Evaluate the effect of UVB radiation on the growth and physiology of Lepidium meyenii crops by biochemical, biophysical and physiological approaches

UNIVERSITY OF PISA

Department of Agricultural, Food and

Environment

MSc in Plant & Microbial Biotechnology

Evaluate the effect of UVB radiation on the growth

and physiology of Lepidium meyenii crops by

biochemical, biophysical and physiological

approaches

CANDIDATE: Gaia Crestani SUPERVISOR: Lorenzo Guglielminetti

ASSISTANT SUPERVISOR: Andrea Scartazza

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Summary

2. Aim of the thesis ... 13

3. Material and Methods ... 15

3.1 Plant material and growth condition ... 15

3.2 Biometric analysis ... 16

3.3 Extraction of plant material ... 16

3.4 Photosynthetic pigments ... 16 3.4.1 Exctraction ... 16 3.4.2 Spectrophotometric quantification ... 17 3.4.3 HPLC ... 17 3.5 Chlorophyll a fluorescence ... 18 3.6 Carbohydrates content ... 19 3.7 Glucosinolates ... 20 3.8 Phenolic compounds ... 21 3.9 Gas exchange ... 22 3.10 Starch quantification ... 23 3.11 Statistical analysis ... 23 4 Results ... 25 4.1 Biometric Analysis ... 25 4.2 Photosynthetic pigments ... 26 4.3 Chlorophyll a fluorescence ... 28

4.4 Total Soluble Sugars (TSS) ... 29

4.5 Glucosinolates ... 30

4.7 Gas exchanges ... 31

4 5. Discussion and Conclusion ... 35

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Abstract

UV-B solar radiation (UV-B) has an extensive impact on the biosphere. Most of the UV that reach the Earth are adsorbed by the stratospheric ozone layer, and therefore UV-B wavelengths (280 to 315 nm) are only a minor component of solar radiation at the Earth’s surface. The ambient level of UV-B is actually very variable and is affected by several factors. In particular, the latitude, season, and time of day affect the solar angle and hence the thickness of the atmosphere that UV-B must penetrate. In addition, the release of chlorofluorocarbons has caused depletion of the ozone layer, resulting in local elevations in UV-B. Lepidium meyenii Walp. (maca) is a Peruvian native plant, that growth especially around Junin District at about 4200 m a.s.l. There, the UV-B radiation is very strong and can reach 0,475 W m-2. A daily exposition to intense UV-B radiation can affected the growth, morphology and synthesis of metabolites as starch, sugar and secondary metabolites as well as the photosynthesis. In this work, have been investigate how maca plants response under B stress condition, comparing the control with UV-B cut off exposed plants after 4 or 7 months after sowing (UV-UV-B light was blocked by OROPLUS® PLASTIC film). We investigate on repartition and concentration of glucosinolates, phenolic compounds, starch, sugars, photosynthetic pigments, on efficiency of PSII and leaves gas exchange. Initially, we found that control plants had more symptoms related to UV-B stress exposure as lower efficiency of PSII and higher concentration of glucosinolates on leaves. After seven months of exposure, control plants, have shown an efficiency response to UV-B. In fact, the processes of senescence were slower, new leaves were produced, and this, can be related to a higher efficiency of PSII and a higher concentration of sugars. Furthermore, it was detected a translocation of glucosinolates from leaves to hypogeal organs and a reduction of starch concentration. The inhibitory effect of UV-B stress on L. meyenii Walp. plants, is modulate through the synthesis of UV-B absorbing compounds and the photosynthetic activity is restored by new leaves formation. In summary, these results suggest the existence of adaptation mechanisms to withstand prolonged UV-B radiation in maca plants.

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1. Introduction

1.1 Maca

Lepidium meyenii Walpers, commonly known as maca, is a native Peruvian Andean crop

belonging to the Brassicaceae family (Jin, et al., 2016). The plant grows exclusively at 3500-4500 m altitude, a zone characterized by areas of barren, rocky terrain, intense sunlight, fierce winds, and freezing temperatures (Wang, et al., 2007).

In Peru, maca is traditionally grown in the “meseta del Bombón”, a plateau situated at 4200 m a.s.l. around the lake Chinchaycocha in the Departments of Junín and Pasco. (Clément, et al., 2010).

Maca presents three mayor phenotypes (fig. 1), red, yellow and black based on their hypocotyl and stem coloration (Esparza, et al., 2015)

Fig. 1. three mayor phenotypes of maca yellow (left), red (centre)and black (right). Source: Esparza et al., 2015

Botanical description

L. meyenii is an herbaceous perennial plant, growing 12-20 cm. It is a rosette of frilly

leaves an enlarged freshly underground organ formed by a taproot and hypocotyl. These parts of the plant swell during growth, forming a storage organ resembling a turnip. The foliage forms a mat, growing in close contact with the ground. The leaves exhibit dimorphism, being larger in the vegetative phase and reduced in the reproductive cycle. Generally, leaves present a scarious margin and oblong form (7-12 cm in length and 1.5 to 2.5 cm wide). Furthermore, leaves are highly resistant to frost and show the typical stomata of the Cruciferae.

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Flowers are hermaphrodite, actinomorphic, pedicellate, 4 free green sepals persistent, 4 free white petals, alternating with the sepals. Androecium with 4 small staminodes and 2 fertile stamens with filaments thickened. The fruits are dry siliquae longer than wide (4-5 mm long by 2 -3 mm wide), longitudinal dehiscence. The seeds are ovoid, 2 to 2.5 mm and reddish grey.

This species is an octoploid with 2n= 8x = 64 chromosomes (Quirós & Aliaga Cárdenas, 1997) (Lock, et al., 2016) (Anon., 1989).

Constituent

The various compositions were considered closely related to health effect of maca. Especially, primary metabolites correspond to the nutritional component of the hypocotyls, and the secondary metabolites to compound with biological and medicinal properties.

Some researchers showed that maca is rich in proteins, amino acids, lipids, carbohydrates and fibre. Free fatty acids are also present in maca, the most abundant being linoleic, palmitic, and oleic acids. Saturated fatty acids represent 40.1% whereas unsaturated fatty acids are present at 52.7%. Minerals reportedly found in maca are iron, calcium, copper, zinc and potassium.

L. meyenii contain several secondary metabolites. The secondary metabolites macaridina,

macaene, macamides and maca alkaloids are only found in this plant. Macaenes are unsaturated fatty acids.Other compounds include sterols as beta-sitosterol, campesterol, and stigmasterol.

Different glucosinolates as the aromatic glucosinolate, glucotropaeolin have been described within maca. Benzyl glucosinolate has been suggested as chemical marker for maca biological activity. However, this has been discarded since glucosinolates may easily metabolize to isothiocyanates and these in other smaller metabolites (Gonzales, 2012) (Chen, et al., 2017).

A variety of factors can cause composition changes in maca, for example, maca planting environment (including altitude, climate and soil fertility), different colours of phenotypes as well as the process of drying especially the activity of hydrolytic enzymes as myrosinases (Chen, et al., 2017) (Esparza, et al., 2015).

9 Uses and properties

The tuber of maca is very well appreciated in pre-Inca and Inca times. This plant was domesticated at last two centuries ago in the Andean mountains where natives used its tubers as food and as a folk medicine (Piacente, et al., 2002). Evidences of early cultivation of this plant have been found dating back as far as 1600 B.C., and nowadays the tuberous hypocotyl is still cultivated for its high nutritional values by the local populations (Ganzera, et al., 2002).

Andean people consume the tuber of maca fresh or dried mixed in food preparation or beverages (Quirós & Aliaga Cárdenas, 1997) but, the flour is also used in the rest part of the world as dietary supplement for sexual dysfunctions and for antifatigue properties (Muhammad, et al., 2002).

Empirical researches revealed that maca exhibits many biological activities, including anti- oxidation, anti- tumour, fertility improvement, memory and learning enhancement and anti- inflammation (Tang W1, et al., 2017).The plant is also reputed to regulate hormonal secretion, stimulate metabolism, improve energy and modulate the response against oxidative stress. Due to its wide spectrum of putative qualities, maca is sometimes called Peruvian ginseng (Zhao, et al., 2012).

10 1.2 UV-B radiation

Plants use sunlight as an energy source and as an important environmental signal to regulate growth and development. Higher plants use sunlight signals to regulate a whole range of developmental processes and adaptations including germination, shade avoidance, stomatal development, circadian rhythm, and flowering. Beside regulation of growth and development, now termed “normal” photomorphogenesis, high light, and particular its integral ultraviolet (UV) part can induce stress responses in plants (Müller-Xing, et al., 2014). Ultraviolet radiation (UV) is a part of the non-ionizing region of the electromagnetic spectrum which comprises approximately 8–9% of the total solar radiation. UV is traditionally divided into three wavelength ranges: UV-C (200–280 nm) is extremely harmful to organisms, but not relevant under natural conditions of solar irradiation; UV-B (280–315 nm) is of particular interest because this wavelength represents only approximately 1.5% of the total spectrum, but can induce a variety of damaging effects in plants; UV-A (315–400 nm) represents approximately 6.3% of the incoming solar radiation and is the less hazardous part of UV radiation (Hollósy, 2002).

Figure 2. electromagnetic spectrum. Source: http://www.drb-mattech.co.uk/uv%20spectrum.html

Global changes in the chemical composition of the atmosphere with a substantial reduction of the protective ozone (O3) layer, results from emission of halogenate

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chemicals such as chlorofluorocarbons, have led to a notable increase in the solar UV-B reaching the earth surface (Kataria, et al., 2014).

The resulting increase of solar UV-B in the biosphere is predicted to be minor in comparison with seasonal variations in UV-B flux. Nonetheless, a statistically significant trend of increasing UV-B photon flux has been measured. Thinning of the ozone layer also results in a shift of the spectral UV-composition towards shorter wavelengths. In general, biological damage is exacerbated as the wavelength becomes shorter. Thus, even modest increases in total UV-B are likely to cause significant biological damage (Jansen, et al., 1998).

The type of stress response induced by light is determined by the fluence rate, exposure time, and whether plants have been acclimated by prior exposure to light.

The deleterious effects of UV-B on plants can be divided in three types of damage: morphological, physiological/biochemical and molecular.

Figure 3. Effects of UV-B on plants. Source: Kataria et al. 2014.

The morphological effects of UV-B include many changes, such as the reduction of plant height and leaf length/ area, increased auxiliary branching, leaf bronze, glazing, chlorosis and necrotic spot. In general, UV- B radiation reduces the dimensions of different plant organs.

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The physiological effects caused by UV-B includes reduction in photosynthetic activity mainly related to the degradation of PS II protein, destruction of chlorophyll and carotenoids, reduced Rubisco activity and effects on stomatal functions. The biochemical effects include flavonoids accumulation in the epidermis that protect plant from UV-B radiation. At high fluence rates UV-B acts as a damaging agent causing damage to biomolecules by generating reactive oxygen species (ROS), which can cause oxidation of lipid and protein and damage DNA and enhancement in lipid peroxidation. In order to lessen the impact of ROS generated by UV-B exposure, plant produces antioxidants such as ascorbic acid and alpha tocopherol and evokes antioxidant enzymes such as superoxide dismutase, ascorbic acid peroxidase, glutathione reductase and guaiacol peroxidase (Kataria, et al., 2014).

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2. Aim of the thesis

The potential impacts of an increase in solar UV-B radiation reaching the Earth’s surface have been investigated by numerous research groups during the past decades. Much of this research has focused on the effect of plant growth and physiology under artificial UV-B irradiation supplied to plants in growth chamber or greenhouse. Valid prediction of UV-B effects can only be made using environmental conditions (Hollósy, 2002). Commonly, plants exposed to an intense and prolonged UV-B radiation, combined with other stress condition, are characterized by faster senescence processes.

Highland Andean plants, such as maca, grow in an unhospitable place characterized by force wind, frequent droughts, great fluctuations in daily temperatures and intense solar radiation. Due to high altitude, the solar radiation is stronger than in other areas.

Probably, these plants are better equipped to survive the damaging effects caused by ultraviolet-B radiation.

This work aimed to assess the impacts of UV-B radiation on L. meyenii Walp. plants growing in field. We tried to understand the mechanism that plants use to survive in an adverse environment, checking a variety of parameters considered important in plant protection against UV-B. Changes in pigments, in secondary defence metabolites, starch, sugars, and photosynthetic activity were analysed in plants five and seven months old sowed in the field at 4200 m a.s.l. Control plants were exposed to UV and PAR, while treated plants were maintained under a UVB cut-off film.

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3. Material and Methods

3.1 Plant material and growth condition

Plants of L. meyenii were collected in April and June 2017 from a field experiment in Peru (UTM 18 L 391976m E, 8768061m S) at 4128 m.

Seeds obtained from local farmers, associated to Ecoandino (Lima, Peru) were sowed in December 2016 and divided in two different treatments: 3 tunnels of control (white film) and 3 tunnels of UVB cut-off (yellow film) OROPLUS® PLASTIK.

The dimensions of each tunnel were 3 m length x 1.5 m width x 1.5 m height.

The growth condition like temperature, humidity and solar radiation were very similar in both treatments but varied widely in line with the environmental conditions. UV- B radiation has reached ~ 0,375 W m-2 in April and ~ 0,310 W m-2 in June. The average temperature was around 6 ° C in April and 4° C in June. The relative humidity can reach 100 %.

Two samples were taken from each tunnel and immediately frozen in liquid nitrogen before lyophilization. After six days lyophilization, samples were weighed again and used for biochemical analysis.

Figure 4 experimental field. Yellow tunnels: OROPLASTIC PLUS FILM (UV-B cut off). White tunnels: CONTROL FILM

16 3.2 Biometric analysis

During the sampling, we analysed different biometrical parameters on fresh plants to assess the impact of UV-B radiation on plant growth.

The analysis consisted in the measurement of roots and hypocotyl length (cm), the diameter of leaf rosette (cm), the weight of roots and hypocotyl (g) and the number of green and yellow leaves.

3.3 Extraction of plant material

0.05 g of lyophilized and powdered maca leaves and hypocotyls were added to 0,5 ml of 70% methanol at 70°C in 2 ml eppendorf. Before closing the tubes, gaseous nitrogen was applied to prevent oxidation phenomena. The samples were placed in a water bath at 80 °C for 10 minutes, sonicated in an ultrasonic bath at a temperature of 50 ° C for 30 minutes and centrifuged at 10000 rpm for 15 minutes, the supernatant was recovered using a Mobicol 1 ml column with 10 μm pore filter and facilitating filtration with nitrogen flow. The pellet was resuspended in 0,5 ml of MeOH and the extraction procedure was repeated twice. However, the last time, samples were not centrifuged, but only filtered and the respective supernatants combined (final volume 1,5 ml). (Esparza, et al., 2015).

3.4 Photosynthetic pigments 3.4.1 Exctraction

Chlorophyll and carotenoids were extracted using a modified protocol of Carnagie (Asner, 2011). Maca leaves (0,02 g) were added to 0,01 g di magnesium carbonate and then, homogenized using a pestle in 2 mL eppendorf.

The powder was sonicated in icy water with 0,5 mL of 99,5 % cold acetone for 5 minutes. Samples were then centrifuged (Eppendorf 5417 C centrifuge, Marshall Scientific) at 1000 X g for 10 minutes and the supernatant was separated. The pellets were resuspended in 0,5 mL of cold acetone, sonicated and centrifuged again1 and the supernatants were combined. All the extraction phases were conducted in a green light chamber.

17 3.4.2 Spectrophotometric quantification

The extracts were diluted with acetone (1:25) and the content of chlorophyll was determined at 470, 662,645, 710 nm (if at 710 nm the absorbance is > 0,05 the samples need to be centrifuge again for the presence of too much sediments).

From measured absorbance it is possible to calculate chlorophyll A, chlorophyll B and carotenoids concentration following this equation (Lichtenthaler & Buschmann, 2001):

Chl A (µg ml-1_{) = [11.24*(A662 – A710) – 2.04*(A645-A710)]*dilution factor}

Chl B concentration (μg ml-1_{) = [20.13*(A645-A710) – 4.19*(A662-A710)] * dilution}

factor

Bulk Carotenoids (μg ml-1_{) = [(1000*(A470-A710) – 1.90*Chl A conc – 63.14*Chl}

Bconc)/214] * dilution factor

3.4.3 HPLC

For HPLC analysis, 200 µL of extract was diluted with 800 µL of 70 % methanol and analysed using Agilent 1200 binary pump HPLC system coupled to a 1260 Agilent Infinity diode array detector.

The column used was Poroshell SB-RP18, 150 x 2.1 mm, 2,7 µm, oven temperature 30 °C and a 0,350 mL min-1 flow rate. The gradient program use MilliQ® water (solvent A), acetonitrile (solvent B), methanol (solvent C),methanol:acetone (4:1) (solvent D) and was as follows:

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Timetable

Time A B C D Flow Pressure min % % % % mL/min bar --- 2.00 18.00 50.00 32.00 0.00 0.350 800.00 9.00 7.20 80.00 12.80 0.00 0.350 800.00 11.00 7.20 80.00 12.80 0.00 0.350 800.00 20.00 1.80 95.00 3.20 0.00 0.350 800.00 22.00 1.80 95.00 3.20 0.00 0.350 800.00 32.00 0.00 0.00 0.00 100.00 0.350 800.00 35.00 0.00 0.00 0.00 100.00 0.350 800.00 40.00 18.00 50.00 32.00 0.00 0.350 800.00 45.00 18.00 50.00 32.00 0.00 0.350 800.00 3.5 Chlorophyll a fluorescence

Chlorophyll a fluorescence measurement was conducted using LI- 6800 portable photosynthesis system (Li-cor, Lincol, NE, USA) implemented with Multiphase Flash™ Fluorometer (6800-01).

The leaves were covered by aluminium foil for 30 minutes to adapt them to darkness before starting the fluorescence measurements (Fv / Fm). Other leaves were adapted to the light to perform PSII efficiency measurements ΦPSII (ΔF / Fm ').

Potential photochemical efficiency of PSII in dark adapted leaves (Fv/ Fm) was calculate as follow: Fv/Fm = (Fm-Fo)/Fm, where Fo and Fm are the minimum and maximum fluorescence yield emitted by the leaves in the dark-adapted state.

Actual photochemical efficiency of PSII in light adapted leaves ΦPSII (ΔF / Fm ') was determinate as ΦPSII = (Fm’-F’)/Fm’ where Fm’ is maximum fluorescence yield in light adapted leaves obtained under exposition to actinic light in condition of saturating light and F’ is the fluorescence of ΦPSII following exposure to actinic light.

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ΦPSII was performed at CO2 concentration of 400 μmol mol-1, relative humidity of 50%, flow of 500 µmol m⁻² s⁻¹, actinic light of 1600 µmol m⁻² s⁻¹, light saturation pulse of 8000 μmol m-2 s-1 and duration of 1000 ms.

Non-photochemical quenching (NPQ) was determinate with this equation: NPQ=Fm/Fm’-1 (Bilger & Björkman, 1990).

3.6 Carbohydrates content

Leaves, hypocotyls and roots (0,02 g DW) were assayed using enzymatic method to determinate glucose, sucrose and fructose.

Previously, samples were extracted using the following protocol which can be divided into three steps.

The first one, consists in an acid extraction through 200 µL of 5.5% HClO4 and stored for 1 hour in ice.

Samples were then centrifuged (14000 rpm for 10 minutes) and the supernatant was neutralized with K2CO3 1M until reaching a neutral pH and stored for 1 hour in ice. In the last step, the volume was determinate picking up the supernatant, after centrifugation (14000 rpm for 10 minutes), and samples were stored at -20° C.

The enzymatic assay requires the preparation of four different solutions: solution A (1 mL Na-Acetate 50mM, pH 4,6 and 8000 U invertase), solution B (1 mL Na-Acetate 50mM, pH 4,6), solution C (4 ml Tris-HCl 300mM, pH 7,6 , 3 ml MgCl2 10mM , 15 mg ATP , 6 mg NADP , 5 U hexokinase , 3 U glucose – 6P- dehydrogenase) and solution D ( 100 Tris-HCl 300mM, pH 7,6 ,10 U glucose – 6P – isomerase).

Samples (40 µL) were then diluted up to final volume of 200 µL in duplicate series. In the first one, 100 µL of solution A have been added and incubated for 30 minutes. The second one was prepared adding 100 µL of solution B. Then, every samples and the standard were incubated with 700 µL of solution C for 30 minutes at 37 °C. The absorbance of each sample was measured at 340 nm using a spectrophotometer. Finally, 10 µL solution D were added to the series treated with solution B, incubated for 15 minutes at 37 °C and then read again at 340 nm. The samples reacted with solution A were employed to determinate free glucose and glucose originated from sucrose, the

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samples with solution B were used to determinate free glucose and the addition of solution D has allowed to quantify free glucose and free fructose.

The standard solution was prepared to build the calibration curve and subsequently to quantify soluble sugar content. 1 mg of glucose monohydrate was dissolved in 1 mL of water and then, different aliquots (0, 5, 10, 20, 40, 80, 160 µL) were diluted in water, up to 300 µL, corresponding to different nmoles of glucose (0, 28, 56, 112, 224, 448, 896) (Huarancca Reyes, et al., 2016).

3.7 Glucosinolates

Benzylglucosinolate was analyzed as its desulfated counterpart following the protocol of Esparza et al. (2015) with modifications.

First step was conditioning Bond Elut™ SAX 100 mg (Agilent Technology) columns positioned on Vaccum Manifold (Agilent technology) with 5 mL of 70% methanol to wet and activate the cartridges. 100 µL of samples (see extraction protocol pt. 3.3) were loaded onto columns with 200 µL of H2O MilliQ. Control 1 was prepared using 100 µL of 70 % methanol with 3 mM sinigrine and 200 µL of water, control 2 contains in addition 5 µL of benzylglucosinolate.

The cartridges were washed with 1 mL of 70% methanol and 1 mL of MilliQ H2O, conditioned with 200 µL of MES buffer (pH 5,2, 0,02 M) and 100 µL of sulfatase ( H-1

Helix pomatia, Sigma), and incubated in the darkness, overnight at room temperature.

The following day, glucosinolate were recovered with 800 µL of 70% methanol and 800 µL of MillQ water.

The extracts were filtered using IC Millex® 13 mm ion filter chromatography certified syringe filter unit (Merk Millipore) and then analysed with HPLC.

The column used was Merk LiChosper 100 RP-18 (250 mm X 4.6 mm, 5 µL), pressure300 bar, detector DAD 190-400 nm, oven temperature 30 °C and 1 mL min-1 flow rate. The gradient program used water (solvent A) and acetonitrile (solvent B).

21 Time (min) A B C D 6 95 5 0 0 8 93 7 0 0 18 79 21 0 0 23 71 29 0 0 25 0 100 0 0 29 0 100 0 0 33 98 2 0 0 40 98 2 0 0 3.8 Phenolic compounds

The protocol used for the quantification of total phenols was the Folin-Ciocalteau one (Waterhouse, 2002). In 2 mL eppendorf, 100 µL of each sample (obtained through the extraction protocol) was mixed with 1,5 mL of MilliQ® and 100 µL Folin-Ciocalteau reagent. After 5 minutes at room temperature, 300 µL of sodium carbonate 1M was added. After two hours in the darkness, the content of phenols was determinate by measuring the absorbance at 760 nm by spectrophotometer. The calibration curve was realized in the same way, using different stock solutions of gallic acid in different concentration: 20, 50, 100, 200, 500 and 1000 µg/µL.

22 Figure 5. gallic acid calibration curve

3.9 Gas exchange

Leaf gas exchange measurement were conducted using a LI- 6800 portable photosynthesis system (Li-cor, Lincol, NE, USA).

The measurements were conducted on central fully expanded leaflet, in April and June with 3 biological replicates, between 7:00 a.m. and 4:30 p.m.

Leaves dimension did not cover the area of measuring chamber, so it was measured with leaves scanner (CI-202 Portable Laser Leaf Area Meter) and then, the data were recalculate obtaining the values.

Measurement of CO2 assimilation rate (A), intercellular CO2 concentration (Ci),

transpiration rate (E) and stomatal conductance (gs) were obtained at CO2 concentration

of 400 µmol mol-1_{, relative humidity around 50-60%, leaf temperature of 25 ºC and at} light intensity of 1600 µmol m-2 _s-1 _{(Huarancca Reyes, et al., 2018).}

The A/Ci curves (CO2 assimilation, A, versus internal CO2 concentration) were obtained

at CO2 range from 0 to 2200 µmol m⁻² s⁻¹ and Photosynthetic Photon Flux Density (PPFD) at 1500 µmol m⁻² s⁻¹, that is the light saturation value.

Maximal carboxylation rate (Vcmax) and maximal rate of electron transport (Jmax) were determinate using the CO2 curves.

Light curves were performed at PPFD of 0, 50, 100, 400, 700, 1000, 1500, 1800, 2000 µmol m⁻² s⁻1_. y = 0,0029x - 0,0074 R² = 0,9985 -0,1 0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0 50 100 150 200 250

23 3.10 Starch quantification

0,01 g of dry weight maca hypocotyls were extracted with 1 mL of KOH 10 mM. The solution was boiled for 1 minute and then, it was transferred in plastic tubes to cool down. 10 ml of HCl 1N were added to samples.

The standard solution was obtained solubilizing 100 mg of starch in 100 mL of DW. After boiling for 1 minute, the calibration curve was prepared diluting 0, 20, 40 ,60, 80, 100 µL of starch solution up to final volume of 100 µL. Then, 50 µL of neutralized solution (100 ml of KOH 10 mM solution + 1 mL of HCl 1N) were added. The volume of standards corresponding to their quantity expressed in µg.

100 µL of water were added to 50 µL of each sample. Finally, 1 mL of iodine solution (2,5 mL of iodine into 25 mL of water) was added to standard dilutions and samples and read at 595 nm.

3.11 Statistical analysis

Experiments were performed in triplicate. Differences between groups were determinate using one-way ANOVA and the statistical significance was obtained using Turkey student. P values below to P < 0,05 were considered statistically significant.

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4 Results

4.1 Biometric Analysis

UV-B radiation did not show large differences in biometric parameters. Statistical tests showed no significant differences comparing fresh weight (g) of rosette, hypocotyl and root. The weights of hypocotyls and rosettes of control plants decreased in June, while in UV-B cut-off plants these parameters were increased. (Fig. 6a). Results of dry weight (Fig. 6b) demonstrated significant variations in roots of plants 4 months old, where values of UV-B cut-off (0.150 g) were higher compared to control (0.0816 g) and in rosettes of plants 7 months old, where the average weight of control (1.6025 g) plants was higher than UV-B cut-off (0.968 g).

Figure 6. Differences in fresh weight (a) and dry weight (b) in control and UV-B cut-off plants in April and June. Asterisk is shown when values were different with p<0.05

0 2 4 6 8 10

Rosette Hypocotyl Root Rosette Hypocotyl Root

Fre sh W ei gh t (g )

Control UVB cut-off

April June a 0 1 2 3

Rosette Hypocotyl Root Rosette Hypocotyl Root

Dr y W ei gh t (g )

Control UVB cut-off

April June

*

*

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The number of leaves in April was the same for both control and UV-B cut-off plants. On the contrary, in June the number of leaves was higher in control plants (29.25) than in UV-B cut-off plants (19.83) as well as the number of yellow senescent leaves (11 in control compared to 0.62 in UV-B cut-off) (Fig. 7)

Figure 7. Number of leaves in control and UV-B cut-off plants. Asterisk is shown when values were different with p<0.05

4.2 Photosynthetic pigments

UV-B radiation did not affect in a significant way photosynthetic pigments. Total chlorophyll, Chl a, and Chl b (Fig.8 a, b, c) had similar values in both treatments. Furthermore, the values remained constantly between April and June. Carotenoids content was statistically relevant in June, where the concentration in UV-B cut-off plants (1.030 mg g-1 DW) was higher than in control plants (0.774 mg g-1 DW) (Fig. 8 d). Carotenoids/total Chl ratio showed significant differences in April, when it was higher in UV-B cut-off plants compared to control. In June the carotenoids/total Chl, ratio increased in both treatment, but non-significant differences were found (Fig. 8 e).

Chl a/ Chl b ratio decreased in control 7 months-old plants (2.993) compared to UV-B cut-off (3.240), while in April the values were very similar (Fig. 8 d).

0 10 20 30

Total Green Yellow Total Green Yellow

Lea

ves N

u

mb

er

Control UVB cut-off

April June

* *

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Figure 8. Effect of UV-B radiation on photosynthetic pigments in control and UV-B cut-off plants in April and June. Total chlorophyll l(a), Chl a (b), Chl b (c), carotenoids (d),carotenoids to total chlorophyll(e) and the ratios of Chl a/b

(f). Asterisks indicate significant differences with p< 0,05.

0 1 2 3 4 April June m g To tal Chl g -1DW

Control UVB cut-off

0 1 2 3 April June m g C hl a g -1DW

Control UVB cut-off

b 0 1 2 April June m g C hl b g -1DW

Control UVB cut-off _c

0 1 2 April June m g C ar o te no ids g -1DW

Control UVB cut-off

* 0 0,2 0,4 0,6 April June C ar o te no ids / T o tal Chl

Control UVB cut-off

* e 0 1 2 3 4 April June C hl a / C hl b

Control UVB cut-off

*

f a

A

28 4.3 Chlorophyll a fluorescence

No variation in Chlorophyll a fluorescence using LI- 6800 portable photosynthesis system (Li-cor, Lincol, NE, USA) were detected. These values were related to efficiency of PSII and they remained constant between stress and control conditions.

Furthermore, the same results were obtained in plants 4and 7 months-old. (Fig. 9)

Figure 9. Chlorophyll a fluorescence. Maximum quantum efficiency of PSII, Fv/Fm (Fv/ Fm) under UV-B light exposure (control) and UV-b cut-off plants. .Asterisks indicate significant differences with p<0.05.

No statistically relevant differences were found in the efficiency of PSII between the control and the treatment. An increased capacity of PSII was detected in control plants 7 months-old when compared with control plants 4 months-old (Fig. 10 a, b)

0 0,2 0,4 0,6 0,8 1 April June Fv / Fm

Control UVB cut-off

0 0,2 0,4 0,6 0,8 0 300 600 900 1200 1500 ΦP SI I PPFD (µmol photons m-2_s-1₎ Control film UVB cut-off

a

b

29 Figure 10. Light response curve of ΦPSII and NPQ. Effects of UV-B radiation exposure (control) or UV-B cut-off (treated) on photochemical efficiency of PSII in April (a) and in June (b). Non-photochemical quenchingin April (c)

and June (d) (NPQ=Fm/Fm’-1) under different Photosynthetic Photon Flux Density (PPFD): µmol m⁻² s⁻¹

The determination of Non Photochemical Quenching (NPQ) showed similar values when plants exposed to UV-B radiation were compared to UV-B cut-off plants in both periods (Fig 10 c, d) These results were in accordance with results of efficiency of PSII, previoulsy described.

4.4 Total Soluble Sugars (TSS)

Results showed different repartition between source and sink organs of TSS after four and seven months (Fig. 11). In UV-B cut-off plants, the concentration of TSS in hypocotyl, was similar in April and in June while they decreased in leaves.

In control plants, TSS hypocotyls level increased considerably, between April and June and it was not changed in leaves.

In April, UV-B cut-off hypocotyl showed higher values (1148.95 µmol organ -1_{) of TSS} compared with control (992.56 µmol organ -1).

In June, a significant variation was found in leaves comparing control (1335.49 µmol organ -1)and treatment (346.43 µmol organ -1).

In both cases the concentration of TSS in roots was significantly higher in UV-B cut off compared to the control (Fig. 11).

-1 0 1 2 0 300 600 900 1200 1500 NP Q PPFD (µmol photons m-2_s-1₎ c -1 0 1 2 0 300 600 900 1200 1500 NP Q PPFD (µmol photons m-2_s-1₎

d

30

Figure 11. TSS levels. The graph compared the differences between control and UV-B cut-off in rosette, hypocotyl and root in April (left) and in June (right). Asterisks indicate significant differences (p< 0,05)

determinate by analysis of variance (ANOVA) and T-test.

4.5 Glucosinolates

Results shown a different allocation in glucosinolates content between source and sink organs under UV-B stress condition.

In April, data on glucosinolates had demonstrate that, rosette concentration of benzylglucosinolate was higer in control (11.21 µmol g-1 DW) compared with UV-B cut-off (6.70 µmol g-1 _{DW). Conversely, the amount of glucosinolates in hypocotyls} was higher in UV-B cut-off (16.10 µmol g-1 _{DW) compared to control maca (7.25 µmol} g-1 _DW).

In June, no statistically relevant differences in glucosinolates concentration were found between control and UV-B cut-off but concentration increased in both organs (Fig. 12)

0 300 600 900 1200 1500 1800

Rosette Hypocotyl Root Rosette Hypocotyl Root

µmo l T otal h ex ose eq u iv ale n t or gan -1

Control UVB cut-off

April June

*

* *

31

Figure 12. different repartition of benzylglucosinolate between source and sink organs in control and UV-B cut off. Differences were detected in April (left) and June (right). ). Asterisks indicate significant differences (p< 0,05)

determinate by analysis of variance (ANOVA) and T-test

4.7 Gas exchanges

Gas exchanges measurement were conducted using LI- 6800 portable photosynthesis system (Li-cor, Lincol, NE, USA).

The intercellular concentration of CO2 was higher in plants exposed to UV-B cut off radiation than the control, leading to major values of Maximal Rubisco carboxylation rate (Vcmax) and Maximum rate of electron transport (Jmax) (Fig 13 a).

0 10 20 30 40 50

Rosette Hypocotyl Rosette Hypocotyl

µmo l B en zyl glu cosin ola te g -1DW

Control UVB cut-off

* April June * -5 -1 3 7 11 15 19 23 0 200 400 600 800 1000 1200 CO 2 as si m ilat io n rat e (µm o l m -2s -1) Intercelullar CO2concentration (µmol mol-1₎ Control film UVB cut-off (Vcmax=23.70; Jmax=64.05) (Vcmax=47.24; Jmax=89.46) a

32

Figure 13.The response of CO2 assimilation rate to intercellular CO2 measured in leaves (A/CI curve) in April (a) and

in June (b) CO2 concentration 400 µmol mol-1_{, relative humidity 50-60%, leaf temperature of 25 ºC and at light} intensity of 1600 µmol m-2 _s-1_{(saturation value).}

Instead, in June A / Ci curve shown similar values between control and treatment (Fig. 13 b)

4.8 Starch quantification

The content of starch in hypocotyls 4 months-old and 7-months old, was statistically higher in UV-B cut of plants compared to the control.

In UV-B cut-off the concentration remained relatively stable in April (756.16 mg organ -1_{) and June (720.10 mg organ}-1_{) while it decreased in control plants. Initially, the average} concentration was 598.84 mg organ-1 to decrease to 456.40 mg organ-1 in maca seven months old. -10 10 30 50 70 0 300 600 900 1200 1500 CO 2 assi mi lati o n r ate (µ mo l m -2s -1) Intercelullar CO2concentration (µmol mol-1₎ Control film UVB cut-off (Vcmax= 108.4 ± 2.1 ; Jmax= 310.2 ± 22.7) (Vcmax= 112.2 ± 2.6 ; Jmax= 334.6 ± 7.7) b

33

Figure 14. Starch concentration. Differences in hypocotyl starch content were determinated comparing control and UV-B cut-off in April (left) and June ( right). ). Asterisks indicate significant differences (p< 0,05) determinate by

analysis of variance (ANOVA) and T-test

0 200 400 600 800 April June mg St ar ch or gan -1

Control UVB cut-off

35

5. Discussion and Conclusion

Due to restricted cultivation area, maca shows robust endurance to extreme stresses including temperatures variation and severe UV-B exposure (Zhang, et al., 2017)

In this thesis, were analysed the mechanisms involved in the response to UV-B radiation exposure on Lepidium meyenii Walp. in comparison with plants protected by a filter film for UV-B.

The results demonstrated the incredible capabilities of maca plants to mitigate effectively UV-B damage. It has been shown how this kind of radiation, which can reach 0,475 W m-2 in Junín district, may influence the growth, different synthesis and translocation of secondary metabolites between source and sink organs.

Furthermore, the stress response has been evaluated in terms of efficiency of PSII and leaf gas exchanges.

In April (4 months after the sowing), there has been reduction of ΦPSII caused by an intense UV-B exposure of control plants. At the same time, Vcmax and Jmax values derived

from CO2 (A/Ci) curve were lower too. The decrease in ΦPSII and gas exchange was

mainly due to the reduction in photosynthesis under UV-B stress condition.

Into UV-B cut-off plants, less damage in ΦPSII is probably related with a higher concentration of free sugars found in hypocotyls and roots, because more sugars were synthetized by photosynthesis process and, immediately translocated to hypogeal organs. UV-mediated effects on glucosinolates are conceivable, since they are involved in the common plant defence response regulated by the signalling pathways involved in perception of UVB (Rozema, et al., 1997). It evidences a different allocation of these secondary metabolites between source and sink organs.

Indeed, a higher synthesis of glucosinolates inside the leaves such as to constitute a defensive barrier in plants under UV-B irradiance, was detected. On the contrary, in plants not exposed to UV-B, the synthesis of glucosinolates into the hypocotyls was higher than in the leaves. This, can probably related to the presence of OROPLUS® PLASTIK film that blocking UV-B radiation and, for this reason plants do not need to synthetize glucosinolates in leaves.

36

In plants 7 months-old the course of the senescence is different: in control plant, the ageing leaves was faster but new leaves were emerged. Indeed, the number of yellow leaves and green leaves increased at the same time.

On the contrary, in UV-B cut-off plants the senescence process is slower and new leaves were not formed. In accordance with the senescence, also a higher concentration of carotenoids was detected.

New leaves in control plants, were probably related to an increased efficiency of PSII and also related with a higher concentration of free sugars. In opposition, in plants under OROPLUS® PLASTIK film, the content of free sugars was decreased and mainly localized into hypogeal organs as glucosinolates and starch and into leaves as glucosinolates.

Differences in the levels of adaptation or acclimation to UV-B determine the response to UV-B exposure. Following the acclimation, the amplitude of response to UV-B is reduced because a certain level of protection is already in place. Indeed, plants grown in light lacking UV-B are more likely to suffer stress on first exposure whereas plants grown in UV-B are more likely to tolerate an increase in dose. Plants that are exposed to elevated UV-B express genes that help the plant to counter any stress effect, repair damage, and establish increased protection (Jenkins, 2009). This, can explain how Lepidium meyenii Walp., by adapting to the environment of the Puna, is able to limit the severe damage of intense UV-B exposure, when compared to other crops.

37

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