a maximum temperature of 30.2°C and a minimum of 6.3°C, a rainfall of 1.28 mm and a relative humidity of 72.7%.

piper aduncum
Several experiments were conducted at the Federal University Lavras of Brazil with the seed of this species and obtained several results.

Altered environmental light

The treatments were characterize by growing plants for 120 days under four altered environmental lights, produced by shaded greenhouses with 70% and 50% of natural incident irradiance, two colored shading nets, in red (RN) and blue (BN), blocking 50% of the incident radiation and a full sun treatment. With the help of a portable spectroradiometer (USB650 Red Tide) coupled to a DT-MINI electromagnetic radiation source (200 to 2000 nm) and a R400-7-VIS-NIR reflective probe (US BioSolutions Ocean Optics®), the radiation spectrum of different environments is evaluate, with a spectral resolution of 1 nm.

The normalized irradiances observed in the 50% treatment were 6.54 W/m², 13.08 W/m² in the 70% treatment, 15.42 W/m² of 100%, 8.86 W/m² in BN and 9.07 W/m² in BN. Each environment presented the highest values in terms of amount and size of the spectrum for the atmosphere with 100% irradiance, followed by the NR atmosphere. Where the value found was 70%, and 50% irradiance for the environment with BN. It is also observ that the blue grid provided irradiances of approximately 450-550 nm, and the red grid between 490 and 690 nm.

Growth analysis

Grow is evaluate in twenty plants of each treatment and the following morphological variables were take as a basis: height, stem diameter, leaf area (LA), number of leaves, leaf dry mass (LDM), stem dry mass (SDM), root dry mass (RDM) and total dry mass (TDM).

The following ratios were also calculate: root/aerial parts (R/AP = RDM/ (LDM + TDM)), specific leaf area (SLA = LA/LDM), leaf area ratio (LAR = LA/TDM), leaf weight ratio (LWR = LDM/TDM) and root weight ratio (RWR = RDM/TDM), using equations according to Benincasa (2003). Height, stem diameter, number of leaves and leaf area were measure in 15 days. Height is measure with a graduate species, an accuracy of 1.0 mm. And distances from the surface to the ground were measure. Stem diameter is measure accurately with a 0.01 mm digital caliper at the soil surface.

The variables leaf dry mass, stem dry mass, root dry mass and total dry mass were obtain at the end of the period. (120 days). The dry mass is obtain by drying the leaves; the stem and roots. Previously separate, were dry in a forced air oven, at 70°C, at constant weight.

hoja de matico
It had excellent growth based on its development.

Secondary metabolites

Secondary compounds were analyze 150 days after transplanting and the contents of total phenolic compounds, lignin and flavonoids were quantified. In addition, phenylalanine ammonia-lyase (PAL) activity is also check. Two leaves were collect, fully expanded, located between the second and third node of five plants for each treatment;

Compositional analyses were perform in triplicate. Total phenolic acid and lignin contents are determine according to Zieslin and Ben-Zaken (1993) with modifications. For extraction, tissues were crush in liquid nitrogen and then freeze-dried for six hours. The lyophilized material is weigh (30 mg) and transfer to a 2-ml tube, 1.5 ml of 80% methanol is add and keep under agitation for 15 h at room temperature and in the dark. Also the extract is centrifuged at 12,000 x g for 15 min;

The supernatant is use for total phenolics quantification and the precipitate for lignin extraction. For quantification of total phenolic acids, 30 µL of Folin-Ciocalteu reagent is mix with methanol extract (0.25 N) in an ELISA plate and allowed to stand for 5 minutes. Then, 30 µL of 1M sodium carbonate is add and homogenized for 10 minutes.

Characterization of Piper aduncum

Completed 160 µL of distilled water, rested for 1 hour and centrifuged at 2,250 x g for 5 minutes. Then, 180 µL of this mixture is collect and deposit on another ELISA plate. The whole procedure is perform at room temperature. Readings are take at 725 nm. Phenolic acid content is calculate as a function of catechol standard curve and expressed as µg catechol per gram of dry matter. Lignin is extract from the resuspended precipitate in 1.5 ml of 80% methanol and centrifuged at 12,000 x g for 10 min. The supernatant is discard and the precipitate is dry at 65°C for four hours. The dried precipitate is resuspend in 1.5 ml of a 1:10 solution of thioglycolic acid and 2N hydrochloric acid and homogenize in a water bath.

Piper aduncum reading time

Again, the supernatant is discard and the precipitate is resuspend in 1.5 ml of distilled water and centrifuged at 12,000 x g for 10 min at 4°C. The dry precipitate is suspend in 1.5 ml of a 1.10 solution of thioglycolic acid and 2N hydrochloric acid and homogenized in a water bath. The supernatant is transfer to a new 2-ml tube and 200 µL of concentrated hydrochloric acid is add. It is then keep on ice for four hours and centrifuged at 12,000 x g for 10 minutes at 4°C. The supernatant is discard and the precipitate is resuspend in 2 mL of 0.5 M sodium hydroxide and homogenized. 200 µL of the extract is collect and deposit on an ELISA plate.

Reading results

Readings are take at 280 nm. Lignin content was calculated based on the lignin standard curve and the value was expressed in µg of lignin per gram of dry matter. Total flavonoids were extracted from leaves according to a method adapted from Santos and Blatt (1998). Approximately 250 mg of dry matter was kept in 70% (v/v) methanol for 24 hours. A 50 µl aliquot part of the supernatant was transferred to test tubes containing 1.8 ml of 70% methanol. A 50 µl aliquot part of the supernatant was transferred to test tubes containing 1.8 ml of 70% methanol. Then, 130 µL of an aluminum chloride solution (5 g of aluminum chloride in 100 mL of 70% methanol) and 6.7 ml of 70% methanol were added and shaken vigorously.

piper aduncum
The secondary metabolism of the plant was that its phenolic and flavonoid compounds were quantified.

Statistical analysis of Piper aduncum

The experimental design was completely randomized. In which 20 replicates per treatment were used for growth evaluations and 5 replicates for biochemical analyses. Data obtained over an extended time (quantitative) were analyzed by polynomial regression (p ≤0.05). While qualitative data were subjected to analysis of variance, and measurements were compared by Tukey’s test (p ≤0.05), using the SAEG program (SAEG-2007).

Table 1. Ratio of roots/aerial parts (R/AP), specific leaf area (SLA), leaf area ratio (LAR), leaf weight ratio (LWR) and root weight ratio (RWR) of Piper aduncum grown under different irradiance conditions.

tabla Tukey mallas rojas y mallas azules

Fig 1. Height (A), stem diameter (B), number of leaves (C) and leaf area (D) of Piper aduncum grown under different irradiances (50, 70, 100% irradiance and red netting – RN and blue netting – BN), at 120 DAT (days after transplanting). Lavras, Brazil.

Fig 2. Leaf dry mass (LDM), stem dry mass (SDM), root dry mass (RDM) and total dry mass (TDM) of Piper aduncum grown under different irradiance conditions (50, 70, 100% irradiance and red meshes – RN and blue meshes -BN). Means followed by the same letter in the columns do not differ according to Tukey’s test (p≤0.05). Lavras, Brazil.

Fig 3. Total phenolic acids (A), lignin (B), flavonoids (C) and PAL activity (D) in leaves of Piper aduncum L. grown under different irradiance conditions (50, 70, 100% irradiance and red meshes – RN and blue meshes -BN). *Means followed by the same letter in the columns do not differ according to Tukey’s test (p≤0.05) .Lavras, Brazil.

Conclusions

Growth and synthesis of secondary compounds (total phenolic acids, lignin and flavonoids) of Piper aduncum were influenced by different irradiance conditions. Plants grown under blue photoselective shading screens showed higher growth and dry mass production. The highest amount of secondary compounds was obtained in plants grown under blue shading screens. Therefore, given that this is the species that produces bioactive compounds with commercial potential, its cultivation in environments supplemented with blue light is recommended.

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