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Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: Augmentation of secondary metabolites and antioxidants
Accepted Manuscript Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: augmentation of secondary metabolites and antioxidants Swabha Takshak, S.B. Agrawal PII:
S0981-9428(15)30120-0
DOI:
10.1016/j.plaphy.2015.09.018
Reference:
PLAPHY 4292
To appear in:
Plant Physiology and Biochemistry
Received Date: 29 July 2015 Revised Date:
30 September 2015
Accepted Date: 30 September 2015
Please cite this article as: S. Takshak, S.B. Agrawal, Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: augmentation of secondary metabolites and antioxidants, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental
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ultraviolet-B radiation: augmentation of secondary metabolites and antioxidants
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Swabha Takshak, S.B. Agrawal*
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Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu
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University, Varanasi-221 005, India
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Abstract
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Supplementary ultraviolet-B (ambient+3.6kJ m-2 day-1) induced changes on morphological,
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physiological, and biochemical characteristics (specifically the defence strategies: UV-B
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protective compounds and antioxidants) of Coleus forskohlii were investigated under field
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conditions at 30, 60, and 90 days after transplantation. Levels of secondary metabolites increased
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under s-UV-B stress; flavonoids and phenolics (primary UV-B screening agents) were recorded
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to be higher in leaves which are directly exposed to s-UV-B. This was also verified by enhanced
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activities of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase (PAL), cinnamyl
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alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone–flavanone isomerase
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(CHI), and dihydroflavonol reductase (DFR). Antioxidants, both enzymatic (ascorbate
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peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide
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dismutase) and non-enzymatic (ascorbic acid and α-tocopherol) also increased in the treated
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organs of the test plant, higher contents being recorded in roots except for ascorbic acid. On the
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contrary, protein and chlorophyll content (directly implicated in regulating plant growth and
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development) declined under s-UV-B. These alterations in plant biochemistry led the plant to
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compromise on its photosynthate allocation towards growth and biomass production as
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evidenced by a reduction in its height and biomass. The study concludes that s-UV-B is a potent
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stimulating factor in increasing the concentrations of defense compounds and antioxidants in C.
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forskohlii to optimize its performance under stress.
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Key words: antioxidants; Coleus forskohlii; oxidative stress; secondary metabolites; s-UV-B
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Abbreviations: APX- ascorbate peroxidase; BSA- bovine serum albumin; CAD- cinnamyl
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alcohol dehydrogenase; CHI- chalcone flavanone isomerase; CAT- catalase; Ci- internal CO2;
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DAT- days after transplantation; DCPIP- 2, 6-Dichlorophenol indophenol; DFR- dihydroflavanol
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reductase; DTNB- 5,5’-Dithiobis-(2-nitrobenzoic acid); EDTA- ethylene diamine tetraacetic
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acid; LPO- lipid peroxidation; F0- initial fluorescence; Fm- maximum fluorescence; Fv- variable
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fluorescence; GR- glutathione reductase; Gs- stomatal conductance; H2O2- hydrogen peroxide;
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IAA- indole acetic acid; MDA- malondialdehyde; ̇O2- - superoxide radical; PAL- phenylalanine
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ammonia lyase; POX- peroxidase; PPO- polyphenol oxidase; Ps- photosynthetic rate; PS I-
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photosystem I; PS II- photosystem II; PVP- polyvinylpyrrolidone; ROS- reactive oxygen
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species; SOD- superoxide dismutase; s-UV-B- supplemental ultraviolet-B; TBA- thiobarbituric
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acid; TCA- trichloroacetic acid; UV- ultraviolet; UV-BBE- biologically effective UV-B; WUE-
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water use efficiency
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*
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E-mail address: [email protected] (S.B. Agrawal)
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Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174.
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1. Introduction In recent years stratospheric ozone (O3) depletion, has been largely attributed to rapidly
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changing climatic conditions, altered land-use patterns, and newly discovered O3 depleting
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substances (Anderson et al., 2012; Laube et al., 2014) and is directly responsible for increased
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penetration of biologically active UV-B radiation (280-315 nm) reaching the Earth’s surface.
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The impacts of UV-B have been widely reported on crop plants, covering morphological and
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physiological (Yang et al., 2005), biochemical (Choudhary and Agrawal, 2014a, b), antioxidative
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defense system (Agrawal et al., 2009), and genetic level (Tripathi et al., 2011) parameters. Plants
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have adapted two basic strategies to optimize their growth and development under s-UV-B
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stress: (i) biosynthesis of higher concentrations of secondary metabolites which function as
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antioxidants, enzyme inhibitors, chemical signals, growth regulators, and UV-B screens
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(Julkunen-Tiitto et al., 2005) and (ii) increased production of enzymatic- and non-enzymatic
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antioxidants primarily to counteract the damaging effects of ROS. The former include
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superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase
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(CAT), peroxidase (POX), and polyphenol oxidase (PPO) among others (Mittler et al., 2004),
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while in the latter category, ascorbic acid and α-tocopherol qualify as two major and widely
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studied candidates (Jaleel, 2009). Thiol and proline also serve as antioxidants under stress while
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also regulating transcriptional and translational level changes (Saradhi et al., 1995; Deneke,
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2000). These defence strategies providing tolerance and resistance to the plant come at the cost
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of reallocation of photosynthates towards new pathways. This may/ may not cause the plant to
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compromise on its biomass/ yield, and photosynthetic efficiency and functioning, depending
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upon the extent and efficacy of these defensive and adaptive strategies.
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The effects of various abiotic stress factors on the morphological, physiological, and
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biochemical characteristics of medicinal plants are few and far-between. Coleus forskohlii
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(Lamiaceae) has been used as an ancient root drug in Ayurvedic medicine for treating asthma,
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bronchitis, insomnia, epilepsy, angina, and psoriasis. Roots have been used to allay burning and
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for the treatment of worms and other skin diseases. Leaves have been used as expectorant,
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diuretic, for treating intestinal disorders, and as a condiment. Forskolin, a labdane diterpenoid,
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isolated from the plant, is used in the treatment of asthma, glaucoma, hypertension, cancer, heart
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diseases, diabetes, and obesity (Khatun, 2011; Singh et al., 2011). In view of the above 3
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mentioned medicinal importance of the test plant, it becomes important to determine the effects
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of s-UV-B radiation on its overall performance. The ultimate objective of the plant is to optimize its performance under adverse stress
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(biotic/abiotic) conditions and ensure its survival. This requires an alteration in its architectural
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and physiological aspects, which in turn, can be attributed to the allocation and partitioning of
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assimilated carbon and/or mobilization of storage carbon reserves (Geiger and Servaites, 1991).
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Chronic exposure of plant to any stress will lead it to develop new physiological capabilities
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which might manifest themselves in terms of altered plant morphology. If a plant responds
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successfully to its stress environment, it will indicate altered plant metabolism and biochemistry
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(or both) via altered gene expression without compromising on its biomass/yield (Geiger and
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Servaites, 1991). Hence, the present work was based on the hypothesis that under s-UV-B, the
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test plant, C. forskohlii, will combat the oxidative stress by increasing the concentrations of UV-
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B absorbing compounds and antioxidative defence system and will consequently be able to
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maintain its morphological traits and biomass. The hypothesis was tested by computing the
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morphological, physiological and biochemical parameters in the leaves and roots of the test plant
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(leaves, because they are directly subjected to s-UV-B radiation and roots, because they are
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economically important plant organs, being the source of medicinally important essential oil).
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2. Materials and methods
2.1. Experimental site and experimental design
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The experimental site was located in Botanical Garden, Department of Botany, Banaras
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Hindu University, Varanasi (25°80’N, 82°03’E, and 76 m above mean sea level), India. The
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meteorological parameters throughout the experimental period (month-wise) are given in Table
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1. The average maximum and minimum temperatures were 33.3°C and 7.6°C respectively,
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relative humidity ranged from 58.2% to 92.4%, while the total rainfall amounted to 78.0 mm.
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The soil texture was sandy loam (with sand, silt, and clay being 45, 28, and 27% respectively)
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having a slightly alkaline pH (7.1). C. forskohlii plants (one month old) obtained from the
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nursery were transplanted in experimental plots (1m×1m). Three rows in each plot were planted
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with 4 plants in each row (a total of 12 plants per plot). The planting conditions were as follows:
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distance between the ridges: 30 cm, distance between ridges and plot border: 15 cm, and distance
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between plants: 20 cm. The plots were prepared in triplicate for each type of treatment set out in
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a randomized block design (RBD). Recommended dose of fertilizers were supplemented as 40,
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60, and 50 kg ha-1 of NPK, respectively. Half the dose of N and full doses of P and K were
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applied as a basal dose during field preparation, while the remaining dose of nitrogen was
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provided as top dressing at 30 DAT (Paul et al., 2013). The plants were irrigated at regular
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intervals as per the requirements.
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2.2. s-UV-B treatment
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Once the plants were established in the field, they were subjected to s-UV-B radiation via
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UV-B lamps (Q Panel UV-B 313 40W fluorescent lamps, Q panel Inc., Cleveland, OH, USA)
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mounted on steel frames at a distance of 30 cm directly above the plant canopy; this distance was
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kept constant throughout the experimental period. The lamps were covered with 0.13 mm
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cellulose diacetate filter (Cadillac Plastic Co., Baltimore, MD, USA; transmission down to 280
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nm) for s-UV-B treatment while for control 0.13 mm polyester filter (Cadillac Plastic Co.; which
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absorbs radiation below 320 nm) was used. Though the filters allowed the transmission of UV-A
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radiation (320-400 nm) as well, its amount was very low (<10%) compared to the UV-A
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irradiance received from sunlight. Thus, the effects on plants from this radiation were presumed
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to be negligible. Hence, control plants received ambient UV-B dose (5.8 kJ m-2 day-1) while
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plants under UV-B lamps experienced ambient+3.6 kJ m-2 d-1 UV-B (biologically effective UV-
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B (UV-BBE) as weighted by Caldwell (1971) generalised plant action spectrum normalised at 300
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nm. As the filters are degraded by UV-B, they were replaced each week. s-UV-B treatment was
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given to the plants during the solar noon period (11:00 to 14:00 h). UV-B irradiance and UV-BBE
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were measured using Ultraviolet Intensity Meter (UVP Inc., San Gabriel, CA, USA) and
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Spectropower-meter (Scientech, Boulder, USA), respectively.
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2.3. Plant sampling and analysis
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Plants were randomly sampled from the triplicate plots for each treatment for the
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analysis. They were dug out in the form of monoliths with roots intact, thoroughly washed with
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running water to remove the debris, and plant parts separated. All metabolites were analysed in
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the fresh tissue (both leaves and roots). Sampling was done at 3 ages (30, 60, and 90 DAT; days
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after transplantation). Five plants of each treatment were up-rooted at each of the sampling ages 5
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for the measurement of growth traits. For the analysis of plant metabolites and enzyme activities,
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three plants were randomly selected (i.e. three each from control as well as s-UV-B-treated ones)
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and three samples per plant were further processed, making a total of nine replicates for each
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treatment. After final calculations, two of the outliers were rejected, while seven observations
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were retained on which further statistical tests were applied.
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2.4. Measurement of growth traits:
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To determine total biomass, plants were collected, plant-parts separated, and oven-dried
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at 80°C till the attainment of constant weight. Total biomass was calculated by adding the dry
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weight of different plant parts. It was expressed in terms of g plant-1. Other growth
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characteristics such as total plant height, number of leaves and leaf area, were also determined,
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the latter being measured via portable leaf area meter (Model Li-3100, Li-COR, Inc., USA). Five
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plants at each of the sampling ages were used for these measurements.
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2.5. Determination of Physiological Parameters:
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Physiological parameters were determined on five randomly selected plants from control
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plots as well as s-UV-B treated plots with three sets of observations being recorded for all
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parameters from each plant, thus amounting to a total of fifteen replications per treatment. The
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plants were tagged at the initial sampling age and subsequent measurements were made on the
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same plants. Photosynthetic rate (Ps), stomatal conductance (gs), and internal CO2 (Ci) were
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measured using a portable photosynthetic system (Model LI6400 XT, Version 6.2, Lincoln,
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Nebraska, USA) at the three sampling ages (30, 60, and 90 DAT) while water use efficiency
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(WUE) was calculated as the ratio of photosynthetic rate to transpiration. The measurements
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were made between 9:00 and 10:30 h on the third fully expanded leaf from top of randomly
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selected plants of each treatment. Chlorophyll fluorescence (initial and maximum, F0 and Fm
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respectively) were measured using plant efficiency analyzer (Model PEA, MK2-9414, Hansatech
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Instruments Ltd., UK) on the same leaves on which photosynthesis was measured during the
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same hours. From F0 and Fm, variable fluorescence (Fv) and photochemical efficiency (Fv/Fm)
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were calculated. The leaves were dark-adapted on the adaxial side for 30 minutes, then irradiated
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with red light and fluorescence signal collected at excitation irradiance set at 3000 µmol m-2 s-1.
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2.6. Determination of IAA oxidase activity:
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The extract preparation was carried out following the method of Dash et al. (2011) by
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homogenizing 1 g plant tissue in 10 ml potassium phosphate buffer (50 mM, pH 6.0) and
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subjecting it to centrifugation at 20 000 × g for 20 min. Supernatant was mixed with cold acetone
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to a final concentration of 70% and re-centrifuged at 20 000 × g for 15 min. The precipitate was
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re-suspended in the same buffer and again centrifuged at at 20 000 × g for 20 min. The resulting
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supernatant was used for enzyme assay. IAA oxidase activity was determined as per Beffa et al.
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(1990) using Salkowski reagent. IAA was calculated by measuring the change in absorbance ∆A
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at 535 nm and expressed as mg IAA degraded min-1 mg protein-1.
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2.7. Determination of plant metabolites:
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Protein content was determined by the method of Lowry et al. (1951) using BSA (bovine
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serum albumin) for the preparation of the standard curve. 0.5 g of fresh tissue, homogenized in
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5ml tris buffer (0.1 M, pH 6.8) was centrifuged at 5000 × g for 5 min; 5ml 10% TCA
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(trichloroacetic acid) was added to the supernatant and allowed to react for 5 min. The mixture
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was again centrifuged at 6000 × g for 10 min. The pellets were dried and dissolved in 5ml 0.1 N
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NaOH and solution was centrifuged at 6000 × g for 10 min. To 1 ml of the supernatant 5 ml of
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alkaline solution [prepared by mixing 50ml of alkaline sodium carbonate (2% (Na2CO3) in 0.1 N
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NaOH] and 1 ml of 0.5% copper sulphate in 1% potassium tartarate solution] was added and
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kept for 10 min at room temperature. To this, 0.5 ml of Folin’s reagent (1 N) was added and kept
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for additional 30 min. Absorbance of the blue-colored complex was recorded at 650 nm on a
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double beam spectrophotometer (Model-2203, Systronics, India).
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Protein content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of protein read from standard curve (µg ml-1), W is weight
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of leaf sample (g), V is volume of extract (ml) and v is volume of supernatant taken for analysis
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(ml).
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Thiol was estimated as per Fahey et al. (1978). 0.1 g tissue was homogenized in 80% v/v
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ethanol, boiled in 10 ml of ethanol (80%) at 80 °C for 15 min, cooled and centrifuged at 10 000
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× g for 10 min. To 1 ml of supernatant, 5 ml Ellman’s reagent (DTNB) (60 µM 5, 5’-Dithiobis-
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(2-nitrobenzoic acid) in phosphate buffer (0.1 M, pH 7.5)) was added and the reaction mixture
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was allowed to stand for 5 min. The absorbance of the yellow colour developed was measured at
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412 nm.
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Thiol content (mg g-1 FW) = (0.22×V×OD) / (W×v×1000) Where, W is weight of leaf sample (g), V is total volume of sample (ml), v is volume of
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the supernatant taken for analysis (ml) and 0.22 is correction factor.
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Proline content was determined following the method of Bates et al. (1973). 0.5 g tissue was
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homogenized in 10 ml sulfosalicylic acid (3% w/v) and centrifuged at 10 000 × g for 10 min. 2
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ml supernatant was incubated at 100 °C for 60 min with 2 ml ninhydrin reagent (1.2 g ninhydrin
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dissolved in 30 ml glacial acetic acid and 20 ml orthophosphoric acid). 2 ml of glacial acetic acid
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was added to the resulting solution. The reaction was terminated by placing the solutions in ice
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bath, extracted with 4 ml of toluene, and mixed vigorously for 10 min. The absorbance of the
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chromophore-containing toluene was recorded at 520 nm. Standard curve was prepared with
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known concentrations of proline using above methodology.
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Proline content (mg g-1 FW) = (C×V) / (115.5×W)
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Where, C is concentration of proline read from standard curve (µg), V is volume of
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toluene (ml), W is weight of leaf sample (mg) and 115.5 is molecular weight of toluene. Secondary metabolites except phenolics (alkaloids, anthocyanins, carotenoids, lycopene,
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β-carotene, flavonoids, lignin, phytosterols, saponins, and tannins) were assessed according to
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the methods described in Takshak and Agrawal (2014b). Total phenol was determined as per
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Bray and Thorpe (1954) using Folin’s reagent and catechol as standard compound. 0.1 g fresh
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tissue was homogenized with 10 ml 70% acetone and centrifuged at 6000 × g for 10 min. To 1
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ml of supernatant, 1 ml Folin’s reagent (1 N) and 2 ml 2% Na2CO3 (w/v) was added and final
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volume was made up to 10 ml with distilled water. Mixture was heated on boiling water bath till
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the appearance of blue colour. Solution was allowed to cool and absorbance of blue colour
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recorded at 650 nm on a double beam spectrophotometer (Model-2203, Systronics, India).
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Phenol content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of phenol read from standard curve (µg ml-1), W is weight
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of leaf sample (g), V is volume of extract (ml) and v is volume of supernatant taken for analysis
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(ml).
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2.8. Determination of phenylpropanoid pathway enzymes’ activities:
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0.4 g leaf tissue was homogenised in 4 ml sodium borate buffer (50 mM; pH 8.7)
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containing 5 mM β-mercaptoethanol, 1 mM EDTA and 2% PVP (w/v). The mixture was
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centrifuged at 20 000 × g for 15 min (twice); the resulting supernatant was used for the
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determination of PAL activity. The extraction buffer for CAD, 4CL, and CHI comprised of 0.2
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M Tris–HCl (pH 7.5), 8 mM MgCl2, 2% PVP, 5 mM DTT, 0.1% Triton X-100, and 1 mM
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PMSF. 1 g of plant tissue was homogenised in 10 ml extraction buffer and subjected to
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centrifugation at 18 000 × g for 20 min. The supernatant collected was re-centrifuged at 15 000 ×
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g for 15 min. Resulting supernatant was used for enzyme assays. All the enzyme extractions
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were carried out at 4° C. For DFR assay, 1 g sample was homogenized in phosphate buffer saline
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(PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4; pH 7.4) and
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centrifuged at 15 000 × g for 15 min. The supernatant was re-centrifuged for 10 min at 10 000 ×
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g, decanted and used as substrate for enzyme analysis. The enzyme activities of PAL, CAD,
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4CL, CHI, and DFR were measured as per the protocols already described by Takshak and
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Agrawal (2014b).
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2.9. Determination of chlorophyll content:
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Leaf tissue (0.1 g) immersed in 10 ml 80% acetone was kept in a stoppered conical flask
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overnight at 4 °C. It was then homogenized, centrifuged at 5 000 × g for 15 min, and final
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volume made up to 25 ml with 80% acetone. Absorbance of the solution was recorded at 645 nm
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and 663 nm. Chlorophyll content was calculated using the formulae by Maclachlan and Zalik
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(1963).
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Chlorophyll a (mg g-1 FW) = [(12.3 OD663-0.86 OD645) × V] / [1000 × W × d]
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Chlorophyll b (mg g-1 FW) = [(19.6 OD645-3.6 OD663) × V] / [1000 × W × d]
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Total chlorophyll (mg g-1 FW) = Chlorophyll a + Chlorophyll b
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2.10. Determination of ROS and LPO: H2O2 content was determined using the protocol of Alexieva et al. (2001). The reaction
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mixture contained 0.5 ml leaf extract supernatant (0.1g tissue extracted in 0.1% TCA at 4°C),
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potassium phosphate buffer (10 mM), and potassium iodide (KI, 1 M). The reaction mixture was
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kept in dark for 1 hour. The absorbance was recorded at 390 nm. The amount of hydrogen
6
peroxide was calculated using standard curve prepared with known concentrations of H2O2.
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Determination of •O2− production rate (as per Elstner and Hupel, 1976) involved the extraction of
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0.5 g tissue in 65 mM phosphate buffer at 4°C. 0.5 ml supernatant was incubated in dark at 25°C
9
for 20 min after adding 65 mM phosphate buffer and 10 mM hydroxylamine hydrochloride. 8.5
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mM sulphanilamide and 3.0 mM α-naphthylamine were added to the solution (still in dark), and
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again left at 25°C for 20 min. The absorbance was recorded at 530 nm. The superoxide radical
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production rate was calculated from the standard graph prepared using potassium nitrite (KNO2).
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Lipid peroxidation was measured in terms of malondialdehyde (MDA) content (Heath and
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Packer, 1968). Fresh tissue (0.5 g) was homogenized in 5% TCA (trichloroacetic acid) and
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centrifuged at 10 000 × g for 10 min. To 1 ml of supernatant, 4 ml 0.5% TBA (thiobarbituric
16
acid) prepared in 20% TCA was added. The mixture was heated on boiling water bath for 30
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min, and cooled immediately on ice bath. Again the mixture was centrifuged for 10 minutes at 10
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000 × g. The absorbance of the yellow colour developed was recorded at 532 nm and 600 nm on
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double beam spectrophotometer (Model 2203, Systronics, India). The MDA content was
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calculated using the following formula:
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MDA (nmol mg-1 FW) = (O.D.600-O.D.532)* 106/155000
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Where, 155000 is the extinction coefficient of MDA.
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2.11. Determination of Enzymatic Antioxidants: 400 mg fresh tissue was homogenized in 10 ml of sodium phosphate buffer (0.1 M, pH
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7.0) containing 0.1 % (m/v) Triton X-100 and 0.2 g of polyvinylpyrrolidone (PVP) under ice-
26
cold conditions. The homogenate was centrifuged at 10 000 g for 20 min and supernatant was re-
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centrifuged at 13 500 × g and 4 °C for another 15 minutes. The supernatant was collected, stored
28
at 4°C and used for the analysis of all enzymatic antioxidants (APX, CAT, GR, POX, PPO, and
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SOD). APX was assayed as per the method of Nakano and Asada (1981) after supplementing the 10
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extraction buffer with 1 mM ascorbate and using the coefficient of absorbance of 2.8 mM-1 cm-1.
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The decrease in absorbance was recorded at 290 nm and enzyme activity expressed in terms of
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mM min-1 mg protein-1. CAT activity was determined by the method of Abei (1984). The
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enzyme activity was determined by measuring the rate of decrease of H2O2 at 240 nm for 1 min,
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calculated using the extinction coefficient of 0.036 mM-1 cm-1 and was expressed as µmol H2O2
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oxidized min-1 mg protein-1. GR activity (as per Anderson, 1996) was determined using
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coefficient of absorbance of 6.22 mM-1 cm-1 and expressed as mM min-1 mg protein-1. Peroxidase
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activity (POX) was determined using the method described by Britton and Mehley (1955).
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Extinction coefficient of 2.47 mM-1 cm-1 was used to calculate the enzyme activity and the latter
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was expressed as µM purpurogallin formed min-1 mg protein-1. PPO activity was determined as
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per Kumar and Khan (1982) and expressed in Units mg protein-1 where one unit represented 0.1
12
change in absorbance per minute. Protocol of Fridovich (1974) was followed for determining
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SOD activity with minor modifications. The absorbance was recorded at 560 nm and the activity
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was expressed as Units mg protein-1. One unit of enzyme activity was defined as the amount of
15
enzyme required for 50% inhibition of the reduction of NBT.
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2.12. Determination of Non-enzymatic Antioxidants:
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Ascorbic acid was estimated using 2, 6-dichlorophenol indophenol (DCPIP) reduction method of
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Keller and Schwager (1977). 20 ml of ice cold extracting solution (50 mg oxalic acid and 75 mg
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EDTA dissolved in 100 ml distilled water) was used to homogenize 0.5 g fresh plant tissue. The
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homogenate was centrifuged at 6000 × g for 15 min; 1 ml supernatant was mixed with 5 ml
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DCPIP (20 µg ml-1) with constant shaking. Absorbance of the pink colored solution was
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recorded at 520 nm (Es) using UV- VIS spectrophotometer (Model 119, Systronics, India). The
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pink colour was then bleached by adding one drop of 1% ascorbic acid and again the absorbance
24
was recorded at the same wavelength (Et). 1 ml extracting solution mixed with 5 ml DCPIP
25
solution served as blank and its absorbance was also recorded at the same wavelength.
26
Calibration curve was prepared using known concentrations of ascorbic acid. Ascorbic acid
27
content was calculated as follows:
28
Ascorbic acid content (mg g-1 FW) = (C×V) / W×1000×v
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Where, C [= Eo-(Es-Et)] is the concentration of the ascorbic acid read from the standard
2
curve (µg), W is weight of leaf sample (g), V is volume of extract (ml) and v is volume of
3
supernatant taken for analysis (ml).
4
α-tocopherol content was measured as per the method of Jaleel (2009). 0.5 g fresh tissue
5
homogenized in 10 ml petroleum ether and ethanol in the ratio 2:1.6 was was centrifuged at 10
6
000 × g for 20 min. To 1 ml supernatant, 0.2 ml 2% dipyridil in ethyl alcohol (w/v) was mixed
7
thoroughly, and kept in dark for 10 min. 4 ml distilled water was added to the resulting solution
8
and mixed well. The solution was kept still at room temperature for 10 min; the absorbance of
9
the solution was recorded at 520 nm. Known concentrations of α-tocopherol were used for the
10
preparation of standard curve using the above methodology. The solution was allowed to stand
11
for another 10 min at room temperature. The absorbance of the resulting solution was recorded at
12
520 nm. Standard graph was prepared using known concentrations of α-tocopherol.
13
α-tocopherol content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of α-tocopherol read from standard curve (µg ml-1), W is
15
weight of leaf sample (g), V is volume of water (ml) and v is volume of supernatant taken for
16
analysis (ml).
17
2.13. Statistical analysis:
18
The means of various parameters between control and treated plants were compared via
19
Student’s t-test. The individual as well as interactive effects of plant age and s-UV-B treatment
20
were computed via two-way ANOVA. All statistical analyses were performed using the SPSS
21
software v.16.
23
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3. Results
3.1. Growth characteristics:
24
Total plant biomass was found to be significantly reduced at all sampling ages (Table 2),
25
maximum reduction being observed at 90 DAT (20.6%). Plant height also reduced significantly
26
at all ages by 8.0, 19.7, and 23.1% at 30, 60, and 90 DAT respectively (Table 2) under s-UV-B.
27
The treatment also caused a significant decline in number of leaves and leaf area (Table 2). Plant 12
ACCEPTED MANUSCRIPT
biomass, plant height, and leaf area were significantly affected by age, treatment, and their
2
interactions, while leaf number was significantly affected only by the individual factors as per
3
the results of two-way ANOVA (Table S1).
4
3.2. Physiological parameters:
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Both Ps and Gs were negatively affected under s-UV-B at all sampling ages (Table 2).
6
Maximum decline in Ps was recorded at 90 DAT (31.0%) and in Gs at 60 DAT (30.9%). Two-
7
way ANOVA results showed significant effects of age, treatment and their interactions on both
8
these parameters (Table S1). Ci increased significantly at all three ages under s-UV-B (Table 2)
9
and was also significantly affected by individual factors and their interactions (Table S1). WUE
10
declined by 1.4% at 30 DAT and increased by 1.8% at 60 DAT under s-UV-B; however these
11
changes were not significant (Table 2). The only significant decline in WUE was observed at 90
12
DAT (15.7%; Table 2). As depicted by the results of two-way ANOVA, WUE was significantly
13
affected by age (p<0.001) and the interactive effects of age and treatment (p<0.05), while
14
treatment alone did not affect it significantly (Table S1). s-UV-B caused F0 to increase by 12.5%,
15
28.8%, and 23.9% and Fm to decline by ~4%, 7.2%, and 10.0% at 30, 60, and 90 DAT,
16
respectively (Table 2). Hence, Fv (=Fm-F0) also declined significantly at all ages. Fv/Fm (a
17
measurement of quantum yield) also reduced significantly under s-UV-B treatment by 5.0%,
18
10.5%, and 10.4% at 30, 60, and 90 DAT, respectively (Table 2). Age, treatment, and their
19
interactions significantly affected all these parameters (F0, Fm, Fv, and Fv/Fm; Table S1).
20
3.3. IAA Oxidase activity:
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IAA oxidase activity increased significantly in treated leaves at all ages by 22.8%,
22
127.6%, and 93.5% at 30, 60, and 90 DAT, respectively (p<0.001). In roots, IAA oxidase
23
activity decreased under treatment at 30 DAT by 0.5% (not significant) while it increased at 60
24
and 90 DAT by 13.5%, and 41.7%, respectively (p<0.001) (Tables 3, 4). IAA oxidase activity
25
was significantly affected in both organs of the test plant due to plant age, s-UV-B treatment, as
26
well as their interactions (Tables S2).
27
3.4. Plant metabolites:
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In both leaves and roots, protein content reduced significantly under s-UV-B at all ages,
2
the decline being greater in leaves compared to roots (Tables 3, 4). An increment in thiol content
3
was observed in both plant organs treated with s-UV-B at all ages; leaves showed maximum
4
increase at 30 DAT (40.8%; p<0.001) while roots at 90 DAT (40.5%; p<0.001) (Tables 3, 4).
5
Proline increased significantly in leaves under s-UV-B at all ages. Maximal increase was
6
observed at 60 DAT (173.3%, p<0.001). Similar trend was also found in roots with maximal
7
increase being recorded at 90 DAT (119.0%, p<0.001). The increase was higher in leaves
8
compared to roots at 30 and 60 DAT, while it was higher in roots at the final sampling age
9
(Tables 3, 4). As per the results of two-way ANOVA, plant age, UV-B treatment, and their
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interactions significantly affected protein, thiol, and proline contents (Table S2). Alkaloid concentrations increased significantly under s-UV-B treatment at all sampling
12
ages in leaves and roots of the test plant with maximum increase being recorded in leaves at 90
13
DAT (125.8%, p<0.001) and in roots at 60 DAT (153.9%, p<0.001) (Tables 3, 4). Anthocyanins
14
showed a trend similar to that of alkaloids. Higher increase was observed in roots compared to
15
the leaves (Tables 3, 4). Individual factors of age and treatment as well as their interactions
16
affected both alkaloid- and anthocyanin contents significantly in both plant organs (Table S2).
17
Total carotenoid content as well as individual carotenoids, lycopene and β-carotene, recorded an
18
increase in leaves and roots at all three sampling ages (Tables 3, 4). The increment in carotenoids
19
was higher in roots compared to leaves, the increase being maximum at 30 DAT (96.1%,
20
p<0.001). Lycopene showed maximum increase in leaves at 30 DAT (26.8%, p<0.01) and in
21
roots at 90 DAT (17.3%, p<0.001). The increase in β-carotene was not significant at 30 DAT
22
(4.6%) and maximum at 60 DAT (24.2%, p<0.01). In roots, however, maximal increment was
23
recorded at 90 DAT (24.4%, p<0.001). Carotenoids, lycopene, and β-carotene were all
24
significantly affected by age and treatment in both leaves and roots while their interactive effects
25
did not affect β-carotene significantly in leaves and carotenoids and β-carotene in roots (Table
26
S2).
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Flavonoid profiles of both leaves and roots recorded an increase under UV-B treatment
28
with increase being more prominent in the former (Tables 3, 4). Two-way ANOVA results
29
showed significant variations in flavonoid contents at all wavelengths at different plant ages,
30
treatments, and their interactions (Table S2). In s-UV-B treated leaves, lignin content increased 14
ACCEPTED MANUSCRIPT
by 83.2%, 42.4%, and 49.1% at 30, 60, and 90 DAT respectively (p<0.001) (Table 3). Lignin
2
also increased in roots at all three ages, however, % increase declined at subsequent ages (Table
3
4). Phenolics increased in both leaves and roots under s-UV-B at all ages compared to their
4
respective controls; maximum increase was observed in leaves at 30 DAT (88.1%, p<0.001) and
5
in roots at 60 DAT (69.1%, p<0.001). Phytosterols showed an enhancement in their
6
concentrations in s-UV-B treated leaves (38.4%, 19.8%, and 49.9% at 30, 60, and 90 DAT
7
respectively, p<0.001) (Table 3). In roots, however, the increase was significant only at 60 DAT
8
(12.4%, p<0.05) and 90 DAT (49.5%, p<0.001) (Table 4). Both saponin and tannin contents
9
recorded an increase in their concentrations in both the organs of the treated plant. Saponins
10
increased by 12.9%, 9.8% (p<0.05), and 78.1% (p<0.001) in leaves and by 10.8%, 14.9%
11
(p<0.01) and 24.8% (p<0.001) in roots at 30, 60, and 90 DAT, respectively (Tables 3, 4).
12
Tannins in leaves increased at all three sampling ages (Table 3). In roots, tannins increased at
13
first two sampling ages, while at 90 DAT, they reduced significantly by 17.3% (p<0.01) (Table
14
4). Two-way ANOVA showed significant variations in lignin-, phenol-, phytosterols-, saponins-,
15
and tannin contents with respect to age, treatment, and their interactions (Table S2).
16
3.5. Phenylpropanoid pathway enzymes:
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All five enzymes of the phenylpropanoid pathway showed a general trend of increment
18
under s-UV-B. PAL increased in both the plant organs at all three sampling ages, the increase
19
being maximum in leaves at 60 DAT (Table S2). CAD activity showed maximum increment in
20
leaves as well as roots at 90 DAT (317.8% and 559.4%, p<0.001, respectively) (Tables 3, 4). The
21
plant age, s-UV-B treatment, and their interactions significantly affected PAL as well as CAD
22
activity (Table S2). Three substrates were used to determine 4CL activity, p-coumaric acid,
23
ferulic acid, and caffeic acid. 4CL activity, with all substrates was found to be significantly
24
enhanced in s-UV-B treated leaves (Table 3). Maximum increments with p-coumaric acid and
25
ferulic acid were recorded at 90 DAT (8.9% and 53.9%, p<0.001, respectively) while with
26
caffeic acid it was recorded at 30 DAT (195.9%, p<0.001) (Table 3). In roots, 4CL activity with
27
p-coumaric acid as substrate increased at the first two sampling ages by 5.5% and 5.3% (at 30
28
and 60 DAT respectively, p<0.001). However, final sampling age exhibited a significant decline
29
of 8.1% (p<0.001) (Table 4). Using ferulic acid as the substrate, 4CL activity increased at 30 and
30
90 DAT by 14.0% and 19.6% (p<0.001) respectively while at 60 DAT it declined significantly
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by 19.2% (p<0.001). An increase of 129.1%, 79.9%, and 41.8% (p<0.001) was observed in 4CL
2
activity in roots of plants under s-UV-B radiation at 30, 60 and 90 DAT respectively when
3
caffeic acid was used as the substrate (Table 4). The age, treatment, and their interactive effects
4
affected all 4CL activities significantly for all the substrates of 4CL in both the plant parts except
5
for the treatment using p-coumaric acid as substrate in roots (Table S2). CHI and DFR activities
6
increased in both s-UV-B treated plant organs with the exception of CHI at 90 DAT which
7
recorded a decrease of 7.5% (p<0.05) in roots. Maximum increment in CHI activity was
8
observed in leaves at 60 DAT (178.5%, p<0.001) and in roots at 30 DAT (222.7%, p<0.001).
9
Increase in DFR activity was found to be maximum in leaves at 60 DAT (112.3%, p<0.001) and
10
in roots at 90 DAT (85.7%, p<0.001) (Tables 3, 4). In both leaves and roots, CHI activity varied
11
significantly with age, treatment and their interactions (Table S2). In leaves, DFR activity was
12
affected significantly only by s-UV-B treatment while in roots there was a significant variation in
13
DFR activity at different plant ages, treatment, and their interactions (Table S2).
14
3.6. Chlorophyll content:
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Chlorophyll a content declined at all ages though the decrease was not significant at 30
16
DAT (1.1%). Chlorophyll b on the other hand, increased by ~3% at 30 DAT (non-significant
17
increase) and declined at subsequent ages by 44.7% (p<0.001) and 24.3% (p<0.05) at 60 and 90
18
DAT respectively. Consequently, total chlorophyll also declined at all three ages, the decrease
19
being non-significant at 30 DAT (0.17) (Fig. 1). Results of two-way ANOVA show that while
20
chlorophyll a, -b, and total chlorophyll, were significantly affected by age and treatment, only
21
chlorophyll b was significantly affected by their interactions as well (Table S2).
22
3.7. ROS and LPO:
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Amongst ROS, s-UV-B treated leaves recorded an increase in H2O2 content compared to
24
the control ones at all sampling ages, the maximum increment being recorded at 60 DAT (82.9%,
25
p<0.001) (Fig. 2A). Roots showed a similar trend of H2O2 increment by 17.0%, 34.5%, and
26
40.1% at 30, 60, and 90 DAT respectively (Fig. 2B). ˙O2- production rate increased in s-UV-B
27
treated leaves compared to the control ones by 2.3% (p<0.05) at 30 DAT and 0.5% at 60 DAT
28
(non-significant). At 90 DAT, it decreased by 1.6% (p<0.05) (Fig. 2A). In roots, a decrease in
29
˙O2- production rate was observed at all three sampling ages (p<0.01) (Fig. 2B). H2O2 varied 16
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significantly in both plant organs with age, treatment, as well as their interactions while ˙O2-
2
production rate was affected significantly only by age in leaves and by age and treatment in roots
3
(Table S2). LPO increased in leaves under s-UV-B radiation as compared to the control at all
4
sampling ages, though there was a progressive decline in percentage increment (Fig. 2A). Roots
5
also depicted a similar trend of increment in LPO at all sampling ages, the increase being non-
6
significant at 30 DAT (18.8%) and maximal at 60 DAT (67.4%, p<0.01) (Fig. 2B). In both plant
7
organs, LPO was significantly affected by age, treatment, and their interactions, except in leaves
8
for their interactive effects (Table S2).
9
3.8. Enzymatic antioxidants:
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Amongst antioxidative enzymes, APX activity increased initially at 30 and 60 DAT while
11
at 90 DAT it decreased by 22.9% (p<0.001) (Fig. 3A). In roots, it increased at all three sampling
12
ages, maximal increase being recorded at 60 DAT (177.4%, p<0.001) (Fig. 3B). CAT activity
13
increased in s-UV-B treated leaves and roots at the first two sampling ages, while at the final
14
sampling age, it declined by 34.4% in leaves (p<0.001) and 47.4% in roots (p<0.001) (Fig. 3A,
15
B). Leaves as well as roots of treated plants showed an increment in GR and POX activity at all
16
three ages. Both GR and POX activities were found to be higher in roots compared to the leaves
17
in both control and treated organs. Maximum increment in GR activity was found in roots at 90
18
DAT (111.1%, p<0.001) and in POX activity in leaves at 90 DAT (29.0%, p<0.001) (Fig. 3A,
19
B). Both PPO and SOD increased at all sampling ages under UV-B in leaves, though the increase
20
was not significant in PPO at 30 DAT (13.5%). Roots also depicted a similar trend of increment
21
for both PPO and SOD activities (Fig. 3A, B). Maximum increment in roots’ PPO activity was
22
observed at 90 DAT (90.5%, p<0.001) and in SOD activity at 60 DAT (43.7%, p<0.001) (Fig.
23
3B). All the antioxidative enzymes were significantly affected by individual factors of age and
24
treatment as well as their interactive effects in both plant organs; CAT was not significantly
25
affected by treatment in leaves (Table S2).
26
3.9. Non-enzymatic antioxidants:
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Ascorbic acid was found to be reduced significantly in s-UV-B treated leaves and roots of
28
C. forskohlii at all sampling ages (Fig. 4A, B). Maximum decline was observed in leaves at 30
29
DAT (72.3%, p<0.001) and in roots at 90 DAT (59.6%, p<0.001) (Fig. 4A, B). α-tocopherol 17
ACCEPTED MANUSCRIPT
content increased by 167.5%, 37.0%, and 23.7% in leaves and by 92.8%, 52.0%, and 44.3% in
2
roots at 30, 60, and 90 DAT, respectively under s-UV-B (Fig. 4A, B). Both these non-enzymatic
3
antioxidants were significantly influenced by age, treatment, and their interactive effects as
4
shown by the results of two-way ANOVA (Table S2).
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4. Discussion
6
Biomass reductions, corroborating the results of the present study, were observed in other
7
plants grown under UV-B such as Cymbopogon citratus (Kumari and Agrawal, 2010) and
8
Triticum aestivum (Yang et al., 2013) suggesting reduced photosynthesis rates (Kumari and
9
Agrawal, 2010) and/or diversion of plant photosynthates towards the synthesis of enhanced
10
concentrations of antioxidative defence compounds and enzymes to counteract the effects of
11
stress (Zhang and Björn, 2009). Reduced plant height observed in A. calamus (Kumari et al.,
12
2009) and Phyllanthus amarus (Indrajith and Ravindran, 2009) might be due to shortening of
13
internodes (Zhao et al., 2003) and/or IAA photo-oxidation and formation of growth-inhibiting
14
photo-products by UV-B. The latter phenomenon has been observed in UV-sensitive ecotype of
15
Spirodela punctata (Jansen et al., 2001). Increase in IAA oxidase activity under s-UV-B treated
16
plant organs has been earlier reported in Oryza sativa (Huang et al., 1997) indicating a decline in
17
IAA content and can be directly correlated with reductions in shoot length, root length and hence
18
plant height (i.e. an alteration in plant architecture). Reduction in number of leaves and leaf area
19
observed in C. forskohlii in the present study might be due to inhibition of cell division and cell
20
expansion and has also been reported by Choudhary and Agrawal (2014a) in pea under s-UV-B.
21
This has been designated as an adaptive strategy to lower the radiation absorbance (Choudhary
22
and Agrawal, 2014a).
AC C
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5
23
Photosynthetic apparatus is one of the primary molecular targets of UV-B radiation which
24
generally leads to its impairment (Jansen et al., 1998). In the present study, reduction in Ps and
25
decrease in total chlorophyll content can be directly correlated, except at 30 DAT where minimal
26
reduction in Ps was observed compared to the subsequent sampling ages and no change was
27
observed in chlorophyll a, -b, and total chlorophyll contents. This might be an adaptation
28
strategy by the plant to maintain the stability of the photosynthetic mechanism (Gratani et al.,
29
1998) under initial stress conditions. This also means that at this age, photosynthetic apparatus of
30
the plant remains undamaged during UV-B stress. Under chronic UV-B exposure, however, 18
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chlorophyll reductions (leading to a consequent decline in Ps) may occur due to reduced
2
synthesis/ destruction of chlorophyll pigment complexes (Jordan et al., 1994) or via excess ROS
3
generation (Peiser and Yang, 1978). Reduction in photosynthetic rate might also be linked to
4
decline in stomatal conductance. Similar studies on C. citratus (Kumari and Agrawal, 2010) and
5
Vigna radiata (Choudhary and Agrawal, 2014b) verify our results. Carbon assimilation was
6
inhibited under s-UV-B resulting in an increase in internal CO2 concentration and WUE also
7
reduced probably because of decreased photosynthesis rates (Maxwell and Johnson, 2000;
8
Kumari and Agrawal, 2010). As in the present study, F0 was found to be increased in V. radiata
9
cultivars (Choudhary and Agrawal, 2014b) after s-UV-B treatment. This might be because of the
10
reduced exciton transfer in the antenna pigment molecules or an increase in antenna cross section
11
(Tevini et al., 1989). Increment in F0 indicates damage to PS II reaction centres, while reduction
12
in Fv and Fm indicate the damage to thylakoid membrane and inhibition of PS II activity (Bjerke
13
et al., 2005). Consequent decrease in Fv/Fm also indicates detrimental effects to PS II activity and
14
retardation of photochemical efficiency (Choudhary and Agrawal, 2014b).
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Similar to our results, a reduction in protein content was reported by Choudhary and Agrawal
16
(2014a, b) in pea and mung bean. Protein degradation under s-UV-B may be direct (via
17
destruction or modification of amino acid residues) or indirect (via oxidative damage due to
18
increased ROS production or due to modified DNA and RNA structures that interfere with
19
transcription and replication resulting in decreased protein synthesis; Sharma et al., 2012). The
20
phenomenon can be correlated with reduced plant growth under stress to optimize plant
21
performance. Increased thiol and proline contents have been previously reported in A. calamus
22
(Kumari et al., 2010) and C. citratus (Kumari and Agrawal, 2010). Both metabolites serve as
23
antioxidants, scavenging free radicals and counteracting peroxidation-induced damage (Saradhi
24
et al., 1995; Deneke, 2000).
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25
Increment in plant’s secondary metabolites’ concentrations under s-UV-B suggests a trade-
26
off between the plant’s primary- and secondary metabolism with more resources being allocated
27
to the biosynthetic pathways of the latter, in all probability to alleviate the effects of s-UV-B
28
induced stress. UV light induced the formation of terpenoid indole alkaloids and their precursors
29
in Cathranthus roseus (Ramani and Jayabaskaran, 2008). UV-B has been known to induce the
30
expression of anthocyanin-biosynthetic pathway genes; anthocyanins are regarded as UV screens 19
ACCEPTED MANUSCRIPT
and protect the photosynthetic apparatus from oxidative damage (Fuglevand et al., 1996). Similar
2
functions have been attributed to carotenoids and these are implicated in the protection of
3
photosynthetic apparatus in leaves; however, their increment in the present study was not
4
sufficient to prevent the decline of chlorophyll at the later sampling ages under s-UV-B. Their
5
concentrations were found to be induced under this stress in Withania somnifera (Takshak and
6
Agrawal, 2014b). Lycopene and β-carotene increased in both s-UV-B treated test plant organs.
7
Giuntini et al. (2005), however, found that lycopene content in tomato cultivar HP1 grown under
8
UV-B was lower than in those grown under its absence. β-carotene is predominant in PS I and its
9
increased content in the leaves of treated plants may protect PS I from oxidative damage (Kakani
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et al., 2003).
Flavonoids act as screening pigments protecting plants against various stresses including
12
UV-B (Agati et al., 2012). Our results are corroborated by other similar studies on medicinal
13
plants (Kumari and Agrawal, 2010; Takshak and Agrawal, 2014b). Lignin, which acts as a
14
mechanical support and defence mechanism in plants was found to be increased in C. forskohlii
15
leaves and roots in accordance with studies on W. somnifera (Takshak and Agrawal, 2014b).
16
Phenolics also protect photosensitive targets against oxidative stress. They are enhanced upon
17
UV-B exposure and are instrumental in inhibiting plant growth (Kumari and Agrawal, 2010).
18
The inverse relation between protein and phenol contents observed in the present study might be
19
explained on the basis of amino acid phenylalanine acting as a common precursor for these
20
metabolites; the competition between these metabolites for limiting precursor phenylalanine
21
results in a trade-off between the rates of their biosynthesis, reversing the relationship between
22
their concentrations and allocation (Jones and Hartley, 1999). Increased phytosterols in C.
23
forskohlii indicate higher membrane integrity (Berli et al., 2010) and can be correlated with
24
lower degree of LPO observed in the leaves of the test plant especially at 60 and 90 DAT. Gil et
25
al. (2012) have reported an increase in sterol in Vitis vinifera leaves at low UV-B doses.
26
Increased saponin concentrations, similar to our results, were reported by Afreen et al. (2005) in
27
Glycyrrhiza uralensis where glycyrrhizic acid content increased at optimum UV-B treatment.
28
Tannin content also increased in the test plant organs under s-UV-B; similar results were
29
obtained by Germ et al. (2010) in the leaves of Hypericum perforatum and Takshak and Agrawal
30
(2014b) in the leaves and roots of W. somnifera. The differential regulation of tannin-
31
biosynthetic pathway-specific genes under this stress might be responsible for its enhancement in
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1
leaves as opposed to a decline in roots at 90 DAT. Reduced tannin content under prolonged UV-
2
B exposure was observed by Kumari and Prasad (2014) in C. aromaticus. The responses of phenylpropanoid pathway genes have been studied widely under various
4
abiotic stresses including s-UV-B. Park et al. (2007) demonstrated a coordinated increase in the
5
activities of phenylpropanoid pathway enzymes (PAL, CHS, CHI, and DFR) with a concomitant
6
increase in the concentration of flavonoids under s-UV-B stress. Earlier studies (Jordan et al.,
7
1994; Mackerness et al., 1997) demonstrated an increase in CHS, PAL, and 4CL expression
8
followed by an increase in the concentration of protective pigments in pea and Arabidopsis.
9
Arabidopsis thaliana tt mutants were found to be more sensitive to UV-B radiation as these
10
lacked normal biosynthetic genes for CHS, CHI, and DFR and consequently were not able to
11
synthesize the requisite level of flavonoids (Li et al., 1993). Present study also revealed an
12
increase in the activities of PAL, CAD, 4CL, CHI, and DFR with parallel increment in the
13
concentrations of flavonoids, anthocyanins, tannins, and lignin contents in the test organs of C.
14
forskohlii. Enhanced 4CL under s-UV-B in C. forskohlii was also recorded for Isatis indigotica
15
(Di et al., 2012), and Oryza sativa (Os4CL2: flavonoid biosynthesis; Sun et al., 2013). Increased
16
4CL and CAD activities directly corresponded with increase in lignin content in the test plant
17
while their down-regulation in tobacco (Chabannes et al., 2001) and switchgrass (Fu et al., 2011)
18
decreased the lignin content. An increase in DFR activity (along with PAL1, CHS, and CHI) was
19
reported in Arabidopsis plants under s-UV-B as early as 1992 (Kubasek et al., 1992). Later, Ubi
20
et al. (2006) reported enhanced mRNA levels of CHS, F3H, DFR, ANS, and UFGT induced by
21
UV-B irradiation in apple skin. Our findings also revealed an increase in DFR activity in the
22
treated test plant. Anomalies observed in 4CL activity (4CL1 at 90 DAT and 4CL2 at 60 DAT)
23
might be might be due to the channelization of their substrates towards other enzymes of this
24
enormously complex pathway to regulate the production of other end products and optimize
25
plant performance.
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Amongst ROS, increment in H2O2 and reduction in ̇O2- radical production rate can be
27
correlated with increased SOD activity which directly scavenges ̇O2- radicals converting them to
28
H2O2. SOD sprayed Arabidopsis leaves showed reduced ̇O2- production under UV-B treatment
29
(Mackerness et al., 2001) indicating an inverse relationship between ̇O2- production rate and
30
SOD activity. Increased content of H2O2 under s-UV-B has been reported in other plants as well 21
ACCEPTED MANUSCRIPT
(Choudhary and Agrawal, 2014b). H2O2 and ̇O2- are also used up in transition-metal-catalyzed
2
Haber-Weiss- and Fenton reactions leading to the formation of other ROS, such as ˑ̇OH. ̇O2- can
3
also react with other ROS like NȮ to produce peroxynitrite (OONO-) (Gill and Tuteja, 2010).
4
Hence the extent of lipid peroxidation (measured in terms of MDA content) cannot be very
5
closely correlated with H2O2 and ̇O2- concentrations alone. For instance, a non-significant
6
increase in MDA content at 90 DAT in leaves and 30 DAT in roots might be due to reduced O2-
7
production rate (and consequently low ̇OH) as well as increased activities of antioxidative
8
enzymes. Increased levels of LPO due to excessive ROS generation under UV-B have been
9
reported by Hagh et al. (2012) in sunflower cultivars and by Takshak and Agrawal (2014a) in W.
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somnifera.
Increased activities of enzymatic antioxidants observed under s-UV-B in both leaves and
12
roots of C. forskohlii controlled the production of ROS preventing the plant from excessive
13
oxidative damage. APX (and other peroxidases as well) scavenges H2O2 and maintains the redox
14
status of cells under stress. Increase in APX and POX activities was observed by Hagh et al.
15
(2012) in sunflower cotyledons. However, declined APX activity at the final sampling age in
16
leaves might be due to APX degradation or repression of APX gene expression under prolonged
17
UV-B exposure (Casati et al., 2002). CAT directly dismutates H2O2 into H2O and O2 (Gill and
18
Tuteja, 2010). It was found to be increased under s-UV-B by Hagh et al. (2012). Being
19
susceptible to photoinactivation and degradation, its activity is reduced under prolonged and
20
intense light conditions as observed in the final sampling age in both the leaves and roots of the
21
test plant. Decreased CAT activity could also be due to the destruction of peroxisomes under UV
22
B stress (Indrajith and Ravindran, 2009). Decline in CAT activity with concomitant increase in
23
POX activity was previously reported in A. calamus (Kumari et al., 2010). GR activity
24
(responsible for maintaining cellular GSH pool) was reported to be increased under s-UV-B by
25
Cakirlar et al. (2011). PPO causes oxidation of phenolic compounds to quinones; however, in the
26
present study the increase in PPO activity was not sufficient to oxidize the phenolics which
27
increased under s-UV-B at all ages. Indrajith and Ravindran (2009) reported increased PPO
28
activity under UV-B stress.
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In the present study, ascorbic acid was found to be reduced at all sampling ages in both plant
30
parts primarily due to increased APX activity which utilizes ascorbic acid as a substrate (Gill and 22
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Tuteja, 2010). However, at the final sampling age in leaves APX activity also declines under s-
2
UV-B might be because of its increased utilization in regenerating alpha-tocopherol (Munné-
3
Bosch and Alegre, 2002). Decline in ascorbic acid content under UV-B stress has been
4
previously reported by Kumari et al. (2010) in A. calamus. α-tocopherol protects plants against
5
oxidative stress by acting as singlet oxygen quencher and stabilizing chloroplast membranes
6
(Ervin et al., 2004). Higher levels of this non-enzymatic antioxidant were found to improve the
7
turf quality of Kentucky bluegrass by preventing oxidative damage (Poa pratenis L.; Ervin et al.,
8
2004).
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5. Conclusions
The findings of the present study on the effects of s-UV-B on C. forskohlii reveal that the plant
11
growth and architecture (in terms of its biomass, plant height, number of leaves, and leaf area)
12
were adversely affected. On the other hand, the defence strategies of the plant (secondary
13
metabolites and enzymatic- and non-enzymatic antioxidants) were up-regulated under stress
14
protecting the plant against excessive UV-B damage. This indicates a shift in plant’s metabolite-
15
biosynthesis preferences and can be termed as an adaptive strategy by the plant to counteract
16
oxidative stress. Figure 5 gives a schematic representation of these outcomes. Also, under the
17
present experimental conditions, C. forskohlii can act as a better source of antioxidants and
18
medicinally important compounds. Enhanced activities of phenylpropanoid pathway enzymes
19
indicate the requirement of genetic level studies to promote the enhanced production of their
20
products for the betterment of human health. The results of the present study refute the proposed
21
hypothesis, as the test plant, though able to increase the contents of defensive enzymes and
22
compounds, was unable to completely recuperate from the consequences of oxidative stress as
23
evidenced by the reduction in plant height and biomass under s-UV-B.
24
Acknowledgements
25
The authors are thankful to the Head, Department of Botany and Coordinator, Centre of
26
Advanced Study in Botany, Banaras Hindu University, for providing laboratory facilities, and to
27
the University Grants Commission (UGC), New Delhi, for financial assistance.
28
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Table 1 Meteorological data during the experimental period Temperature
Relative humidity
Total rainfall
Total sunshine
(°C)
(%)
(mm)
(h)
Minimum
Maximum
Minimum
October/2012
33.3
20.1
86.0
68.9
November/2012
28.8
13.2
86.6
69.4
December/2012
23.4
10.0
85.7
65.4
January/2013
22.0
7.6
92.4
58.9
February/2013
25.9
12.6
89.4
58.2
M AN U TE D EP AC C 31
15.2
252.7
-
207.1
-
176.8
0.4
189.2
62.4
190.5
SC
Maximum
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Month/Year
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Table 2
TE D
6.61±0.151* 0.747±0.019** 226.29±0.937*** 4.72±0.109ns 242.00±1.882*** 910.73±1.631*** 668.73±2.411*** 0.734±0.002***
EP
7.13±0.121 0.851±0.020 175.80±1.377 4.79±0.078 215.13±2.238 948.60±1.379 733.47±2.554 0.773±0.002
SC
70 DAT 100 DAT Control s-UV B Control s-UV B 11.58±0.018 9.38±0.034*** 18.44±0.034 14.64±0.074*** ** 49.86±0.758 40.04±0.507 64.56±0.847 49.66±0.497*** 57.00±2.429 42.00±2.345** 72.40±2.542 52.80±4.352** ** 261.76±5.368 217.78±3.930 339.16±3.384 263.54±4.262***
M AN U
40 DAT Control s-UV B 3.34±0.030 2.87±0.019*** 33.86±0.144 31.14±0.189*** 24.60±1.030 15.40±1.077*** 146.10±4.054 99.76±4.884**
AC C
Parameters Growth Total biomass (g plant-1) Plant height (cm) Number of leaves Leaf area (cm2) Physiological Ps (µmol CO2 m-2 s-1) Gs (mol H2O m-2 s-1) Ci (µmol mol-1) WUE (µmol CO2 m-2 s-1/ mmol m-2 s-1) F0 (milli volt) Fm (milli volt) Fv (milli volt) Fv/Fm
RI PT
The effect of s-UV-B treatment on growth characteristics, and physiological parameters in Coleus forskohlii at three sampling ages. Means±SE, n=5 (Growth parameters); n=15 (Physiological parameters) Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation. F0: Initial fluorescence; Fm: Maximum fluorescence; Fv: Variable fluorescence; Fv/Fm: Photochemical efficiency; Ps: Photosynthetic rate; Gs: Stomatal conductance; Ci: Internal CO2; WUE: Water use efficiency
32
11.79±0.226 1.402±0.032 207.23±0.988 4.58±0.165 218.60±1.864 1020.53±1.028 801.93±1.761 0.786±0.002
9.72±0.206*** 0.969±0.018*** 277.60±1.230*** 4.66±0.194ns 281.53±0.668*** 947.27±0.848*** 665.73±1.030*** 0.703±0.001***
15.34±0.310 1.800±0.036 282.06±1.697 4.27±0.132 232.80±2.764 1074.07±0.628 841.27±3.048 0.783±0.003
10.58±0.224*** 1.381±0.023*** 341.73±1.219*** 3.60±0.121** 288.53±0.710*** 966.47±0.742*** 677.93±1.093*** 0.701±0.001***
ACCEPTED MANUSCRIPT
Table 3
M AN U
SC
70 DAT Control s-UV-B 0.006±0.000 0.014±0.001*** 7.077±0.076 4.608±0.086*** 6.236±0.021 8.071±0.178*** 0.004±0.000 0.012±0.000*** 1.125±0.010 1.670±0.019*** 0.480±0.004 0.522±0.013** 0.377±0.001 0.533±0.001*** 0.191±0.006 0.222±0.004* 0.136±0.004 0.169±0.007**
100 DAT Control s-UV-B 0.008±0.000 0.015±0.000*** 7.896±0.010 4.893±0.012*** 7.394±0.015 8.844±0.024*** 0.007±0.000 0.013±0.000*** 1.135±0.011 2.562±0.087*** 0.636±0.003 0.814±0.005*** 0.460±0.002 0.592±0.002*** 0.297±0.003 0.342±0.004*** 0.580±0.007 0.616±0.003**
0.853±0.004 0.857±0.001 0.823±0.001 0.841±0.000 0.906±0.002 0.145±0.001 5.040±0.195 0.863±0.015 0.631±0.018 2.206±0.168 8.921±0.028 0.410±0.002
1.776±0.014*** 1.871±0.008*** 1.794±0.007*** 1.788±0.010*** 1.835±0.005*** 0.266±0.006*** 9.483±0.348*** 1.194±0.014*** 0.712±0.019* 4.063±0.121*** 9.719±0.055*** 1.284±0.008***
0.914±0.002 1.156±0.026 1.142±0.002 1.137±0.002 1.159±0.001 0.273±0.009 6.409±0.213 1.141±0.023 0.816±0.010 3.377±0.203 10.307±0.016 0.534±0.008
2.630±0.008*** 2.657±0.006*** 2.250±0.048*** 2.622±0.009*** 2.634±0.009*** 0.389±0.003*** 10.971±0.250*** 3.342±0.046*** 0.896±0.011* 5.549±0.119*** 13.248±0.034*** 1.838±0.006***
1.167±0.020 1.142±0.003 1.171±0.002 2.126±0.006 2.318±0.013 0.815±0.009 8.109±0.254 3.089±0.058 1.194±0.012 6.834±0.106 12.822±0.555 0.604±0.003
2.960±0.018*** 3.169±0.023*** 2.721±0.028*** 3.514±0.068*** 3.386±0.061*** 1.215±0.004*** 12.520±0.283*** 4.630±0.015*** 2.126±0.015*** 7.920±0.087*** 15.543±0.170*** 2.523±0.004***
12.985±0.236 7.359±0.157 1.296±0.210
13.997±0.127*** 8.790±0.184*** 3.834±0.176***
13.612±0.340 7.723±0.123 2.313±0.203
14.783±0.189*** 9.428±0.249*** 4.859±0.258***
15.820±0.149 8.466±0.301 3.762±0.194
17.235±0.192*** 13.030±0.216*** 6.475±0.257***
AC C
EP
IAA Oxidase (mg IAA degraded min-1 mg protein-1) Protein (mg g-1 f.w.) Thiol (mg g-1 f.w.) Proline (mg g-1 f.w.) Alkaloids (mg g-1 f.w.) Anthocyanins (mg g-1 f.w.) Carotenoids (mg g-1 f.w.) Lycopene (µg g-1 f.w.) β-carotene (µg g-1 f.w.) Flavonoids (Absrbance) 280 nm 290 nm 300 nm 310 nm 320 nm Lignin (mg g-1 f.w.) Phenol (mg g-1 f.w.) Phytosterols (mg g-1 f.w.) Saponins (mg g-1 f.w.) Tannins (mg g-1 f.w.) PAL (µM trans-cinnamic acid formed min-1 mg protein-1) CAD (nmol cinnamyl alcohol oxidised min-1 mg protein-1) 4CL1 (nmol p-coumaroyl CoA ester formed min-1 mg protein-1) 4CL2 (nmol feruloyl CoA ester formed min-1 mg protein-1) 4CL3 (nmol caffeoyl CoA ester formed min-1 mg protein-1)
40 DAT Control s-UV-B 0.004±0.000 0.005±0.000*** 6.200± 0.063 3.793±0.063*** 5.582±0.019 7.860±0.020*** 0.002±0.000 0.005±0.000*** 0.917±0.092 1.207±0.105*** 0.202±0.003 0.241±0.002*** 0.192±0.001 0.206±0.001*** 0.126±0.005 0.160±0.005** 0.059±0.009 0.061±0.002ns
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Parameters
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The effects of s-UV-B treatment on IAA oxidase activity, primary and secondary plant metabolites, and phenylpropanoid pathway enzymes in leaves of C. forskohlii at three sampling ages. Means±SE, n=7. Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation.
33
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0.013±0.001 0.377±0.010
0.030±0.002*** 0.620±0.012***
0.023±0.001 0.429±0.007
0.064±0.002*** 0.912±0.318***
AC C
EP
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SC
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CHI (nmol flavanone produced min-1 mg protein-1) DFR(nmol dihydroquercetin reduced min-1 mg protein-1)
34
0.057±0.002 0.477±0.009
0.089±0.002*** 0.775±0.020***
ACCEPTED MANUSCRIPT
Table 4
M AN U
SC
70 DAT Control s-UV-B 0.008±0.000 0.009±0.000*** 9.382±0.519 6.605±0.083*** 4.801±0.016 6.338±0.020*** 0.005±0.000 0.007±0.000*** 1.216±0.020 3.087±0.066*** 0.362±0.002 0.608±0.004*** 0.447±0.006 0.694±0.010*** 0.274±0.004 0.307±0.004** 0.180±0.007 0.197±0.005*
100 DAT Control s-UV-B 0.010±0.000 0.014±0.000*** 11.814±0.011 9.513±0.016*** 5.306±0.018 7.456±0.019*** 0.012±0.000 0.026±0.000*** 2.005±0.018 3.809±0.017*** 0.462±0.003 0.907±0.005*** 0.696±0.004 0.914±0.002*** 0.362±0.003 0.425±0.005*** 0.192±0.006 0.239±0.005**
1.370±0.030 1.619±0.009 1.310±0.006 1.311±0.013 1.544±0.033 0.223±0.024 3.169±0.023 0.744±0.015 0.696±0.012 2.491±0.111 10.307±0.027 0.511±0.033
1.532±0.009*** 1.709±0.017*** 1.460±0.014*** 1.342±0.018** 1.627±0.012** 0.421±0.015*** 4.034±0.038*** 0.791±0.063ns 0.771±0.014** 5.063±0.156*** 11.366±0.038*** 2.071±0.043***
1.650±0.014 1.807±0.004 1.498±0.016 1.664±0.015 1.902±0.012 0.374±0.013 4.226±0.023 1.211±0.091 0.794±0.012 3.663±0.121 12.482±0.017 0.722±0.034
1.793±0.015*** 1.920±0.027** 1.652±0.016*** 1.712±0.023ns 2.340±0.026*** 0.515±0.014*** 7.146±0.034*** 1.361±0.024* 0.913±0.017** 7.063±0.072*** 13.783±0.031*** 3.629±0.047***
1.763±0.014 1.869±0.019 1.595±0.021 1.725±0.007 2.135±0.021 1.595±0.033 5.829±0.018 3.693±0.013 1.895±0.010 9.406±0.130 13.605±0.057 0.888±0.036
1.999±0.013*** 2.613±0.022*** 1.943±0.023*** 2.906±0.005*** 3.216±0.016*** 1.814±0.014*** 9.177±0.025*** 5.523±0.024*** 2.366±0.015*** 7.777±0.173** 15.902±0.044*** 5.857±0.033***
13.350±0.016 6.146±0.214 4.030±0.038
14.091±0.092*** 7.007±0.165*** 9.234±0.040***
14.929±0.026 9.755±0.224 5.703±0.044
15.722±0.033*** 7.885±0.074*** 10.258±0.040***
18.892±0.020 10.130±0.183 7.831±0.026
17.359±0.014*** 12.113±0.236*** 11.104±0.048***
AC C
EP
IAA Oxidase (mg IAA degraded min-1 mg protein-1) Protein (mg g-1 f.w.) Thiol (mg g-1 f.w.) Proline (mg g-1 f.w.) Alkaloids (mg g-1 f.w.) Anthocyanins (mg g-1 f.w.) Carotenoids (mg g-1 f.w.) Lycopene (µg g-1 f.w.) β-carotene (µg g-1 f.w.) Flavonoids (Absorbance) 280 nm 290 nm 300 nm 310 nm 320 nm Lignin (mg g-1 f.w.) Phenol (mg g-1 f.w.) Phytosterols (mg g-1 f.w.) Saponins (mg g-1 f.w.) Tannins (mg g-1 f.w.) PAL (µM trans-cinnamic acid formed min-1 mg protein-1) CAD (nmol cinnamyl alcohol oxidised min-1 mg protein-1) 4CL1 (nmol p-coumaroyl CoA ester formed min-1 mg protein-1) 4CL2 (nmol feruloyl CoA ester formed min-1 mg protein-1) 4CL3 (nmol caffeoyl CoA ester formed min-1 mg protein-1)
40 DAT Control s-UV-B 0.006±0.000 0.006±0.000ns 6.280±0.104 5.035±0.063*** 2.776±0.022 3.217±0.015*** 0.002±0.000 0.002±0.000* 1.175±0.012 2.079±0.017*** 0.289±0.004 0.387±0.002*** 0.242±0.003 0.475±0.009*** 0.174±0.004 0.204±0.004*** 0.174±0.008 0.194±0.007**
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Parameters
RI PT
The effects of s-UV-B treatment on IAA oxidase activity, primary and secondary plant metabolites, and phenylpropanoid pathway enzymes in leaves of C. forskohlii at three sampling ages. Means±SE, n=7. Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation.
35
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0.045±0.001 0.904±0.009
0.147±0.002*** 1.390±0.025***
0.124±0.001 1.074±0.007
0.169±0.002*** 1.766±0.069***
AC C
EP
TE D
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CHI (nmol flavanone produced min-1 mg protein-1) DFR(nmol dihydroquercetin reduced min-1 mg protein-1)
36
0.183±0.001 0.853±0.009
0.169±0.002* 1.585±0.015***
ACCEPTED MANUSCRIPT Fig.1. The effects of s-UV-B on chlorophyll a, chlorophyll b, and total chlorophyll content in leaves of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001). Fig.2. The effects of s-UV-B on H2O2 content, ̇O2- production rate, and lipid peroxidation in leaves ** p<0.01, *** p<0.001).
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(A) and roots (B) of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05,
Fig.3. The effects of s-UV-B on the activities of antioxidative enzymes (APX, CAT, GR, POX, PPO, SOD) in leaves (A) and roots (B) of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns:
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non-significant, * p<0.05, ** p<0.01, *** p<0.001).
Fig.4. The effects of s-UV-B on ascorbic acid and α-tocopherol in leaves (A) and roots (B) of C.
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forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
Fig.5. Schematic representation of the processes at transcriptional and translational levels leading to altered cellular metabolism and biochemistry which in turn leads to changes in plant morphology and physiology under s-UV-B stress. The % changes in plant metabolites and enzymes tested in both leaves and roots under s-UV-B are given here (at 60 DAT). Abbreviations: IAAO: Indole acetic acid
TE D
oxidase; PAL: Phenylalanine ammonia lyase; CAD: Cinnamyl alcohol dehydrogenase; 4CL: 4Coumarate CoA ligase; CHI: Chalcone isomerase; DFR: Dihydroflavanol reductase; APX: Ascorbate peroxidase; CAT: Catalase; GR: Glutathione reductase; POX: Peroxidase; PPO:
EP
Polyphenol oxidase; SOD: Superoxide dismutase; Ps: Photosynthetic rate; Gs: Stomatal
AC C
conductance; Fv/Fm: Photochemical efficiency [↑: increase; ↓: decrease].
37
Fig.1.
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Fig.2.
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Fig.3.
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Fig.4.
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Fig.5.
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Highlights: s-UV-B increases secondary metabolite concentrations in C. forskohlii
•
Phenylpropanoid pathway enzymes’ activities increase under s-UV-B in C. forskohlii
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s-UV-B increases enzymatic and non-enzymatic antioxidants in C. forskohlii
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s-UV-B alters C. forskohlii’s morphology and physiology
•
s-UV-B treated plant parts of C. forskohlii can serve as better source of antioxidants
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•
ACCEPTED MANUSCRIPT Table S1: 2 way ANOVA test to determine the effects of s-UV-B (T) and plant age (A) and their interactions on growth and physiological parameters of C. forskohlii. F ratios and levels of significance (ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
412.1*** 480.3*** 3910*** 20.3*** 155.7*** 3424*** 382.3*** 21.0***
195.1*** 233.8*** 3384*** 3.7ns 1027*** 6567*** 4918*** 1976***
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A×T 884.0*** 60.6*** 2.1ns 8.2**
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T 4437*** 406.6*** 49.2*** 241.2***
49.8*** 26.7*** 30.8*** 4.2* 53.0*** 500.6*** 288.4*** 90.2***
SC
A 57680*** 981.3*** 146.4*** 863.3***
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GROWTH PARAMETERS Total biomass Plant height No. of leaves Leaf area PHYSIOLOGICAL PARAMETERS Ps Gs Ci WUE F0 Fm Fv Fv/Fm
ACCEPTED MANUSCRIPT Table S2: 2 way ANOVA test to determine the effects of s-UV-B (T) and plant age (A) and their interactions on various metabolites and enzymes tested in the leaves and roots of C. forskohlii. F ratios and levels of significance (ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
1663*** 1486*** 391.3*** 1441*** 1673*** 333400*** 6805*** 725000*** 253900*** 481.9*** 8232*** 9.6*** 40840*** 888700*** 29000*** 419.1*** 0.943ns 31.4*** 41.8***
19120*** 15590*** 4162*** 3028*** 2981*** 59360*** 43750*** 497200*** 91610*** 223.0*** 4844*** 104.9*** 17450*** 217300*** 88950*** 423.3*** 10.3** 89.0*** 21.6***
675.9*** 583.0*** 86.6*** 51.3*** 59.7*** 11620*** 4.6* 80780*** 55500*** 7.9** 484.0*** 5.1* 166.8*** 33130*** 43.2*** 23.6*** 0.462ns 2.2ns 12.9***
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EP
52.1*** 1658*** 1068*** 79.0*** 29720*** 36.1*** 23420*** 1680*** 1026*** 9234*** 105400*** 34630***
85.5*** 5318*** 0.008ns 33.4*** 56.3*** 0.267ns 2495*** 4646*** 463.9*** 4638*** 62730*** 1637***
ROOTS
A 4291*** 124200*** 18510*** 14920*** 14230*** 4198*** 2440*** 1341*** 13.3***
T 1173*** 65920*** 8416*** 4876*** 60100*** 7189*** 1983*** 169.5*** 28.0***
A×T 594.0*** 3041*** 1110*** 2521*** 2508*** 1057*** 2.6ns 10.2*** 3.1ns
323.1*** 533.2*** 251.4*** 2301*** 1298*** 69930*** 9789*** 318200*** 642600*** 700.3*** 5400*** 11450*** 221900*** 285100*** 152.8*** 1062*** 41.6*** ---------
166.4*** 464.1*** 241.6*** 1212*** 934.4*** 3103*** 10890*** 250800*** 39520*** 182.8*** 2519*** 77830*** 0.027ns 43670*** 1075*** 944.9*** 630.5*** ---------
4.1* 213.6*** 21.9*** 997.2*** 279.7*** 48.9*** 1131*** 183700*** 12610*** 211.4*** 150.1*** 7730*** 19700*** 54190*** 18.3*** 537.8*** 9.0** ---------
----4595*** 2758*** 67.2*** 18570*** 152.2*** 77.5*** 19.3*** 1547*** 17420*** 576.7*** 10050***
----2477*** 68.9*** 41.2*** 95090*** 5.3* 31.6*** 386.7*** 5047*** 13460*** 10720*** 11860***
----303.7*** 0.364ns 9.3** 5832*** 378.3*** 12.9*** 0.003ns 224.9*** 72.7*** 170.0*** 2826***
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A×T 38.9*** 685.1*** 222.8*** 494.9*** 2883*** 80.0*** 1461*** 41.0*** 2.8ns
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T 216.7*** 132400*** 13400*** 9158*** 13810*** 279.2*** 7659*** 312.0*** 9.7**
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A 157.7*** 12690*** 2662*** 3580*** 5040*** 3216*** 29870*** 858.9*** 49230***
AC C
LEAVES IAA Oxidase Protein Thiol Proline Alkaloids Anthocyanins Carotenoids Lycopene β-carotene Flavonoids 280 nm 290 nm 300 nm 310 nm 320 nm Lignin Phenol Phytosterols Saponins Tannins PAL CAD 4CL1 4CL2 4CL3 CHI DFR Chlorophyll a Chlorophyll b Total chlorophyll H2O2 ̇O2LPO APX CAT GR POX PPO SOD Ascorbic acid α-tocopherol
2.6ns 416.7*** 0.679ns 2.8ns 2172*** 40.0*** 140.0*** 238.4*** 118.2*** 1020*** 5698*** 216.7***
S0981-9428(15)30120-0
DOI:
10.1016/j.plaphy.2015.09.018
Reference:
PLAPHY 4292
To appear in:
Plant Physiology and Biochemistry
Received Date: 29 July 2015 Revised Date:
30 September 2015
Accepted Date: 30 September 2015
Please cite this article as: S. Takshak, S.B. Agrawal, Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental ultraviolet-B radiation: augmentation of secondary metabolites and antioxidants, Plant Physiology et Biochemistry (2015), doi: 10.1016/j.plaphy.2015.09.018. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Defence strategies adopted by the medicinal plant Coleus forskohlii against supplemental
2
ultraviolet-B radiation: augmentation of secondary metabolites and antioxidants
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Swabha Takshak, S.B. Agrawal*
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Laboratory of Air Pollution and Global Climate Change, Department of Botany, Banaras Hindu
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University, Varanasi-221 005, India
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Abstract
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Supplementary ultraviolet-B (ambient+3.6kJ m-2 day-1) induced changes on morphological,
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physiological, and biochemical characteristics (specifically the defence strategies: UV-B
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protective compounds and antioxidants) of Coleus forskohlii were investigated under field
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conditions at 30, 60, and 90 days after transplantation. Levels of secondary metabolites increased
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under s-UV-B stress; flavonoids and phenolics (primary UV-B screening agents) were recorded
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to be higher in leaves which are directly exposed to s-UV-B. This was also verified by enhanced
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activities of phenylpropanoid pathway enzymes: phenylalanine ammonia lyase (PAL), cinnamyl
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alcohol dehydrogenase (CAD), 4-coumarate-CoA ligase (4CL), chalcone–flavanone isomerase
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(CHI), and dihydroflavonol reductase (DFR). Antioxidants, both enzymatic (ascorbate
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peroxidase, catalase, glutathione reductase, peroxidase, polyphenol oxidase, and superoxide
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dismutase) and non-enzymatic (ascorbic acid and α-tocopherol) also increased in the treated
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organs of the test plant, higher contents being recorded in roots except for ascorbic acid. On the
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contrary, protein and chlorophyll content (directly implicated in regulating plant growth and
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development) declined under s-UV-B. These alterations in plant biochemistry led the plant to
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compromise on its photosynthate allocation towards growth and biomass production as
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evidenced by a reduction in its height and biomass. The study concludes that s-UV-B is a potent
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stimulating factor in increasing the concentrations of defense compounds and antioxidants in C.
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forskohlii to optimize its performance under stress.
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Key words: antioxidants; Coleus forskohlii; oxidative stress; secondary metabolites; s-UV-B
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Abbreviations: APX- ascorbate peroxidase; BSA- bovine serum albumin; CAD- cinnamyl
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alcohol dehydrogenase; CHI- chalcone flavanone isomerase; CAT- catalase; Ci- internal CO2;
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DAT- days after transplantation; DCPIP- 2, 6-Dichlorophenol indophenol; DFR- dihydroflavanol
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reductase; DTNB- 5,5’-Dithiobis-(2-nitrobenzoic acid); EDTA- ethylene diamine tetraacetic
2
acid; LPO- lipid peroxidation; F0- initial fluorescence; Fm- maximum fluorescence; Fv- variable
3
fluorescence; GR- glutathione reductase; Gs- stomatal conductance; H2O2- hydrogen peroxide;
4
IAA- indole acetic acid; MDA- malondialdehyde; ̇O2- - superoxide radical; PAL- phenylalanine
5
ammonia lyase; POX- peroxidase; PPO- polyphenol oxidase; Ps- photosynthetic rate; PS I-
6
photosystem I; PS II- photosystem II; PVP- polyvinylpyrrolidone; ROS- reactive oxygen
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species; SOD- superoxide dismutase; s-UV-B- supplemental ultraviolet-B; TBA- thiobarbituric
8
acid; TCA- trichloroacetic acid; UV- ultraviolet; UV-BBE- biologically effective UV-B; WUE-
9
water use efficiency
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*
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E-mail address: [email protected] (S.B. Agrawal)
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Corresponding author. Tel.: +91 542 2368156; fax: +91 542 2368174.
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1. Introduction In recent years stratospheric ozone (O3) depletion, has been largely attributed to rapidly
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changing climatic conditions, altered land-use patterns, and newly discovered O3 depleting
4
substances (Anderson et al., 2012; Laube et al., 2014) and is directly responsible for increased
5
penetration of biologically active UV-B radiation (280-315 nm) reaching the Earth’s surface.
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The impacts of UV-B have been widely reported on crop plants, covering morphological and
7
physiological (Yang et al., 2005), biochemical (Choudhary and Agrawal, 2014a, b), antioxidative
8
defense system (Agrawal et al., 2009), and genetic level (Tripathi et al., 2011) parameters. Plants
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have adapted two basic strategies to optimize their growth and development under s-UV-B
10
stress: (i) biosynthesis of higher concentrations of secondary metabolites which function as
11
antioxidants, enzyme inhibitors, chemical signals, growth regulators, and UV-B screens
12
(Julkunen-Tiitto et al., 2005) and (ii) increased production of enzymatic- and non-enzymatic
13
antioxidants primarily to counteract the damaging effects of ROS. The former include
14
superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), catalase
15
(CAT), peroxidase (POX), and polyphenol oxidase (PPO) among others (Mittler et al., 2004),
16
while in the latter category, ascorbic acid and α-tocopherol qualify as two major and widely
17
studied candidates (Jaleel, 2009). Thiol and proline also serve as antioxidants under stress while
18
also regulating transcriptional and translational level changes (Saradhi et al., 1995; Deneke,
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2000). These defence strategies providing tolerance and resistance to the plant come at the cost
20
of reallocation of photosynthates towards new pathways. This may/ may not cause the plant to
21
compromise on its biomass/ yield, and photosynthetic efficiency and functioning, depending
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upon the extent and efficacy of these defensive and adaptive strategies.
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The effects of various abiotic stress factors on the morphological, physiological, and
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biochemical characteristics of medicinal plants are few and far-between. Coleus forskohlii
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(Lamiaceae) has been used as an ancient root drug in Ayurvedic medicine for treating asthma,
26
bronchitis, insomnia, epilepsy, angina, and psoriasis. Roots have been used to allay burning and
27
for the treatment of worms and other skin diseases. Leaves have been used as expectorant,
28
diuretic, for treating intestinal disorders, and as a condiment. Forskolin, a labdane diterpenoid,
29
isolated from the plant, is used in the treatment of asthma, glaucoma, hypertension, cancer, heart
30
diseases, diabetes, and obesity (Khatun, 2011; Singh et al., 2011). In view of the above 3
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1
mentioned medicinal importance of the test plant, it becomes important to determine the effects
2
of s-UV-B radiation on its overall performance. The ultimate objective of the plant is to optimize its performance under adverse stress
4
(biotic/abiotic) conditions and ensure its survival. This requires an alteration in its architectural
5
and physiological aspects, which in turn, can be attributed to the allocation and partitioning of
6
assimilated carbon and/or mobilization of storage carbon reserves (Geiger and Servaites, 1991).
7
Chronic exposure of plant to any stress will lead it to develop new physiological capabilities
8
which might manifest themselves in terms of altered plant morphology. If a plant responds
9
successfully to its stress environment, it will indicate altered plant metabolism and biochemistry
10
(or both) via altered gene expression without compromising on its biomass/yield (Geiger and
11
Servaites, 1991). Hence, the present work was based on the hypothesis that under s-UV-B, the
12
test plant, C. forskohlii, will combat the oxidative stress by increasing the concentrations of UV-
13
B absorbing compounds and antioxidative defence system and will consequently be able to
14
maintain its morphological traits and biomass. The hypothesis was tested by computing the
15
morphological, physiological and biochemical parameters in the leaves and roots of the test plant
16
(leaves, because they are directly subjected to s-UV-B radiation and roots, because they are
17
economically important plant organs, being the source of medicinally important essential oil).
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2. Materials and methods
2.1. Experimental site and experimental design
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The experimental site was located in Botanical Garden, Department of Botany, Banaras
21
Hindu University, Varanasi (25°80’N, 82°03’E, and 76 m above mean sea level), India. The
22
meteorological parameters throughout the experimental period (month-wise) are given in Table
23
1. The average maximum and minimum temperatures were 33.3°C and 7.6°C respectively,
24
relative humidity ranged from 58.2% to 92.4%, while the total rainfall amounted to 78.0 mm.
25
The soil texture was sandy loam (with sand, silt, and clay being 45, 28, and 27% respectively)
26
having a slightly alkaline pH (7.1). C. forskohlii plants (one month old) obtained from the
27
nursery were transplanted in experimental plots (1m×1m). Three rows in each plot were planted
28
with 4 plants in each row (a total of 12 plants per plot). The planting conditions were as follows:
29
distance between the ridges: 30 cm, distance between ridges and plot border: 15 cm, and distance
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between plants: 20 cm. The plots were prepared in triplicate for each type of treatment set out in
2
a randomized block design (RBD). Recommended dose of fertilizers were supplemented as 40,
3
60, and 50 kg ha-1 of NPK, respectively. Half the dose of N and full doses of P and K were
4
applied as a basal dose during field preparation, while the remaining dose of nitrogen was
5
provided as top dressing at 30 DAT (Paul et al., 2013). The plants were irrigated at regular
6
intervals as per the requirements.
7
2.2. s-UV-B treatment
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Once the plants were established in the field, they were subjected to s-UV-B radiation via
9
UV-B lamps (Q Panel UV-B 313 40W fluorescent lamps, Q panel Inc., Cleveland, OH, USA)
10
mounted on steel frames at a distance of 30 cm directly above the plant canopy; this distance was
11
kept constant throughout the experimental period. The lamps were covered with 0.13 mm
12
cellulose diacetate filter (Cadillac Plastic Co., Baltimore, MD, USA; transmission down to 280
13
nm) for s-UV-B treatment while for control 0.13 mm polyester filter (Cadillac Plastic Co.; which
14
absorbs radiation below 320 nm) was used. Though the filters allowed the transmission of UV-A
15
radiation (320-400 nm) as well, its amount was very low (<10%) compared to the UV-A
16
irradiance received from sunlight. Thus, the effects on plants from this radiation were presumed
17
to be negligible. Hence, control plants received ambient UV-B dose (5.8 kJ m-2 day-1) while
18
plants under UV-B lamps experienced ambient+3.6 kJ m-2 d-1 UV-B (biologically effective UV-
19
B (UV-BBE) as weighted by Caldwell (1971) generalised plant action spectrum normalised at 300
20
nm. As the filters are degraded by UV-B, they were replaced each week. s-UV-B treatment was
21
given to the plants during the solar noon period (11:00 to 14:00 h). UV-B irradiance and UV-BBE
22
were measured using Ultraviolet Intensity Meter (UVP Inc., San Gabriel, CA, USA) and
23
Spectropower-meter (Scientech, Boulder, USA), respectively.
24
2.3. Plant sampling and analysis
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Plants were randomly sampled from the triplicate plots for each treatment for the
26
analysis. They were dug out in the form of monoliths with roots intact, thoroughly washed with
27
running water to remove the debris, and plant parts separated. All metabolites were analysed in
28
the fresh tissue (both leaves and roots). Sampling was done at 3 ages (30, 60, and 90 DAT; days
29
after transplantation). Five plants of each treatment were up-rooted at each of the sampling ages 5
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for the measurement of growth traits. For the analysis of plant metabolites and enzyme activities,
2
three plants were randomly selected (i.e. three each from control as well as s-UV-B-treated ones)
3
and three samples per plant were further processed, making a total of nine replicates for each
4
treatment. After final calculations, two of the outliers were rejected, while seven observations
5
were retained on which further statistical tests were applied.
6
2.4. Measurement of growth traits:
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To determine total biomass, plants were collected, plant-parts separated, and oven-dried
8
at 80°C till the attainment of constant weight. Total biomass was calculated by adding the dry
9
weight of different plant parts. It was expressed in terms of g plant-1. Other growth
10
characteristics such as total plant height, number of leaves and leaf area, were also determined,
11
the latter being measured via portable leaf area meter (Model Li-3100, Li-COR, Inc., USA). Five
12
plants at each of the sampling ages were used for these measurements.
13
2.5. Determination of Physiological Parameters:
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Physiological parameters were determined on five randomly selected plants from control
15
plots as well as s-UV-B treated plots with three sets of observations being recorded for all
16
parameters from each plant, thus amounting to a total of fifteen replications per treatment. The
17
plants were tagged at the initial sampling age and subsequent measurements were made on the
18
same plants. Photosynthetic rate (Ps), stomatal conductance (gs), and internal CO2 (Ci) were
19
measured using a portable photosynthetic system (Model LI6400 XT, Version 6.2, Lincoln,
20
Nebraska, USA) at the three sampling ages (30, 60, and 90 DAT) while water use efficiency
21
(WUE) was calculated as the ratio of photosynthetic rate to transpiration. The measurements
22
were made between 9:00 and 10:30 h on the third fully expanded leaf from top of randomly
23
selected plants of each treatment. Chlorophyll fluorescence (initial and maximum, F0 and Fm
24
respectively) were measured using plant efficiency analyzer (Model PEA, MK2-9414, Hansatech
25
Instruments Ltd., UK) on the same leaves on which photosynthesis was measured during the
26
same hours. From F0 and Fm, variable fluorescence (Fv) and photochemical efficiency (Fv/Fm)
27
were calculated. The leaves were dark-adapted on the adaxial side for 30 minutes, then irradiated
28
with red light and fluorescence signal collected at excitation irradiance set at 3000 µmol m-2 s-1.
29
2.6. Determination of IAA oxidase activity:
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The extract preparation was carried out following the method of Dash et al. (2011) by
2
homogenizing 1 g plant tissue in 10 ml potassium phosphate buffer (50 mM, pH 6.0) and
3
subjecting it to centrifugation at 20 000 × g for 20 min. Supernatant was mixed with cold acetone
4
to a final concentration of 70% and re-centrifuged at 20 000 × g for 15 min. The precipitate was
5
re-suspended in the same buffer and again centrifuged at at 20 000 × g for 20 min. The resulting
6
supernatant was used for enzyme assay. IAA oxidase activity was determined as per Beffa et al.
7
(1990) using Salkowski reagent. IAA was calculated by measuring the change in absorbance ∆A
8
at 535 nm and expressed as mg IAA degraded min-1 mg protein-1.
9
2.7. Determination of plant metabolites:
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Protein content was determined by the method of Lowry et al. (1951) using BSA (bovine
11
serum albumin) for the preparation of the standard curve. 0.5 g of fresh tissue, homogenized in
12
5ml tris buffer (0.1 M, pH 6.8) was centrifuged at 5000 × g for 5 min; 5ml 10% TCA
13
(trichloroacetic acid) was added to the supernatant and allowed to react for 5 min. The mixture
14
was again centrifuged at 6000 × g for 10 min. The pellets were dried and dissolved in 5ml 0.1 N
15
NaOH and solution was centrifuged at 6000 × g for 10 min. To 1 ml of the supernatant 5 ml of
16
alkaline solution [prepared by mixing 50ml of alkaline sodium carbonate (2% (Na2CO3) in 0.1 N
17
NaOH] and 1 ml of 0.5% copper sulphate in 1% potassium tartarate solution] was added and
18
kept for 10 min at room temperature. To this, 0.5 ml of Folin’s reagent (1 N) was added and kept
19
for additional 30 min. Absorbance of the blue-colored complex was recorded at 650 nm on a
20
double beam spectrophotometer (Model-2203, Systronics, India).
21
Protein content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of protein read from standard curve (µg ml-1), W is weight
23
of leaf sample (g), V is volume of extract (ml) and v is volume of supernatant taken for analysis
24
(ml).
25
Thiol was estimated as per Fahey et al. (1978). 0.1 g tissue was homogenized in 80% v/v
26
ethanol, boiled in 10 ml of ethanol (80%) at 80 °C for 15 min, cooled and centrifuged at 10 000
27
× g for 10 min. To 1 ml of supernatant, 5 ml Ellman’s reagent (DTNB) (60 µM 5, 5’-Dithiobis-
28
(2-nitrobenzoic acid) in phosphate buffer (0.1 M, pH 7.5)) was added and the reaction mixture
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was allowed to stand for 5 min. The absorbance of the yellow colour developed was measured at
2
412 nm.
3
Thiol content (mg g-1 FW) = (0.22×V×OD) / (W×v×1000) Where, W is weight of leaf sample (g), V is total volume of sample (ml), v is volume of
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the supernatant taken for analysis (ml) and 0.22 is correction factor.
6
Proline content was determined following the method of Bates et al. (1973). 0.5 g tissue was
7
homogenized in 10 ml sulfosalicylic acid (3% w/v) and centrifuged at 10 000 × g for 10 min. 2
8
ml supernatant was incubated at 100 °C for 60 min with 2 ml ninhydrin reagent (1.2 g ninhydrin
9
dissolved in 30 ml glacial acetic acid and 20 ml orthophosphoric acid). 2 ml of glacial acetic acid
10
was added to the resulting solution. The reaction was terminated by placing the solutions in ice
11
bath, extracted with 4 ml of toluene, and mixed vigorously for 10 min. The absorbance of the
12
chromophore-containing toluene was recorded at 520 nm. Standard curve was prepared with
13
known concentrations of proline using above methodology.
14
Proline content (mg g-1 FW) = (C×V) / (115.5×W)
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Where, C is concentration of proline read from standard curve (µg), V is volume of
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toluene (ml), W is weight of leaf sample (mg) and 115.5 is molecular weight of toluene. Secondary metabolites except phenolics (alkaloids, anthocyanins, carotenoids, lycopene,
18
β-carotene, flavonoids, lignin, phytosterols, saponins, and tannins) were assessed according to
19
the methods described in Takshak and Agrawal (2014b). Total phenol was determined as per
20
Bray and Thorpe (1954) using Folin’s reagent and catechol as standard compound. 0.1 g fresh
21
tissue was homogenized with 10 ml 70% acetone and centrifuged at 6000 × g for 10 min. To 1
22
ml of supernatant, 1 ml Folin’s reagent (1 N) and 2 ml 2% Na2CO3 (w/v) was added and final
23
volume was made up to 10 ml with distilled water. Mixture was heated on boiling water bath till
24
the appearance of blue colour. Solution was allowed to cool and absorbance of blue colour
25
recorded at 650 nm on a double beam spectrophotometer (Model-2203, Systronics, India).
26
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Phenol content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of phenol read from standard curve (µg ml-1), W is weight
2
of leaf sample (g), V is volume of extract (ml) and v is volume of supernatant taken for analysis
3
(ml).
4
2.8. Determination of phenylpropanoid pathway enzymes’ activities:
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0.4 g leaf tissue was homogenised in 4 ml sodium borate buffer (50 mM; pH 8.7)
6
containing 5 mM β-mercaptoethanol, 1 mM EDTA and 2% PVP (w/v). The mixture was
7
centrifuged at 20 000 × g for 15 min (twice); the resulting supernatant was used for the
8
determination of PAL activity. The extraction buffer for CAD, 4CL, and CHI comprised of 0.2
9
M Tris–HCl (pH 7.5), 8 mM MgCl2, 2% PVP, 5 mM DTT, 0.1% Triton X-100, and 1 mM
10
PMSF. 1 g of plant tissue was homogenised in 10 ml extraction buffer and subjected to
11
centrifugation at 18 000 × g for 20 min. The supernatant collected was re-centrifuged at 15 000 ×
12
g for 15 min. Resulting supernatant was used for enzyme assays. All the enzyme extractions
13
were carried out at 4° C. For DFR assay, 1 g sample was homogenized in phosphate buffer saline
14
(PBS; 137 mM NaCl, 8.1 mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4; pH 7.4) and
15
centrifuged at 15 000 × g for 15 min. The supernatant was re-centrifuged for 10 min at 10 000 ×
16
g, decanted and used as substrate for enzyme analysis. The enzyme activities of PAL, CAD,
17
4CL, CHI, and DFR were measured as per the protocols already described by Takshak and
18
Agrawal (2014b).
19
2.9. Determination of chlorophyll content:
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Leaf tissue (0.1 g) immersed in 10 ml 80% acetone was kept in a stoppered conical flask
21
overnight at 4 °C. It was then homogenized, centrifuged at 5 000 × g for 15 min, and final
22
volume made up to 25 ml with 80% acetone. Absorbance of the solution was recorded at 645 nm
23
and 663 nm. Chlorophyll content was calculated using the formulae by Maclachlan and Zalik
24
(1963).
25
Chlorophyll a (mg g-1 FW) = [(12.3 OD663-0.86 OD645) × V] / [1000 × W × d]
26
Chlorophyll b (mg g-1 FW) = [(19.6 OD645-3.6 OD663) × V] / [1000 × W × d]
27
Total chlorophyll (mg g-1 FW) = Chlorophyll a + Chlorophyll b
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2.10. Determination of ROS and LPO: H2O2 content was determined using the protocol of Alexieva et al. (2001). The reaction
3
mixture contained 0.5 ml leaf extract supernatant (0.1g tissue extracted in 0.1% TCA at 4°C),
4
potassium phosphate buffer (10 mM), and potassium iodide (KI, 1 M). The reaction mixture was
5
kept in dark for 1 hour. The absorbance was recorded at 390 nm. The amount of hydrogen
6
peroxide was calculated using standard curve prepared with known concentrations of H2O2.
7
Determination of •O2− production rate (as per Elstner and Hupel, 1976) involved the extraction of
8
0.5 g tissue in 65 mM phosphate buffer at 4°C. 0.5 ml supernatant was incubated in dark at 25°C
9
for 20 min after adding 65 mM phosphate buffer and 10 mM hydroxylamine hydrochloride. 8.5
10
mM sulphanilamide and 3.0 mM α-naphthylamine were added to the solution (still in dark), and
11
again left at 25°C for 20 min. The absorbance was recorded at 530 nm. The superoxide radical
12
production rate was calculated from the standard graph prepared using potassium nitrite (KNO2).
13
Lipid peroxidation was measured in terms of malondialdehyde (MDA) content (Heath and
14
Packer, 1968). Fresh tissue (0.5 g) was homogenized in 5% TCA (trichloroacetic acid) and
15
centrifuged at 10 000 × g for 10 min. To 1 ml of supernatant, 4 ml 0.5% TBA (thiobarbituric
16
acid) prepared in 20% TCA was added. The mixture was heated on boiling water bath for 30
17
min, and cooled immediately on ice bath. Again the mixture was centrifuged for 10 minutes at 10
18
000 × g. The absorbance of the yellow colour developed was recorded at 532 nm and 600 nm on
19
double beam spectrophotometer (Model 2203, Systronics, India). The MDA content was
20
calculated using the following formula:
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MDA (nmol mg-1 FW) = (O.D.600-O.D.532)* 106/155000
22
Where, 155000 is the extinction coefficient of MDA.
23 24
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2.11. Determination of Enzymatic Antioxidants: 400 mg fresh tissue was homogenized in 10 ml of sodium phosphate buffer (0.1 M, pH
25
7.0) containing 0.1 % (m/v) Triton X-100 and 0.2 g of polyvinylpyrrolidone (PVP) under ice-
26
cold conditions. The homogenate was centrifuged at 10 000 g for 20 min and supernatant was re-
27
centrifuged at 13 500 × g and 4 °C for another 15 minutes. The supernatant was collected, stored
28
at 4°C and used for the analysis of all enzymatic antioxidants (APX, CAT, GR, POX, PPO, and
29
SOD). APX was assayed as per the method of Nakano and Asada (1981) after supplementing the 10
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extraction buffer with 1 mM ascorbate and using the coefficient of absorbance of 2.8 mM-1 cm-1.
2
The decrease in absorbance was recorded at 290 nm and enzyme activity expressed in terms of
3
mM min-1 mg protein-1. CAT activity was determined by the method of Abei (1984). The
4
enzyme activity was determined by measuring the rate of decrease of H2O2 at 240 nm for 1 min,
5
calculated using the extinction coefficient of 0.036 mM-1 cm-1 and was expressed as µmol H2O2
6
oxidized min-1 mg protein-1. GR activity (as per Anderson, 1996) was determined using
7
coefficient of absorbance of 6.22 mM-1 cm-1 and expressed as mM min-1 mg protein-1. Peroxidase
8
activity (POX) was determined using the method described by Britton and Mehley (1955).
9
Extinction coefficient of 2.47 mM-1 cm-1 was used to calculate the enzyme activity and the latter
10
was expressed as µM purpurogallin formed min-1 mg protein-1. PPO activity was determined as
11
per Kumar and Khan (1982) and expressed in Units mg protein-1 where one unit represented 0.1
12
change in absorbance per minute. Protocol of Fridovich (1974) was followed for determining
13
SOD activity with minor modifications. The absorbance was recorded at 560 nm and the activity
14
was expressed as Units mg protein-1. One unit of enzyme activity was defined as the amount of
15
enzyme required for 50% inhibition of the reduction of NBT.
16
2.12. Determination of Non-enzymatic Antioxidants:
17
Ascorbic acid was estimated using 2, 6-dichlorophenol indophenol (DCPIP) reduction method of
18
Keller and Schwager (1977). 20 ml of ice cold extracting solution (50 mg oxalic acid and 75 mg
19
EDTA dissolved in 100 ml distilled water) was used to homogenize 0.5 g fresh plant tissue. The
20
homogenate was centrifuged at 6000 × g for 15 min; 1 ml supernatant was mixed with 5 ml
21
DCPIP (20 µg ml-1) with constant shaking. Absorbance of the pink colored solution was
22
recorded at 520 nm (Es) using UV- VIS spectrophotometer (Model 119, Systronics, India). The
23
pink colour was then bleached by adding one drop of 1% ascorbic acid and again the absorbance
24
was recorded at the same wavelength (Et). 1 ml extracting solution mixed with 5 ml DCPIP
25
solution served as blank and its absorbance was also recorded at the same wavelength.
26
Calibration curve was prepared using known concentrations of ascorbic acid. Ascorbic acid
27
content was calculated as follows:
28
Ascorbic acid content (mg g-1 FW) = (C×V) / W×1000×v
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Where, C [= Eo-(Es-Et)] is the concentration of the ascorbic acid read from the standard
2
curve (µg), W is weight of leaf sample (g), V is volume of extract (ml) and v is volume of
3
supernatant taken for analysis (ml).
4
α-tocopherol content was measured as per the method of Jaleel (2009). 0.5 g fresh tissue
5
homogenized in 10 ml petroleum ether and ethanol in the ratio 2:1.6 was was centrifuged at 10
6
000 × g for 20 min. To 1 ml supernatant, 0.2 ml 2% dipyridil in ethyl alcohol (w/v) was mixed
7
thoroughly, and kept in dark for 10 min. 4 ml distilled water was added to the resulting solution
8
and mixed well. The solution was kept still at room temperature for 10 min; the absorbance of
9
the solution was recorded at 520 nm. Known concentrations of α-tocopherol were used for the
10
preparation of standard curve using the above methodology. The solution was allowed to stand
11
for another 10 min at room temperature. The absorbance of the resulting solution was recorded at
12
520 nm. Standard graph was prepared using known concentrations of α-tocopherol.
13
α-tocopherol content (mg g-1 FW) = (C×V) / (W×1000×v)
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Where C is the concentration of α-tocopherol read from standard curve (µg ml-1), W is
15
weight of leaf sample (g), V is volume of water (ml) and v is volume of supernatant taken for
16
analysis (ml).
17
2.13. Statistical analysis:
18
The means of various parameters between control and treated plants were compared via
19
Student’s t-test. The individual as well as interactive effects of plant age and s-UV-B treatment
20
were computed via two-way ANOVA. All statistical analyses were performed using the SPSS
21
software v.16.
23
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3. Results
3.1. Growth characteristics:
24
Total plant biomass was found to be significantly reduced at all sampling ages (Table 2),
25
maximum reduction being observed at 90 DAT (20.6%). Plant height also reduced significantly
26
at all ages by 8.0, 19.7, and 23.1% at 30, 60, and 90 DAT respectively (Table 2) under s-UV-B.
27
The treatment also caused a significant decline in number of leaves and leaf area (Table 2). Plant 12
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biomass, plant height, and leaf area were significantly affected by age, treatment, and their
2
interactions, while leaf number was significantly affected only by the individual factors as per
3
the results of two-way ANOVA (Table S1).
4
3.2. Physiological parameters:
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Both Ps and Gs were negatively affected under s-UV-B at all sampling ages (Table 2).
6
Maximum decline in Ps was recorded at 90 DAT (31.0%) and in Gs at 60 DAT (30.9%). Two-
7
way ANOVA results showed significant effects of age, treatment and their interactions on both
8
these parameters (Table S1). Ci increased significantly at all three ages under s-UV-B (Table 2)
9
and was also significantly affected by individual factors and their interactions (Table S1). WUE
10
declined by 1.4% at 30 DAT and increased by 1.8% at 60 DAT under s-UV-B; however these
11
changes were not significant (Table 2). The only significant decline in WUE was observed at 90
12
DAT (15.7%; Table 2). As depicted by the results of two-way ANOVA, WUE was significantly
13
affected by age (p<0.001) and the interactive effects of age and treatment (p<0.05), while
14
treatment alone did not affect it significantly (Table S1). s-UV-B caused F0 to increase by 12.5%,
15
28.8%, and 23.9% and Fm to decline by ~4%, 7.2%, and 10.0% at 30, 60, and 90 DAT,
16
respectively (Table 2). Hence, Fv (=Fm-F0) also declined significantly at all ages. Fv/Fm (a
17
measurement of quantum yield) also reduced significantly under s-UV-B treatment by 5.0%,
18
10.5%, and 10.4% at 30, 60, and 90 DAT, respectively (Table 2). Age, treatment, and their
19
interactions significantly affected all these parameters (F0, Fm, Fv, and Fv/Fm; Table S1).
20
3.3. IAA Oxidase activity:
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IAA oxidase activity increased significantly in treated leaves at all ages by 22.8%,
22
127.6%, and 93.5% at 30, 60, and 90 DAT, respectively (p<0.001). In roots, IAA oxidase
23
activity decreased under treatment at 30 DAT by 0.5% (not significant) while it increased at 60
24
and 90 DAT by 13.5%, and 41.7%, respectively (p<0.001) (Tables 3, 4). IAA oxidase activity
25
was significantly affected in both organs of the test plant due to plant age, s-UV-B treatment, as
26
well as their interactions (Tables S2).
27
3.4. Plant metabolites:
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In both leaves and roots, protein content reduced significantly under s-UV-B at all ages,
2
the decline being greater in leaves compared to roots (Tables 3, 4). An increment in thiol content
3
was observed in both plant organs treated with s-UV-B at all ages; leaves showed maximum
4
increase at 30 DAT (40.8%; p<0.001) while roots at 90 DAT (40.5%; p<0.001) (Tables 3, 4).
5
Proline increased significantly in leaves under s-UV-B at all ages. Maximal increase was
6
observed at 60 DAT (173.3%, p<0.001). Similar trend was also found in roots with maximal
7
increase being recorded at 90 DAT (119.0%, p<0.001). The increase was higher in leaves
8
compared to roots at 30 and 60 DAT, while it was higher in roots at the final sampling age
9
(Tables 3, 4). As per the results of two-way ANOVA, plant age, UV-B treatment, and their
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interactions significantly affected protein, thiol, and proline contents (Table S2). Alkaloid concentrations increased significantly under s-UV-B treatment at all sampling
12
ages in leaves and roots of the test plant with maximum increase being recorded in leaves at 90
13
DAT (125.8%, p<0.001) and in roots at 60 DAT (153.9%, p<0.001) (Tables 3, 4). Anthocyanins
14
showed a trend similar to that of alkaloids. Higher increase was observed in roots compared to
15
the leaves (Tables 3, 4). Individual factors of age and treatment as well as their interactions
16
affected both alkaloid- and anthocyanin contents significantly in both plant organs (Table S2).
17
Total carotenoid content as well as individual carotenoids, lycopene and β-carotene, recorded an
18
increase in leaves and roots at all three sampling ages (Tables 3, 4). The increment in carotenoids
19
was higher in roots compared to leaves, the increase being maximum at 30 DAT (96.1%,
20
p<0.001). Lycopene showed maximum increase in leaves at 30 DAT (26.8%, p<0.01) and in
21
roots at 90 DAT (17.3%, p<0.001). The increase in β-carotene was not significant at 30 DAT
22
(4.6%) and maximum at 60 DAT (24.2%, p<0.01). In roots, however, maximal increment was
23
recorded at 90 DAT (24.4%, p<0.001). Carotenoids, lycopene, and β-carotene were all
24
significantly affected by age and treatment in both leaves and roots while their interactive effects
25
did not affect β-carotene significantly in leaves and carotenoids and β-carotene in roots (Table
26
S2).
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Flavonoid profiles of both leaves and roots recorded an increase under UV-B treatment
28
with increase being more prominent in the former (Tables 3, 4). Two-way ANOVA results
29
showed significant variations in flavonoid contents at all wavelengths at different plant ages,
30
treatments, and their interactions (Table S2). In s-UV-B treated leaves, lignin content increased 14
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by 83.2%, 42.4%, and 49.1% at 30, 60, and 90 DAT respectively (p<0.001) (Table 3). Lignin
2
also increased in roots at all three ages, however, % increase declined at subsequent ages (Table
3
4). Phenolics increased in both leaves and roots under s-UV-B at all ages compared to their
4
respective controls; maximum increase was observed in leaves at 30 DAT (88.1%, p<0.001) and
5
in roots at 60 DAT (69.1%, p<0.001). Phytosterols showed an enhancement in their
6
concentrations in s-UV-B treated leaves (38.4%, 19.8%, and 49.9% at 30, 60, and 90 DAT
7
respectively, p<0.001) (Table 3). In roots, however, the increase was significant only at 60 DAT
8
(12.4%, p<0.05) and 90 DAT (49.5%, p<0.001) (Table 4). Both saponin and tannin contents
9
recorded an increase in their concentrations in both the organs of the treated plant. Saponins
10
increased by 12.9%, 9.8% (p<0.05), and 78.1% (p<0.001) in leaves and by 10.8%, 14.9%
11
(p<0.01) and 24.8% (p<0.001) in roots at 30, 60, and 90 DAT, respectively (Tables 3, 4).
12
Tannins in leaves increased at all three sampling ages (Table 3). In roots, tannins increased at
13
first two sampling ages, while at 90 DAT, they reduced significantly by 17.3% (p<0.01) (Table
14
4). Two-way ANOVA showed significant variations in lignin-, phenol-, phytosterols-, saponins-,
15
and tannin contents with respect to age, treatment, and their interactions (Table S2).
16
3.5. Phenylpropanoid pathway enzymes:
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All five enzymes of the phenylpropanoid pathway showed a general trend of increment
18
under s-UV-B. PAL increased in both the plant organs at all three sampling ages, the increase
19
being maximum in leaves at 60 DAT (Table S2). CAD activity showed maximum increment in
20
leaves as well as roots at 90 DAT (317.8% and 559.4%, p<0.001, respectively) (Tables 3, 4). The
21
plant age, s-UV-B treatment, and their interactions significantly affected PAL as well as CAD
22
activity (Table S2). Three substrates were used to determine 4CL activity, p-coumaric acid,
23
ferulic acid, and caffeic acid. 4CL activity, with all substrates was found to be significantly
24
enhanced in s-UV-B treated leaves (Table 3). Maximum increments with p-coumaric acid and
25
ferulic acid were recorded at 90 DAT (8.9% and 53.9%, p<0.001, respectively) while with
26
caffeic acid it was recorded at 30 DAT (195.9%, p<0.001) (Table 3). In roots, 4CL activity with
27
p-coumaric acid as substrate increased at the first two sampling ages by 5.5% and 5.3% (at 30
28
and 60 DAT respectively, p<0.001). However, final sampling age exhibited a significant decline
29
of 8.1% (p<0.001) (Table 4). Using ferulic acid as the substrate, 4CL activity increased at 30 and
30
90 DAT by 14.0% and 19.6% (p<0.001) respectively while at 60 DAT it declined significantly
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by 19.2% (p<0.001). An increase of 129.1%, 79.9%, and 41.8% (p<0.001) was observed in 4CL
2
activity in roots of plants under s-UV-B radiation at 30, 60 and 90 DAT respectively when
3
caffeic acid was used as the substrate (Table 4). The age, treatment, and their interactive effects
4
affected all 4CL activities significantly for all the substrates of 4CL in both the plant parts except
5
for the treatment using p-coumaric acid as substrate in roots (Table S2). CHI and DFR activities
6
increased in both s-UV-B treated plant organs with the exception of CHI at 90 DAT which
7
recorded a decrease of 7.5% (p<0.05) in roots. Maximum increment in CHI activity was
8
observed in leaves at 60 DAT (178.5%, p<0.001) and in roots at 30 DAT (222.7%, p<0.001).
9
Increase in DFR activity was found to be maximum in leaves at 60 DAT (112.3%, p<0.001) and
10
in roots at 90 DAT (85.7%, p<0.001) (Tables 3, 4). In both leaves and roots, CHI activity varied
11
significantly with age, treatment and their interactions (Table S2). In leaves, DFR activity was
12
affected significantly only by s-UV-B treatment while in roots there was a significant variation in
13
DFR activity at different plant ages, treatment, and their interactions (Table S2).
14
3.6. Chlorophyll content:
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Chlorophyll a content declined at all ages though the decrease was not significant at 30
16
DAT (1.1%). Chlorophyll b on the other hand, increased by ~3% at 30 DAT (non-significant
17
increase) and declined at subsequent ages by 44.7% (p<0.001) and 24.3% (p<0.05) at 60 and 90
18
DAT respectively. Consequently, total chlorophyll also declined at all three ages, the decrease
19
being non-significant at 30 DAT (0.17) (Fig. 1). Results of two-way ANOVA show that while
20
chlorophyll a, -b, and total chlorophyll, were significantly affected by age and treatment, only
21
chlorophyll b was significantly affected by their interactions as well (Table S2).
22
3.7. ROS and LPO:
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Amongst ROS, s-UV-B treated leaves recorded an increase in H2O2 content compared to
24
the control ones at all sampling ages, the maximum increment being recorded at 60 DAT (82.9%,
25
p<0.001) (Fig. 2A). Roots showed a similar trend of H2O2 increment by 17.0%, 34.5%, and
26
40.1% at 30, 60, and 90 DAT respectively (Fig. 2B). ˙O2- production rate increased in s-UV-B
27
treated leaves compared to the control ones by 2.3% (p<0.05) at 30 DAT and 0.5% at 60 DAT
28
(non-significant). At 90 DAT, it decreased by 1.6% (p<0.05) (Fig. 2A). In roots, a decrease in
29
˙O2- production rate was observed at all three sampling ages (p<0.01) (Fig. 2B). H2O2 varied 16
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significantly in both plant organs with age, treatment, as well as their interactions while ˙O2-
2
production rate was affected significantly only by age in leaves and by age and treatment in roots
3
(Table S2). LPO increased in leaves under s-UV-B radiation as compared to the control at all
4
sampling ages, though there was a progressive decline in percentage increment (Fig. 2A). Roots
5
also depicted a similar trend of increment in LPO at all sampling ages, the increase being non-
6
significant at 30 DAT (18.8%) and maximal at 60 DAT (67.4%, p<0.01) (Fig. 2B). In both plant
7
organs, LPO was significantly affected by age, treatment, and their interactions, except in leaves
8
for their interactive effects (Table S2).
9
3.8. Enzymatic antioxidants:
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Amongst antioxidative enzymes, APX activity increased initially at 30 and 60 DAT while
11
at 90 DAT it decreased by 22.9% (p<0.001) (Fig. 3A). In roots, it increased at all three sampling
12
ages, maximal increase being recorded at 60 DAT (177.4%, p<0.001) (Fig. 3B). CAT activity
13
increased in s-UV-B treated leaves and roots at the first two sampling ages, while at the final
14
sampling age, it declined by 34.4% in leaves (p<0.001) and 47.4% in roots (p<0.001) (Fig. 3A,
15
B). Leaves as well as roots of treated plants showed an increment in GR and POX activity at all
16
three ages. Both GR and POX activities were found to be higher in roots compared to the leaves
17
in both control and treated organs. Maximum increment in GR activity was found in roots at 90
18
DAT (111.1%, p<0.001) and in POX activity in leaves at 90 DAT (29.0%, p<0.001) (Fig. 3A,
19
B). Both PPO and SOD increased at all sampling ages under UV-B in leaves, though the increase
20
was not significant in PPO at 30 DAT (13.5%). Roots also depicted a similar trend of increment
21
for both PPO and SOD activities (Fig. 3A, B). Maximum increment in roots’ PPO activity was
22
observed at 90 DAT (90.5%, p<0.001) and in SOD activity at 60 DAT (43.7%, p<0.001) (Fig.
23
3B). All the antioxidative enzymes were significantly affected by individual factors of age and
24
treatment as well as their interactive effects in both plant organs; CAT was not significantly
25
affected by treatment in leaves (Table S2).
26
3.9. Non-enzymatic antioxidants:
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Ascorbic acid was found to be reduced significantly in s-UV-B treated leaves and roots of
28
C. forskohlii at all sampling ages (Fig. 4A, B). Maximum decline was observed in leaves at 30
29
DAT (72.3%, p<0.001) and in roots at 90 DAT (59.6%, p<0.001) (Fig. 4A, B). α-tocopherol 17
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content increased by 167.5%, 37.0%, and 23.7% in leaves and by 92.8%, 52.0%, and 44.3% in
2
roots at 30, 60, and 90 DAT, respectively under s-UV-B (Fig. 4A, B). Both these non-enzymatic
3
antioxidants were significantly influenced by age, treatment, and their interactive effects as
4
shown by the results of two-way ANOVA (Table S2).
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4. Discussion
6
Biomass reductions, corroborating the results of the present study, were observed in other
7
plants grown under UV-B such as Cymbopogon citratus (Kumari and Agrawal, 2010) and
8
Triticum aestivum (Yang et al., 2013) suggesting reduced photosynthesis rates (Kumari and
9
Agrawal, 2010) and/or diversion of plant photosynthates towards the synthesis of enhanced
10
concentrations of antioxidative defence compounds and enzymes to counteract the effects of
11
stress (Zhang and Björn, 2009). Reduced plant height observed in A. calamus (Kumari et al.,
12
2009) and Phyllanthus amarus (Indrajith and Ravindran, 2009) might be due to shortening of
13
internodes (Zhao et al., 2003) and/or IAA photo-oxidation and formation of growth-inhibiting
14
photo-products by UV-B. The latter phenomenon has been observed in UV-sensitive ecotype of
15
Spirodela punctata (Jansen et al., 2001). Increase in IAA oxidase activity under s-UV-B treated
16
plant organs has been earlier reported in Oryza sativa (Huang et al., 1997) indicating a decline in
17
IAA content and can be directly correlated with reductions in shoot length, root length and hence
18
plant height (i.e. an alteration in plant architecture). Reduction in number of leaves and leaf area
19
observed in C. forskohlii in the present study might be due to inhibition of cell division and cell
20
expansion and has also been reported by Choudhary and Agrawal (2014a) in pea under s-UV-B.
21
This has been designated as an adaptive strategy to lower the radiation absorbance (Choudhary
22
and Agrawal, 2014a).
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23
Photosynthetic apparatus is one of the primary molecular targets of UV-B radiation which
24
generally leads to its impairment (Jansen et al., 1998). In the present study, reduction in Ps and
25
decrease in total chlorophyll content can be directly correlated, except at 30 DAT where minimal
26
reduction in Ps was observed compared to the subsequent sampling ages and no change was
27
observed in chlorophyll a, -b, and total chlorophyll contents. This might be an adaptation
28
strategy by the plant to maintain the stability of the photosynthetic mechanism (Gratani et al.,
29
1998) under initial stress conditions. This also means that at this age, photosynthetic apparatus of
30
the plant remains undamaged during UV-B stress. Under chronic UV-B exposure, however, 18
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chlorophyll reductions (leading to a consequent decline in Ps) may occur due to reduced
2
synthesis/ destruction of chlorophyll pigment complexes (Jordan et al., 1994) or via excess ROS
3
generation (Peiser and Yang, 1978). Reduction in photosynthetic rate might also be linked to
4
decline in stomatal conductance. Similar studies on C. citratus (Kumari and Agrawal, 2010) and
5
Vigna radiata (Choudhary and Agrawal, 2014b) verify our results. Carbon assimilation was
6
inhibited under s-UV-B resulting in an increase in internal CO2 concentration and WUE also
7
reduced probably because of decreased photosynthesis rates (Maxwell and Johnson, 2000;
8
Kumari and Agrawal, 2010). As in the present study, F0 was found to be increased in V. radiata
9
cultivars (Choudhary and Agrawal, 2014b) after s-UV-B treatment. This might be because of the
10
reduced exciton transfer in the antenna pigment molecules or an increase in antenna cross section
11
(Tevini et al., 1989). Increment in F0 indicates damage to PS II reaction centres, while reduction
12
in Fv and Fm indicate the damage to thylakoid membrane and inhibition of PS II activity (Bjerke
13
et al., 2005). Consequent decrease in Fv/Fm also indicates detrimental effects to PS II activity and
14
retardation of photochemical efficiency (Choudhary and Agrawal, 2014b).
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Similar to our results, a reduction in protein content was reported by Choudhary and Agrawal
16
(2014a, b) in pea and mung bean. Protein degradation under s-UV-B may be direct (via
17
destruction or modification of amino acid residues) or indirect (via oxidative damage due to
18
increased ROS production or due to modified DNA and RNA structures that interfere with
19
transcription and replication resulting in decreased protein synthesis; Sharma et al., 2012). The
20
phenomenon can be correlated with reduced plant growth under stress to optimize plant
21
performance. Increased thiol and proline contents have been previously reported in A. calamus
22
(Kumari et al., 2010) and C. citratus (Kumari and Agrawal, 2010). Both metabolites serve as
23
antioxidants, scavenging free radicals and counteracting peroxidation-induced damage (Saradhi
24
et al., 1995; Deneke, 2000).
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Increment in plant’s secondary metabolites’ concentrations under s-UV-B suggests a trade-
26
off between the plant’s primary- and secondary metabolism with more resources being allocated
27
to the biosynthetic pathways of the latter, in all probability to alleviate the effects of s-UV-B
28
induced stress. UV light induced the formation of terpenoid indole alkaloids and their precursors
29
in Cathranthus roseus (Ramani and Jayabaskaran, 2008). UV-B has been known to induce the
30
expression of anthocyanin-biosynthetic pathway genes; anthocyanins are regarded as UV screens 19
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and protect the photosynthetic apparatus from oxidative damage (Fuglevand et al., 1996). Similar
2
functions have been attributed to carotenoids and these are implicated in the protection of
3
photosynthetic apparatus in leaves; however, their increment in the present study was not
4
sufficient to prevent the decline of chlorophyll at the later sampling ages under s-UV-B. Their
5
concentrations were found to be induced under this stress in Withania somnifera (Takshak and
6
Agrawal, 2014b). Lycopene and β-carotene increased in both s-UV-B treated test plant organs.
7
Giuntini et al. (2005), however, found that lycopene content in tomato cultivar HP1 grown under
8
UV-B was lower than in those grown under its absence. β-carotene is predominant in PS I and its
9
increased content in the leaves of treated plants may protect PS I from oxidative damage (Kakani
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et al., 2003).
Flavonoids act as screening pigments protecting plants against various stresses including
12
UV-B (Agati et al., 2012). Our results are corroborated by other similar studies on medicinal
13
plants (Kumari and Agrawal, 2010; Takshak and Agrawal, 2014b). Lignin, which acts as a
14
mechanical support and defence mechanism in plants was found to be increased in C. forskohlii
15
leaves and roots in accordance with studies on W. somnifera (Takshak and Agrawal, 2014b).
16
Phenolics also protect photosensitive targets against oxidative stress. They are enhanced upon
17
UV-B exposure and are instrumental in inhibiting plant growth (Kumari and Agrawal, 2010).
18
The inverse relation between protein and phenol contents observed in the present study might be
19
explained on the basis of amino acid phenylalanine acting as a common precursor for these
20
metabolites; the competition between these metabolites for limiting precursor phenylalanine
21
results in a trade-off between the rates of their biosynthesis, reversing the relationship between
22
their concentrations and allocation (Jones and Hartley, 1999). Increased phytosterols in C.
23
forskohlii indicate higher membrane integrity (Berli et al., 2010) and can be correlated with
24
lower degree of LPO observed in the leaves of the test plant especially at 60 and 90 DAT. Gil et
25
al. (2012) have reported an increase in sterol in Vitis vinifera leaves at low UV-B doses.
26
Increased saponin concentrations, similar to our results, were reported by Afreen et al. (2005) in
27
Glycyrrhiza uralensis where glycyrrhizic acid content increased at optimum UV-B treatment.
28
Tannin content also increased in the test plant organs under s-UV-B; similar results were
29
obtained by Germ et al. (2010) in the leaves of Hypericum perforatum and Takshak and Agrawal
30
(2014b) in the leaves and roots of W. somnifera. The differential regulation of tannin-
31
biosynthetic pathway-specific genes under this stress might be responsible for its enhancement in
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1
leaves as opposed to a decline in roots at 90 DAT. Reduced tannin content under prolonged UV-
2
B exposure was observed by Kumari and Prasad (2014) in C. aromaticus. The responses of phenylpropanoid pathway genes have been studied widely under various
4
abiotic stresses including s-UV-B. Park et al. (2007) demonstrated a coordinated increase in the
5
activities of phenylpropanoid pathway enzymes (PAL, CHS, CHI, and DFR) with a concomitant
6
increase in the concentration of flavonoids under s-UV-B stress. Earlier studies (Jordan et al.,
7
1994; Mackerness et al., 1997) demonstrated an increase in CHS, PAL, and 4CL expression
8
followed by an increase in the concentration of protective pigments in pea and Arabidopsis.
9
Arabidopsis thaliana tt mutants were found to be more sensitive to UV-B radiation as these
10
lacked normal biosynthetic genes for CHS, CHI, and DFR and consequently were not able to
11
synthesize the requisite level of flavonoids (Li et al., 1993). Present study also revealed an
12
increase in the activities of PAL, CAD, 4CL, CHI, and DFR with parallel increment in the
13
concentrations of flavonoids, anthocyanins, tannins, and lignin contents in the test organs of C.
14
forskohlii. Enhanced 4CL under s-UV-B in C. forskohlii was also recorded for Isatis indigotica
15
(Di et al., 2012), and Oryza sativa (Os4CL2: flavonoid biosynthesis; Sun et al., 2013). Increased
16
4CL and CAD activities directly corresponded with increase in lignin content in the test plant
17
while their down-regulation in tobacco (Chabannes et al., 2001) and switchgrass (Fu et al., 2011)
18
decreased the lignin content. An increase in DFR activity (along with PAL1, CHS, and CHI) was
19
reported in Arabidopsis plants under s-UV-B as early as 1992 (Kubasek et al., 1992). Later, Ubi
20
et al. (2006) reported enhanced mRNA levels of CHS, F3H, DFR, ANS, and UFGT induced by
21
UV-B irradiation in apple skin. Our findings also revealed an increase in DFR activity in the
22
treated test plant. Anomalies observed in 4CL activity (4CL1 at 90 DAT and 4CL2 at 60 DAT)
23
might be might be due to the channelization of their substrates towards other enzymes of this
24
enormously complex pathway to regulate the production of other end products and optimize
25
plant performance.
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26
Amongst ROS, increment in H2O2 and reduction in ̇O2- radical production rate can be
27
correlated with increased SOD activity which directly scavenges ̇O2- radicals converting them to
28
H2O2. SOD sprayed Arabidopsis leaves showed reduced ̇O2- production under UV-B treatment
29
(Mackerness et al., 2001) indicating an inverse relationship between ̇O2- production rate and
30
SOD activity. Increased content of H2O2 under s-UV-B has been reported in other plants as well 21
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(Choudhary and Agrawal, 2014b). H2O2 and ̇O2- are also used up in transition-metal-catalyzed
2
Haber-Weiss- and Fenton reactions leading to the formation of other ROS, such as ˑ̇OH. ̇O2- can
3
also react with other ROS like NȮ to produce peroxynitrite (OONO-) (Gill and Tuteja, 2010).
4
Hence the extent of lipid peroxidation (measured in terms of MDA content) cannot be very
5
closely correlated with H2O2 and ̇O2- concentrations alone. For instance, a non-significant
6
increase in MDA content at 90 DAT in leaves and 30 DAT in roots might be due to reduced O2-
7
production rate (and consequently low ̇OH) as well as increased activities of antioxidative
8
enzymes. Increased levels of LPO due to excessive ROS generation under UV-B have been
9
reported by Hagh et al. (2012) in sunflower cultivars and by Takshak and Agrawal (2014a) in W.
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somnifera.
Increased activities of enzymatic antioxidants observed under s-UV-B in both leaves and
12
roots of C. forskohlii controlled the production of ROS preventing the plant from excessive
13
oxidative damage. APX (and other peroxidases as well) scavenges H2O2 and maintains the redox
14
status of cells under stress. Increase in APX and POX activities was observed by Hagh et al.
15
(2012) in sunflower cotyledons. However, declined APX activity at the final sampling age in
16
leaves might be due to APX degradation or repression of APX gene expression under prolonged
17
UV-B exposure (Casati et al., 2002). CAT directly dismutates H2O2 into H2O and O2 (Gill and
18
Tuteja, 2010). It was found to be increased under s-UV-B by Hagh et al. (2012). Being
19
susceptible to photoinactivation and degradation, its activity is reduced under prolonged and
20
intense light conditions as observed in the final sampling age in both the leaves and roots of the
21
test plant. Decreased CAT activity could also be due to the destruction of peroxisomes under UV
22
B stress (Indrajith and Ravindran, 2009). Decline in CAT activity with concomitant increase in
23
POX activity was previously reported in A. calamus (Kumari et al., 2010). GR activity
24
(responsible for maintaining cellular GSH pool) was reported to be increased under s-UV-B by
25
Cakirlar et al. (2011). PPO causes oxidation of phenolic compounds to quinones; however, in the
26
present study the increase in PPO activity was not sufficient to oxidize the phenolics which
27
increased under s-UV-B at all ages. Indrajith and Ravindran (2009) reported increased PPO
28
activity under UV-B stress.
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In the present study, ascorbic acid was found to be reduced at all sampling ages in both plant
30
parts primarily due to increased APX activity which utilizes ascorbic acid as a substrate (Gill and 22
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Tuteja, 2010). However, at the final sampling age in leaves APX activity also declines under s-
2
UV-B might be because of its increased utilization in regenerating alpha-tocopherol (Munné-
3
Bosch and Alegre, 2002). Decline in ascorbic acid content under UV-B stress has been
4
previously reported by Kumari et al. (2010) in A. calamus. α-tocopherol protects plants against
5
oxidative stress by acting as singlet oxygen quencher and stabilizing chloroplast membranes
6
(Ervin et al., 2004). Higher levels of this non-enzymatic antioxidant were found to improve the
7
turf quality of Kentucky bluegrass by preventing oxidative damage (Poa pratenis L.; Ervin et al.,
8
2004).
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5. Conclusions
The findings of the present study on the effects of s-UV-B on C. forskohlii reveal that the plant
11
growth and architecture (in terms of its biomass, plant height, number of leaves, and leaf area)
12
were adversely affected. On the other hand, the defence strategies of the plant (secondary
13
metabolites and enzymatic- and non-enzymatic antioxidants) were up-regulated under stress
14
protecting the plant against excessive UV-B damage. This indicates a shift in plant’s metabolite-
15
biosynthesis preferences and can be termed as an adaptive strategy by the plant to counteract
16
oxidative stress. Figure 5 gives a schematic representation of these outcomes. Also, under the
17
present experimental conditions, C. forskohlii can act as a better source of antioxidants and
18
medicinally important compounds. Enhanced activities of phenylpropanoid pathway enzymes
19
indicate the requirement of genetic level studies to promote the enhanced production of their
20
products for the betterment of human health. The results of the present study refute the proposed
21
hypothesis, as the test plant, though able to increase the contents of defensive enzymes and
22
compounds, was unable to completely recuperate from the consequences of oxidative stress as
23
evidenced by the reduction in plant height and biomass under s-UV-B.
24
Acknowledgements
25
The authors are thankful to the Head, Department of Botany and Coordinator, Centre of
26
Advanced Study in Botany, Banaras Hindu University, for providing laboratory facilities, and to
27
the University Grants Commission (UGC), New Delhi, for financial assistance.
28
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Table 1 Meteorological data during the experimental period Temperature
Relative humidity
Total rainfall
Total sunshine
(°C)
(%)
(mm)
(h)
Minimum
Maximum
Minimum
October/2012
33.3
20.1
86.0
68.9
November/2012
28.8
13.2
86.6
69.4
December/2012
23.4
10.0
85.7
65.4
January/2013
22.0
7.6
92.4
58.9
February/2013
25.9
12.6
89.4
58.2
M AN U TE D EP AC C 31
15.2
252.7
-
207.1
-
176.8
0.4
189.2
62.4
190.5
SC
Maximum
RI PT
Month/Year
ACCEPTED MANUSCRIPT
Table 2
TE D
6.61±0.151* 0.747±0.019** 226.29±0.937*** 4.72±0.109ns 242.00±1.882*** 910.73±1.631*** 668.73±2.411*** 0.734±0.002***
EP
7.13±0.121 0.851±0.020 175.80±1.377 4.79±0.078 215.13±2.238 948.60±1.379 733.47±2.554 0.773±0.002
SC
70 DAT 100 DAT Control s-UV B Control s-UV B 11.58±0.018 9.38±0.034*** 18.44±0.034 14.64±0.074*** ** 49.86±0.758 40.04±0.507 64.56±0.847 49.66±0.497*** 57.00±2.429 42.00±2.345** 72.40±2.542 52.80±4.352** ** 261.76±5.368 217.78±3.930 339.16±3.384 263.54±4.262***
M AN U
40 DAT Control s-UV B 3.34±0.030 2.87±0.019*** 33.86±0.144 31.14±0.189*** 24.60±1.030 15.40±1.077*** 146.10±4.054 99.76±4.884**
AC C
Parameters Growth Total biomass (g plant-1) Plant height (cm) Number of leaves Leaf area (cm2) Physiological Ps (µmol CO2 m-2 s-1) Gs (mol H2O m-2 s-1) Ci (µmol mol-1) WUE (µmol CO2 m-2 s-1/ mmol m-2 s-1) F0 (milli volt) Fm (milli volt) Fv (milli volt) Fv/Fm
RI PT
The effect of s-UV-B treatment on growth characteristics, and physiological parameters in Coleus forskohlii at three sampling ages. Means±SE, n=5 (Growth parameters); n=15 (Physiological parameters) Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation. F0: Initial fluorescence; Fm: Maximum fluorescence; Fv: Variable fluorescence; Fv/Fm: Photochemical efficiency; Ps: Photosynthetic rate; Gs: Stomatal conductance; Ci: Internal CO2; WUE: Water use efficiency
32
11.79±0.226 1.402±0.032 207.23±0.988 4.58±0.165 218.60±1.864 1020.53±1.028 801.93±1.761 0.786±0.002
9.72±0.206*** 0.969±0.018*** 277.60±1.230*** 4.66±0.194ns 281.53±0.668*** 947.27±0.848*** 665.73±1.030*** 0.703±0.001***
15.34±0.310 1.800±0.036 282.06±1.697 4.27±0.132 232.80±2.764 1074.07±0.628 841.27±3.048 0.783±0.003
10.58±0.224*** 1.381±0.023*** 341.73±1.219*** 3.60±0.121** 288.53±0.710*** 966.47±0.742*** 677.93±1.093*** 0.701±0.001***
ACCEPTED MANUSCRIPT
Table 3
M AN U
SC
70 DAT Control s-UV-B 0.006±0.000 0.014±0.001*** 7.077±0.076 4.608±0.086*** 6.236±0.021 8.071±0.178*** 0.004±0.000 0.012±0.000*** 1.125±0.010 1.670±0.019*** 0.480±0.004 0.522±0.013** 0.377±0.001 0.533±0.001*** 0.191±0.006 0.222±0.004* 0.136±0.004 0.169±0.007**
100 DAT Control s-UV-B 0.008±0.000 0.015±0.000*** 7.896±0.010 4.893±0.012*** 7.394±0.015 8.844±0.024*** 0.007±0.000 0.013±0.000*** 1.135±0.011 2.562±0.087*** 0.636±0.003 0.814±0.005*** 0.460±0.002 0.592±0.002*** 0.297±0.003 0.342±0.004*** 0.580±0.007 0.616±0.003**
0.853±0.004 0.857±0.001 0.823±0.001 0.841±0.000 0.906±0.002 0.145±0.001 5.040±0.195 0.863±0.015 0.631±0.018 2.206±0.168 8.921±0.028 0.410±0.002
1.776±0.014*** 1.871±0.008*** 1.794±0.007*** 1.788±0.010*** 1.835±0.005*** 0.266±0.006*** 9.483±0.348*** 1.194±0.014*** 0.712±0.019* 4.063±0.121*** 9.719±0.055*** 1.284±0.008***
0.914±0.002 1.156±0.026 1.142±0.002 1.137±0.002 1.159±0.001 0.273±0.009 6.409±0.213 1.141±0.023 0.816±0.010 3.377±0.203 10.307±0.016 0.534±0.008
2.630±0.008*** 2.657±0.006*** 2.250±0.048*** 2.622±0.009*** 2.634±0.009*** 0.389±0.003*** 10.971±0.250*** 3.342±0.046*** 0.896±0.011* 5.549±0.119*** 13.248±0.034*** 1.838±0.006***
1.167±0.020 1.142±0.003 1.171±0.002 2.126±0.006 2.318±0.013 0.815±0.009 8.109±0.254 3.089±0.058 1.194±0.012 6.834±0.106 12.822±0.555 0.604±0.003
2.960±0.018*** 3.169±0.023*** 2.721±0.028*** 3.514±0.068*** 3.386±0.061*** 1.215±0.004*** 12.520±0.283*** 4.630±0.015*** 2.126±0.015*** 7.920±0.087*** 15.543±0.170*** 2.523±0.004***
12.985±0.236 7.359±0.157 1.296±0.210
13.997±0.127*** 8.790±0.184*** 3.834±0.176***
13.612±0.340 7.723±0.123 2.313±0.203
14.783±0.189*** 9.428±0.249*** 4.859±0.258***
15.820±0.149 8.466±0.301 3.762±0.194
17.235±0.192*** 13.030±0.216*** 6.475±0.257***
AC C
EP
IAA Oxidase (mg IAA degraded min-1 mg protein-1) Protein (mg g-1 f.w.) Thiol (mg g-1 f.w.) Proline (mg g-1 f.w.) Alkaloids (mg g-1 f.w.) Anthocyanins (mg g-1 f.w.) Carotenoids (mg g-1 f.w.) Lycopene (µg g-1 f.w.) β-carotene (µg g-1 f.w.) Flavonoids (Absrbance) 280 nm 290 nm 300 nm 310 nm 320 nm Lignin (mg g-1 f.w.) Phenol (mg g-1 f.w.) Phytosterols (mg g-1 f.w.) Saponins (mg g-1 f.w.) Tannins (mg g-1 f.w.) PAL (µM trans-cinnamic acid formed min-1 mg protein-1) CAD (nmol cinnamyl alcohol oxidised min-1 mg protein-1) 4CL1 (nmol p-coumaroyl CoA ester formed min-1 mg protein-1) 4CL2 (nmol feruloyl CoA ester formed min-1 mg protein-1) 4CL3 (nmol caffeoyl CoA ester formed min-1 mg protein-1)
40 DAT Control s-UV-B 0.004±0.000 0.005±0.000*** 6.200± 0.063 3.793±0.063*** 5.582±0.019 7.860±0.020*** 0.002±0.000 0.005±0.000*** 0.917±0.092 1.207±0.105*** 0.202±0.003 0.241±0.002*** 0.192±0.001 0.206±0.001*** 0.126±0.005 0.160±0.005** 0.059±0.009 0.061±0.002ns
TE D
Parameters
RI PT
The effects of s-UV-B treatment on IAA oxidase activity, primary and secondary plant metabolites, and phenylpropanoid pathway enzymes in leaves of C. forskohlii at three sampling ages. Means±SE, n=7. Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation.
33
ACCEPTED MANUSCRIPT
0.013±0.001 0.377±0.010
0.030±0.002*** 0.620±0.012***
0.023±0.001 0.429±0.007
0.064±0.002*** 0.912±0.318***
AC C
EP
TE D
M AN U
SC
RI PT
CHI (nmol flavanone produced min-1 mg protein-1) DFR(nmol dihydroquercetin reduced min-1 mg protein-1)
34
0.057±0.002 0.477±0.009
0.089±0.002*** 0.775±0.020***
ACCEPTED MANUSCRIPT
Table 4
M AN U
SC
70 DAT Control s-UV-B 0.008±0.000 0.009±0.000*** 9.382±0.519 6.605±0.083*** 4.801±0.016 6.338±0.020*** 0.005±0.000 0.007±0.000*** 1.216±0.020 3.087±0.066*** 0.362±0.002 0.608±0.004*** 0.447±0.006 0.694±0.010*** 0.274±0.004 0.307±0.004** 0.180±0.007 0.197±0.005*
100 DAT Control s-UV-B 0.010±0.000 0.014±0.000*** 11.814±0.011 9.513±0.016*** 5.306±0.018 7.456±0.019*** 0.012±0.000 0.026±0.000*** 2.005±0.018 3.809±0.017*** 0.462±0.003 0.907±0.005*** 0.696±0.004 0.914±0.002*** 0.362±0.003 0.425±0.005*** 0.192±0.006 0.239±0.005**
1.370±0.030 1.619±0.009 1.310±0.006 1.311±0.013 1.544±0.033 0.223±0.024 3.169±0.023 0.744±0.015 0.696±0.012 2.491±0.111 10.307±0.027 0.511±0.033
1.532±0.009*** 1.709±0.017*** 1.460±0.014*** 1.342±0.018** 1.627±0.012** 0.421±0.015*** 4.034±0.038*** 0.791±0.063ns 0.771±0.014** 5.063±0.156*** 11.366±0.038*** 2.071±0.043***
1.650±0.014 1.807±0.004 1.498±0.016 1.664±0.015 1.902±0.012 0.374±0.013 4.226±0.023 1.211±0.091 0.794±0.012 3.663±0.121 12.482±0.017 0.722±0.034
1.793±0.015*** 1.920±0.027** 1.652±0.016*** 1.712±0.023ns 2.340±0.026*** 0.515±0.014*** 7.146±0.034*** 1.361±0.024* 0.913±0.017** 7.063±0.072*** 13.783±0.031*** 3.629±0.047***
1.763±0.014 1.869±0.019 1.595±0.021 1.725±0.007 2.135±0.021 1.595±0.033 5.829±0.018 3.693±0.013 1.895±0.010 9.406±0.130 13.605±0.057 0.888±0.036
1.999±0.013*** 2.613±0.022*** 1.943±0.023*** 2.906±0.005*** 3.216±0.016*** 1.814±0.014*** 9.177±0.025*** 5.523±0.024*** 2.366±0.015*** 7.777±0.173** 15.902±0.044*** 5.857±0.033***
13.350±0.016 6.146±0.214 4.030±0.038
14.091±0.092*** 7.007±0.165*** 9.234±0.040***
14.929±0.026 9.755±0.224 5.703±0.044
15.722±0.033*** 7.885±0.074*** 10.258±0.040***
18.892±0.020 10.130±0.183 7.831±0.026
17.359±0.014*** 12.113±0.236*** 11.104±0.048***
AC C
EP
IAA Oxidase (mg IAA degraded min-1 mg protein-1) Protein (mg g-1 f.w.) Thiol (mg g-1 f.w.) Proline (mg g-1 f.w.) Alkaloids (mg g-1 f.w.) Anthocyanins (mg g-1 f.w.) Carotenoids (mg g-1 f.w.) Lycopene (µg g-1 f.w.) β-carotene (µg g-1 f.w.) Flavonoids (Absorbance) 280 nm 290 nm 300 nm 310 nm 320 nm Lignin (mg g-1 f.w.) Phenol (mg g-1 f.w.) Phytosterols (mg g-1 f.w.) Saponins (mg g-1 f.w.) Tannins (mg g-1 f.w.) PAL (µM trans-cinnamic acid formed min-1 mg protein-1) CAD (nmol cinnamyl alcohol oxidised min-1 mg protein-1) 4CL1 (nmol p-coumaroyl CoA ester formed min-1 mg protein-1) 4CL2 (nmol feruloyl CoA ester formed min-1 mg protein-1) 4CL3 (nmol caffeoyl CoA ester formed min-1 mg protein-1)
40 DAT Control s-UV-B 0.006±0.000 0.006±0.000ns 6.280±0.104 5.035±0.063*** 2.776±0.022 3.217±0.015*** 0.002±0.000 0.002±0.000* 1.175±0.012 2.079±0.017*** 0.289±0.004 0.387±0.002*** 0.242±0.003 0.475±0.009*** 0.174±0.004 0.204±0.004*** 0.174±0.008 0.194±0.007**
TE D
Parameters
RI PT
The effects of s-UV-B treatment on IAA oxidase activity, primary and secondary plant metabolites, and phenylpropanoid pathway enzymes in leaves of C. forskohlii at three sampling ages. Means±SE, n=7. Differences significant at *- P < 0.05, **- P < 0.01, *** - P < 0.001, ns= nonsignificant. DAT- days after transplantation.
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0.045±0.001 0.904±0.009
0.147±0.002*** 1.390±0.025***
0.124±0.001 1.074±0.007
0.169±0.002*** 1.766±0.069***
AC C
EP
TE D
M AN U
SC
RI PT
CHI (nmol flavanone produced min-1 mg protein-1) DFR(nmol dihydroquercetin reduced min-1 mg protein-1)
36
0.183±0.001 0.853±0.009
0.169±0.002* 1.585±0.015***
ACCEPTED MANUSCRIPT Fig.1. The effects of s-UV-B on chlorophyll a, chlorophyll b, and total chlorophyll content in leaves of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001). Fig.2. The effects of s-UV-B on H2O2 content, ̇O2- production rate, and lipid peroxidation in leaves ** p<0.01, *** p<0.001).
RI PT
(A) and roots (B) of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05,
Fig.3. The effects of s-UV-B on the activities of antioxidative enzymes (APX, CAT, GR, POX, PPO, SOD) in leaves (A) and roots (B) of C. forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns:
SC
non-significant, * p<0.05, ** p<0.01, *** p<0.001).
Fig.4. The effects of s-UV-B on ascorbic acid and α-tocopherol in leaves (A) and roots (B) of C.
M AN U
forskohlii 30, 60, and 90 DAT (Mean±SE; n=7; ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
Fig.5. Schematic representation of the processes at transcriptional and translational levels leading to altered cellular metabolism and biochemistry which in turn leads to changes in plant morphology and physiology under s-UV-B stress. The % changes in plant metabolites and enzymes tested in both leaves and roots under s-UV-B are given here (at 60 DAT). Abbreviations: IAAO: Indole acetic acid
TE D
oxidase; PAL: Phenylalanine ammonia lyase; CAD: Cinnamyl alcohol dehydrogenase; 4CL: 4Coumarate CoA ligase; CHI: Chalcone isomerase; DFR: Dihydroflavanol reductase; APX: Ascorbate peroxidase; CAT: Catalase; GR: Glutathione reductase; POX: Peroxidase; PPO:
EP
Polyphenol oxidase; SOD: Superoxide dismutase; Ps: Photosynthetic rate; Gs: Stomatal
AC C
conductance; Fv/Fm: Photochemical efficiency [↑: increase; ↓: decrease].
37
Fig.1.
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Fig.2.
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Fig.3.
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Fig.4.
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Fig.5.
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Highlights: s-UV-B increases secondary metabolite concentrations in C. forskohlii
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Phenylpropanoid pathway enzymes’ activities increase under s-UV-B in C. forskohlii
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s-UV-B increases enzymatic and non-enzymatic antioxidants in C. forskohlii
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s-UV-B alters C. forskohlii’s morphology and physiology
•
s-UV-B treated plant parts of C. forskohlii can serve as better source of antioxidants
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•
ACCEPTED MANUSCRIPT Table S1: 2 way ANOVA test to determine the effects of s-UV-B (T) and plant age (A) and their interactions on growth and physiological parameters of C. forskohlii. F ratios and levels of significance (ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
412.1*** 480.3*** 3910*** 20.3*** 155.7*** 3424*** 382.3*** 21.0***
195.1*** 233.8*** 3384*** 3.7ns 1027*** 6567*** 4918*** 1976***
TE D EP AC C
A×T 884.0*** 60.6*** 2.1ns 8.2**
RI PT
T 4437*** 406.6*** 49.2*** 241.2***
49.8*** 26.7*** 30.8*** 4.2* 53.0*** 500.6*** 288.4*** 90.2***
SC
A 57680*** 981.3*** 146.4*** 863.3***
M AN U
GROWTH PARAMETERS Total biomass Plant height No. of leaves Leaf area PHYSIOLOGICAL PARAMETERS Ps Gs Ci WUE F0 Fm Fv Fv/Fm
ACCEPTED MANUSCRIPT Table S2: 2 way ANOVA test to determine the effects of s-UV-B (T) and plant age (A) and their interactions on various metabolites and enzymes tested in the leaves and roots of C. forskohlii. F ratios and levels of significance (ns: non-significant, * p<0.05, ** p<0.01, *** p<0.001).
1663*** 1486*** 391.3*** 1441*** 1673*** 333400*** 6805*** 725000*** 253900*** 481.9*** 8232*** 9.6*** 40840*** 888700*** 29000*** 419.1*** 0.943ns 31.4*** 41.8***
19120*** 15590*** 4162*** 3028*** 2981*** 59360*** 43750*** 497200*** 91610*** 223.0*** 4844*** 104.9*** 17450*** 217300*** 88950*** 423.3*** 10.3** 89.0*** 21.6***
675.9*** 583.0*** 86.6*** 51.3*** 59.7*** 11620*** 4.6* 80780*** 55500*** 7.9** 484.0*** 5.1* 166.8*** 33130*** 43.2*** 23.6*** 0.462ns 2.2ns 12.9***
TE D
EP
52.1*** 1658*** 1068*** 79.0*** 29720*** 36.1*** 23420*** 1680*** 1026*** 9234*** 105400*** 34630***
85.5*** 5318*** 0.008ns 33.4*** 56.3*** 0.267ns 2495*** 4646*** 463.9*** 4638*** 62730*** 1637***
ROOTS
A 4291*** 124200*** 18510*** 14920*** 14230*** 4198*** 2440*** 1341*** 13.3***
T 1173*** 65920*** 8416*** 4876*** 60100*** 7189*** 1983*** 169.5*** 28.0***
A×T 594.0*** 3041*** 1110*** 2521*** 2508*** 1057*** 2.6ns 10.2*** 3.1ns
323.1*** 533.2*** 251.4*** 2301*** 1298*** 69930*** 9789*** 318200*** 642600*** 700.3*** 5400*** 11450*** 221900*** 285100*** 152.8*** 1062*** 41.6*** ---------
166.4*** 464.1*** 241.6*** 1212*** 934.4*** 3103*** 10890*** 250800*** 39520*** 182.8*** 2519*** 77830*** 0.027ns 43670*** 1075*** 944.9*** 630.5*** ---------
4.1* 213.6*** 21.9*** 997.2*** 279.7*** 48.9*** 1131*** 183700*** 12610*** 211.4*** 150.1*** 7730*** 19700*** 54190*** 18.3*** 537.8*** 9.0** ---------
----4595*** 2758*** 67.2*** 18570*** 152.2*** 77.5*** 19.3*** 1547*** 17420*** 576.7*** 10050***
----2477*** 68.9*** 41.2*** 95090*** 5.3* 31.6*** 386.7*** 5047*** 13460*** 10720*** 11860***
----303.7*** 0.364ns 9.3** 5832*** 378.3*** 12.9*** 0.003ns 224.9*** 72.7*** 170.0*** 2826***
RI PT
A×T 38.9*** 685.1*** 222.8*** 494.9*** 2883*** 80.0*** 1461*** 41.0*** 2.8ns
SC
T 216.7*** 132400*** 13400*** 9158*** 13810*** 279.2*** 7659*** 312.0*** 9.7**
M AN U
A 157.7*** 12690*** 2662*** 3580*** 5040*** 3216*** 29870*** 858.9*** 49230***
AC C
LEAVES IAA Oxidase Protein Thiol Proline Alkaloids Anthocyanins Carotenoids Lycopene β-carotene Flavonoids 280 nm 290 nm 300 nm 310 nm 320 nm Lignin Phenol Phytosterols Saponins Tannins PAL CAD 4CL1 4CL2 4CL3 CHI DFR Chlorophyll a Chlorophyll b Total chlorophyll H2O2 ̇O2LPO APX CAT GR POX PPO SOD Ascorbic acid α-tocopherol
2.6ns 416.7*** 0.679ns 2.8ns 2172*** 40.0*** 140.0*** 238.4*** 118.2*** 1020*** 5698*** 216.7***