Ultraviolet-B induced responses of Psoralea corylifolia L. with special emphasis on: growth, anatomy, physiology, yield, essential oil content and composition

Psoralea corylifolia L. is a traditionally and medicinally important, endangered plant of the family Fabaceae. Seeds obtained from it widely used in treatment of skin diseases like leprosy, psoriasis, leucoderma, vitiligo. The present study was aimed to assess the growth, physiology, anatomy, yield and essential oil content and composition of P. corylifolia in response to elevated UV-B (eUV-B; ambient + 7.2 kJm − 2 day − 1 ). The results showed reductions in the growth and physiological parameters under eUV-B treatment (except chlorophyll a/b ratio, carotenoids content and water use eciency which showed increment) while reverse trend was observed for UV-B absorbing compounds. The total thickness of leaves decreased by 13.8% under eUV-B exposure. Due to eUV-B exposure number of racemes, owers and seeds, and the length of racemes signicantly reduced whereas length of owers, seed size and seed mass (thousand seeds weight) showed non-signicant variations. The essential oil content of seeds showed increment by 46.4% under eUV-B treatment. The GC-MS analysis of essential oil revealed 28 major compounds from control and eUV-B exposed plants. Overall the monoterpenes showed reduction whereas sesquiterpenes and meroterpene showed increment. The metabolites caryophyllene, caryophyllene oxide and bakuchiol (possess anti-cancerous, anti-inammatory activities) were identied as major metabolites of essential oil, which showed increment under eUV-B treatment. The study displayed that eUV-B enhanced the content of essential oil with improvement in the quality of seeds in terms of medicinally important compounds of seeds.


Introduction
Although ultraviolet-B (UV-B) radiation is a minor component of the solar spectrum, it has the ability to signi cantly affect all living organisms on the Earth' surface. Upcoming UV-B radiation is affected by many factors including seasonal, diurnal, meteorological factors, altitude, latitude and atmospheric pollution (Jenkins 2017). Recently it has been reported by various groups of researchers that the changes in incident surface UV-B radiation is highly contributed by climate change, irrespective of the changes in level of stratospheric ozone Bornman et al. 2019). As plants are sessile and photoautotrophic organisms, they constantly exposed to high energy UV-B radiation in their environments that may in uence development, growth and overall physiology and metabolisms of plants. UV-B radiation directly or indirectly, through the enhanced generation of reactive oxygen species (ROS) imposes damaging effects on nucleic acids, proteins, membrane lipids and chloroplasts (Takshak and Agrawal 2019). In addition to indirect effect via inhibition of photosynthesis and thus availability of photo assimilates, UV-B may also directly perturb the reproductive growth. UV-B affects a variety of oral structures or it may also lead to abortion of oral buds and reduction in number of owers, leading to decreased reproductive success (Del Valle et al. 2020). However, nowadays the perspective of UV-B radiation studies have changed from stressor to regulator of plant growth' and as a physic tool to improve the nutraceutical and pharmaceutical qualities of fruits and medicinal plants (Coffey et al. 2017;Mannucci et al. 2020). Hideg et al. (2013) reported that balance between distress and eustress i.e. damaging and regulating effects of UV-B on a plant species depends on several factors including time and dose of UV-B exposure, genetic setup, environmental variables, acclimation strategy and developmental stages of plants.
Psoralea corylifolia L. (syn. Cullen corylifolium L.) commonly known as bakuchi, babchi is a medicinally important, endangered plant species of the family Fabaceae, which is widely distributed in subtropical and tropical region of the world. It has been used for a long time in traditional Chinese and Ayurvedic system of medicine. It is also known as Kushtanashini due to its frequent use since ages in the treatment of skin diseases like leprosy, leucoderma and psoriasis (Khushboo et al. 2010). Whole plant parts, especially seeds and volatile oils obtain from seeds are rich source of several bioactive constituents such as psoralen, bakuchiol, isopsoralen, bavachalcone, isobavachalcone, psoralidin, bavachinin, bavachin, psoralidin, caryophylllene, daidzein, genistein, which belongs to various classes such as furanocoumarins, coumarins, terpenes, meroterpenes, avones and iso avonoids. Due to presence of these important constituents it possess several pharmaceutical activities such as anticancer, anti-in ammatory, anti-HIV, anti-vitiligo, anti-psoriatic, antidepressant, estrogenic, neuroprotective, immunomodulatory and anti-Alzheimer's activities and is widely used in the treatment of various diseases (Agrawal and Pandey 2019;Koul et al. 2019). Its bioactive constituent psoralen along with UV-A radiation used in PUVA therapy for the cure of several skin diseases (Khushboo et al. 2010). Taking into account the medicinal importance of P. corylifolia, there are only few reports available on abiotic stress response of P. corylifolia including, gamma radiation (Bhat et al. 2015;Jan et al. 2015) cadmium (Satdive et al. 2014) SO 2 (Ali et al. 2008) and salt stress (Katare et al. 2012), however there are no reports yet available on the effect of UV-B radiation on P. corylifolia. Considering the literature lacunae, the present study was conducted to unravel the response of P. corylifolia against elevated UV-B radiation with the following objectives: (1) to evaluate the effect of eUV-B on growth, reproductive and anatomical features (ii) to estimate the effect of eUV-B on physiological parameters along with UV-B absorbing compounds under eUV-B (iii) to study the effect of eUV-B on medicinally important plant part i.e seeds and its essential content and composition.

Material And Methods
Experimental area Ten plants per plot were selected randomly and tagged for further observations. The number of racemes and owers were counted. The length of racemes and owers were measured on ten randomly selected owers from separate plants per plot. Ten tagged plants per plot were harvested for assessing yield parameters including number of seeds, diameter and length of seeds and the test weight (1000 seeds weight).

Essential oil extraction, GC-MS analysis and identi cation of compounds
The extraction of essential oil from seeds was performed following the methodologies of Takshak and Agrawal (2018) with few modi cations.
Fresh seeds (250 g) were collected in triplicate from both control and treatment plots at 180 DAT. Samples of seeds were homogenized nely using a grinding machine and then subjected to oil extraction using the hydrodistillation technique in a Clevenger apparatus for 6 hours. The essential oil was collected and stored in a dark glass vials at 4 ºC after drying with anhydrous sodium sulfate (Gautam and Agrawal 2017). The essential oil obtained was measured and expressed as μL of essential oil per 250 g fresh seeds.
The essential oil obtained from seeds was subjected to GC-MS (QP-2010 Plus Shimadzu), having column (Rtx-5Ms capillary column) of 0.25 mm diameter, 30 mm in length and 0.25 μm lm thickness. Initially the temperature of column oven was kept at 50 ºC for 2 min, followed by the increment in temperature to 210 ºC at a rate of 3 ºC min -1 and holding time of 2 min. The temperature was again increased upto 280 ºC at a rate of 8 ºC min -1 with a hold time 4 min, which was kept constant till the end of program. Essential oil (1.0 μL) was injected into the system at 260 ºC with ow rate of 1.21 mL min -1 and helium as a carrier gas. The pressure of carrier gas (helium) was 69 kPa with total ow (16.3 mL min -1 ), column ow (1.21 mL min -1 ), purge ow (3.0 mL min -1 ) and linear velocity (39.9 cm s -1 ). The interface and ion source temperature of MS were 270 ºC and 220 ºC, respectively and the solvent cut time was 3.5 min. The mass scan start at 40 amu and end at 650 amu with scan speed 1428 amu s -1 and event time 0.50 s. The total running time of program was 70.08 min. The metabolites of essential of seeds were identi ed by comparison of their retention indices and mass spectra fragmentation pattern with those of available in libraries (NIST14, NIST14s, WILEY8, FFNSC, and SZTERP) of GC-MS data system.

Statistical analysis
The student's t-test (con dence level of 95%) was performed at each growth stages to nd out the difference between the means of two groups (i.e. control and eUV-B treatment). A two-way analysis of variance (ANOVA) was carried out to evaluate the signi cant effect of ages, treatment, and their interaction. A bivariate Pearson's correlation was performed to identify the correlation between growth, physiological parameters and UV-B absorbing compounds. Further, the identi ed metabolites of essential oil from two groups (control and eUV-B treatment) were subjected to principal component analysis (PCA). PCA was performed by varimax rotation and Kaiser's normalization. All the statistical analysis was conducted by SPSS Inc. version 20.0 (IBM Corp, Armonk, NY).

Growth parameters
Under eUV-B exposure shoot length and root length signi cantly decreased at three growth stages with maximum reduction in shoot length at 120 DAT (11.71%, p ≤ 0.01) and root length at 60 DAT (38%, p ≤ 0.01) as compared to control. The number of leaves and leaf area also reduced under eUV-B exposure with signi cant reduction in number of leaves at 120 DAT (16.48%, p ≤ 0.05) and 180 DAT (14.78%, p ≤ 0.01) and in leaf area by 24.4, 6.2 and 6.6% at 60, 120 and 180 DAT, respectively (Fig. 1). The number of branch and number of nodules showed a signi cant reduction by 20 and 21.1% at 120 DAT and 18.9 and 19.8% at 180 DAT respectively when exposed to eUV-B. Under eUV-B exposure below and above ground biomass signi cantly reduced at all the growth stages with maximum reduction at 60 DAT by 48 (p ≤ 0.001) and 44.3% (p ≤ 0.001) respectively in below and above ground biomass (Fig. 2). As per the result of two way ANOVA all the growth parameters were signi cantly affected by age, treatment and their interaction except root length and leaf area which only varied with the individual factors of age and treatment (Table 1).

Anatomical features
Under eUV-B exposure, total thickness of leaves was signi cantly reduced by 13.8% compared to control ones. The thickness of adaxial and abaxial epidermis also decreased by 41.1 and 34.7%, respectively due to eUV-B exposure ( Table 2). In control plants the thickness of spongy parenchyma was higher as compared to palisade parenchyma whereas opposite trend was observed for eUV-B exposed plants. Due to eUV-B exposure, thickness of palisade parenchyma increased and the cells of palisade parenchyma appear compressed as the proportion of intercellular cavities in palisade parenchyma decreased. However the proportion of intercellular cavities in spongy parenchyma was nonsigni cantly affected by eUV-B exposure ( Table 2). Exposure of plants to eUV-B also increased the number of oil glands as compared to control ( Fig. 3) Gas exchange and chlorophyll uorescence Due to eUV-B exposure photosynthetic rate (Ps), stomatal conductance (gs) and transpiration rate (Tr) reduced with maximum reduction at 60 DAT by 33.9 (p ≤ 0.001), 40.78 (p ≤ 0.001) and 54.6 (p ≤ 0.001), respectively in Ps, gs and Tr. Internal CO 2 concentration (Ci) was only signi cantly reduced by 6.2 (p ≤ 0.01) and 8.5% (p ≤ 0.05) at 60 and 180 DAT respectively. Under eUV-B exposure water use e ciency (WUE) signi cantly increased by 47.18% at 60 DAT whereas this increase was non-signi cant (p > 0.05) at two later growth stages. There was no signi cant variations (p > 0.05) noticed in Fv/ Fm at any of the growth stages of plants under eUV-B exposure (Fig. 4). Ps, gs, Tr and WUE were signi cantly affected by age, treatment and their interaction while Ci and Fv/ Fm only affected by individual factor of age and treatment (Table  1).

Pigments and UV-B absorbing compounds
Total chlorophyll content signi cantly reduced by 10.6 (p ≤ 0.001), 2.6 (p ≤ 0.01) and 13 % (p ≤ 0.001) at 60, 120 and 180 DAT, respectively under eUV-B exposure as compared to control. Chlorophyll a/b ratio and carotenoids content was found to increase under eUV-B exposure with maximum increase of 32.4 (p ≤ 0.001) and 35.2% (p ≤ 0.001) in Chlorophyll a/b ratio and carotenoids content respectively at 60 DAT , however this increase was non-signi cant (p > 0.05) for Chlorophyll a/b ratio and carotenoids content at 120 and 180 DAT, respectively (Fig.  5).
UV-B absorbing compounds like avonoids, anthocyanins and phenols showed signi cant increase under eUV-B exposure at all growth stages with maximum increment in anthocyanins (16.3 %; p ≤ 0.01) and avonoids (15.95%; p ≤ 0.001) at 60 DAT whereas in phenols (41.4; p ≤ 0.001) at 180 DAT (Fig. 5). All the pigments and UV-B absorbing compounds were affected by treatment, age and its interaction except avonoids which was only affected by age and treatment (Table 1).

Reproductive parameters and yield attributes
The number of racemes per plant and the number of owers per plant were signi cantly reduced under eUV-B treatment by 28.2 (p ≤ 0.001) and 8 % (p ≤ 0.001). Further, the length of racemes also signi cantly reduced (14.5 %; p ≤ 0.001) under eUV-B, while the length of owers was not signi cantly affected (p > 0.05) by eUV-B as compared to control (Table 3).
The number of seeds per plant decreased by 9.3 % (p ≤ 0.01), whereas the length and diameter of seeds showed a non-signi cant reduction (p > 0.05). Test weight also showed non-signi cant variation (p > 0.05) under eUV-B exposure as compared to control (Table 3).

Essential oil content and composition
The essential oil of seeds of P. corylifolia was yellowish in color with a pleasant odor and stickiness. The essential oils content increased by 46.4% under eUV-B as compared to control (Fig. 6). The GC-MS analysis of essential oil of seeds displayed a total of 58 compounds (including trace and major compounds of control and eUV-B exposed plant), among which 28 major compounds were identi ed and shown here ( Table  4). The identi ed compounds were categorized in different classes such as monoterpenes, sesquiterpenes, meroterpene and others, that overall contributes to 4.43, 74.37, 20.23 and 0.95%, respectively. Overall the monoterpenes showed reduction (32%), whereas sesquiterpenes (6.8%) and meroterpene (14.2%) showed increment under eUV-B exposure, compared to control ones. Among identi ed metabolites, βcaryophyllene, caryophyllene oxide and bakuchiol were responsible for the major proportion of essential oil and showed increment under eUV-B treatment compared to control (Table 4). The metabolites such as β-myrcene, β-limonene, tetradecynol and cyclosativene were not detected under eUV-B treatment whereas nonadienol and α-amorphene were only detected in eUV-B exposed plants. The eUV-B exposure leads to nonsigni cant changes in some metabolites such as myrtanol, patchoulane, α-and γ-muurolene and cis-calamenene while reduction in β-linalool, β-geraniol and α-pinene oxide. Further some of the metabolites of sesquiterpene class (farnesol, α-humulene, γ-gurjunene, β-selinene and humulene oxide) showed reduction whereas some metabolites (α-copaene, δ-cadinene, aromadendrene, caryophylladienol, caryophyllene epoxide) showed increment under eUV-B treatment as compared to control (Table 4).

Discussion
The adverse effects of UV-B on the growth, biochemistry and physiology of plants are well known however the extent of damage differ among the plant species or the cultivars of same species (Reddy et al. 2013;Choudhary and Agrawal 2014). The results of present study demonstrated that eUV-B negatively affected all the studied growth parameters under eUV-B exposure. Alterations in the plant growth under eUV-B exposure might be due to disturbance in the process of cell elongation and cell division as a consequence of imbalance in the endogenous level of growth regulators (through direct photodegradation by UV-B or by the activation of oxidases) or the alteration in transport of growth regulators (Hopkins et al. 2002;Takshak and Agrawal 2018). In present study number of leaves and leaf area showed reduction under eUV-B exposure and was positively correlated ( Fig. 8; r = 0.989), which might be seen as an adaptive response of plants to reduce the absorbtion of UV-B radiation (Choudhary and Agrawal 2014;Tripathi et al. 2019). Chen et al. (2016) revealed that, the enhanced UV-B radiation signi cantly decreased the shoot height, basal diameter, total leaf area and total dry mass of male and female Morus alba saplings. Takshak and Agrawal. (2018) also reported reduction in root length, shoot length, number of leaves, leaf area and total biomass of Coleus forskohlii under eUV-B condition. The process of nodulation and activation of Nod gene depends on speci c composition of avonoids, which may alter following UV-B exposure (Chimphango et al. 2004). However reduction in number of nodules as noticed in our study might be due to UV-B induced inhibition of photosynthesis that may alter the availability of resources to micro-symbionts, which leads to reduced number and activity of nodules or it might be due to UV-B induced increment in avonoids and phenols content in leaves. This hypothesis was supported by the positive correlation between number of nodules and photosynthetic rate (Ps) (Fig. 8; r = 0.770) and the negative correlation of number of nodules with phenols ( Fig. 8; r = -0.543). Similarly Choudhary and Agrawal (2014) reported reduction in number and fresh weight of nodules in Pisum sativum under UV-B stress.
The biomass of plants represents the long term integration of all the growth, physiological and biochemical aspects (Teramura 1983). The reduction in biomass as observed in present study was corroborated with several other studies on Cymbopogon citratus (Kumari and Agrawal 2010) Coleus forskohlii (Takshak and Agrawal 2015) and Morus alba (Chen et al. 2016) under UV-B stress condition. Tevini and Teramura (1989) reported that reduction in total biomass under UV-B was correlated with reduced plant height and leaf area of plant which was also noticed in our study as reduction in above and below ground biomass of test plant was correlated well with the reduced leaf area ( Fig. 8; r = 0.972 and 0.965) and shoot length ( Fig. 8; r = 0.965 and 0.969). More reduction in below ground biomass and root length as compared to above ground biomass and shoot length under eUV-B exposure in present study suggest the lower allocation of photo-assimilates towards the belowground plant parts. Further the higher reduction in above and belowground biomass of eUV-B exposed plants at initial growth stage showed the plant was more sensitive at early growth stage. This nding was corroborated well with the results of Nazari and Zarinkamar (2020), who reported that Mentha aquatica exposed to UV-B radiation was more sensitive at early vegetative growth stage as compared to later growth stage. The anatomical studies showed the reduction in total thickness of leaves under eUV-B which was associated with reduction in adaxial epidermis, spongy parenchyma and abaxial epidermis. Similarly, Kakani et al. (2003) and Romanatti et al. (2019) also reported reduction in leaf thickness in cotton and eggplant, respectively. The adaxial surface of leaves, that was directly exposed to eUV-B showed highest reduction of 41.16%. The decrease in the thickness of epidermal cells might be related to reduction in division and expansion of cell. Inostroza-Blancheteau et al. (2014) reported that the slower division of cells provides additional time for repair of DNA, which is one of the important strategies adopted by plants to protect against UV-B induced damage. The increased thickness of palisade parenchyma with short, multilayered and compact cells could be seen as a protective strategy of test plant to reduce the penetration of UV-B radiation to spongy parenchyma.
The major photosynthetic processes including photochemical reactions in the thylakoid membranes, enzymatic reactions of the CO 2 xation in Calvin cycle, and the stomatal control of CO 2 diffusion get altered following the exposure of plants to UV-B radiation. However the photosynthetic responses of plants under enhanced UV-B condition varies and depends on plants species, cultivar, UV-B dose and other environmental factors (Zhao et al. 2004). In the present study exposure of plants to eUV-B decreased the Ps at all the growth stages, which was mainly attributed to reduced gs and Ci that was further supported by positive correlation of Ps with gs ( Fig. 8; r = 0.728) and Ci ( Fig. 8; r = 0.856). Under eUV-B exposure reduction in Tr could also be seen as a strategy of plants to conserve water for later use as shown by increased WUE. This hypothesis was strengthened by the negative correlation between Tr and WUE ( Fig. 8; r = -0.637). Our results were in agreement with previous ndings on Curcuma species (Jaiswal and Agrawal 2021) and Cymbopogon citratus (Kumari and Agrawal 2010). However contrasting results (reduction in both Tr and WUE) were reported by Rai and Agrawal (2020) in E. alba under intermittent and continuous UV-B treatment.
Fv/Fm (maximum quantum yield of PSII) is a measurement ratio that represents the e ciency and stability of PSII, a major component of the photosynthetic apparatus. Any alteration in Fv/Fm, re ects the changes in the photochemical conversion e ciency of PSII, due to which Fv/Fm can be seen as a good indicator of photo-inhibition of photosynthesis (Ranjbarfordoei et al. 2011). However in our study non signi cant variations in Fv/Fm under eUV-B treatment was noticed at all the growth stages of plants that re ect the absence of PSII photo-inhibition and the consequent photodamage. Martínez-lüscher et al. (2013) reported that both the short and long term exposure of 5.9 and 9.6 kJ m − 2 d − 1 UV-B radiation did not cause any alteration in Fv/Fm in Vitis vinifera.
Our study displayed that under eUV-B exposure, chlorophyll content reduced that might be the result of inhibition of enzymes involved in chlorophyll biosynthesis pathway or it might be due to destruction of chlorophyll molecule and its precursors (Kataria et al. 2014). The reduction in chlorophyll content might be taken as one of the reasons (in addition to stomatal limitation) for reduction in Ps under eUV-B exposure in our study as both the parameters (i.e. chlorophyll content and Ps) showed positive correlation ( Fig. 8; r = 0.696). An increased chlorophyll a/b ratio under eUV-B exposure as observed in the present study re ects the content of chlorophyll b was affected more than chlorophyll a. Tevini et al. (1981) reported that biosynthesis of chlorophyll b was inhibited more than the biosynthesis of chlorophyll a under UV-B exposure that result in enhanced chlorophyll a/b ratio in bean, radish, barley and corn. Carotenoids act as a quencher that dissipate excess excitation energy and protect chlorophyll molecule from photo-oxidative damage. The increase in carotenoids content in present study with concomitant increase in chlorophyll a/b might have offered protection to P. corylifolia against eUV-B. The reduced content of chlorophyll under eUV-B treatment could also be seen as a strategy of plants to reduce light absorbance that contributes to its photo-protection (Machado et al. 2017), which was evidenced in the present study as absence of any signi cant changes in photochemical e ciency of PSII (Fv/Fm).
The secondary metabolites such as avonoids, anthocyanins and phenols, which are also known as UV-B absorbing compounds accumulate mainly in epidermal and mesophyll cells and help in reducing the penetration of UV-B deep inside the leaves. Further, it may also scavenge the free radicals and provide protection against oxidative damage (Agati et al. 2012;Pandey and Agrawal 2020). In the present study all the UV-B absorbing compounds increased differentially at different growth stages of P. corylifolia which was corroborated well with the results of Jaiswal et al. (2020) observed in Curcuma sp.
Sexual reproduction is a sophisticated process of a plant species' life cycle, and the various stages of reproductive development were noticed to be sensitive against UV-B radiation (Tripathi et al. 2019). However some studies also documented stimulatory effects of UV-B on the reproductive parameters (Grammatikopoulos et al. 1998;Petropoulou et al. 2001). In present investigation, number of seeds reduced under eUV-B exposure and was directly linked to reduce number of racemes and owers, resulting from UV-B induced negative impact on vegetative growth of plants as observed by reduced plant height and number of branches. Gan et al. (2013) reported that branches directly in uence seeds output and reproductive allocation of a plant species, as the branches accounts for 'assurance of material' for the reproduction in varied environments. Our results were in accordance with study of Jan et al. (2011), who noticed reduction in these reproductive parameters of P. corylifolia at higher doses of gamma radiation. Tripathi et al. (2019) also reported reduction in number of heads and achenes due to eUV-B exposure in Helianthus annuus. In present study the length of racemes reduced under eUV-B condition that might be due to disturbance in the architecture of in orescence under stressful condition (Park et al. 2016). Seed size is one of the potential traits of plants' life history and is an important factor that determines the tness of a plant species. It has been reported that there is a trade-off between number and size of seeds, which enable plants to adapt and survive in their varied habitat (Gan et al. 2013). Abeli et al. (2017) reported that under stressful environment plants invest more photosynthates towards reproduction and enhance the provisioning and quality of seeds as an adaptive response to ensure survival of seedlings. This might be happened in our case, where the number of seeds reduced while seeds size and mass did not show any signi cant variation as an adaptive response under stressful environment of UV-B. Similarly Yao et al. (2006) reported reduction in seed yield while test weight was unaffected, at lower dose of enhanced UV-B radiation in autumn buckwheat. Essential oils are formed by plants as a complex mixture of secondary metabolites and are volatile in nature and have a strong odor. Essential oils are known to possess strong potential against several diseases and are use in the preservation of foods from the deteriorating effects of oxidants (Hajlaoui et al. 2009). In present study content of essential oil of seeds increased under eUV-B treatment that might be related with reduced leaf area and higher density of oil glands (Fig. 3). Jaiswal and Agrawal (2021) reported that exposure of plants to eUV-B causes induction of pathway related to essential oil biosynthesis which may leads to increased production and varied composition of essential oils. It has been reported by several authors that the production and composition of essential oil depend on several factors, including environmental conditions, genetic setup of plants, developmental stages, plant tissues and analytical conditions (Ebrahimi et al. 2008;Hajlaoui et al. 2009;Pandey et al. 2021). This might be the probable reason for the observed difference between our study (where caryophyllene, caryophyllene oxide and bakuchiol were observed as major component) with those of the studies of Jan et al. (2015), who reported, α-pinene, psoralen, bakuchiol and caryophyllene as major components of P. corylifolia seeds' essential oils.
Bakuchiol which is one of the major components of P. corylifolia, belongs to meroterpene class of compound and is derived from the phenylpropane and isoprenoid units. Mehta et al. (1966) extracted bakuchiol for the rst time from P. corylifolia. It possesses several pharamacological properties such as anticancer, anti-in ammatory, antidepression, hypoglycemic, neuroprotective, estrogenic, anti-aging activities (Agrawal and Pandey 2019;Xin et al. 2019). Due to eUV-B exposure content of bakuchiol increased, so this trend of increase in major active compounds of P. corylifolia was signi cant, considering the pharmaceutical perspectives. Jan et al. (2015) also reported increased content of bakuchiol in P. corylifolia exposed to variable doses of gamma radiation. Further, the reduction and disappearance of most of the monoterpenes under eUV-B treatment, in our study was might be due to impairment in the chlorophyll containing machinery and thus disturbance in the biosynthesis pathway of monoterpenes, as monoterpenes are mainly synthesized in plastids via methylerythritol pathway (MEP), by using geranyl pyrophosphate (GPP) as a precursor (Rai and Agrawal 2020).
Sesquiterpenes are synthesized in cytosol via mevalonate (MVA) pathway by utilizing farnesyl pyrophosphate (FPP) as precursor. The metabolites α-humulene and β-caryophyllene are the widely occurring and medicinally important sesquiterpenes which possess antiin ammatory, anticarcinogenic activities. Both of these sesquiterpenes utilize a common humulyl intermediate, that itself generated by FPP (Cane 1999). An increment in β-caryophyllene and its derivatives (caryophyllene oxide, caryophyllene epoxide and caryophylladienol) whereas reduction in α-humulene and its derivative (humulene oxide) suggest the involvement of humulyl intermediate towards the caryophyllene and its derivatives synthesis, and might be the reason for increment of β-caryophyllene and its derivatives under eUV-B in present study. This hypothesis was con rmed by the results of PCA where the metabolites β-caryophyllene, caryophyllene oxide, caryophyllene epoxide and caryophylladienol grouped in one cluster (cluster 1) whereas α-humulene and humulene oxide grouped in another cluster (cluster 2) and also the metabolites of two clusters showed negative correlation (Fig. 7).
Further the farnesol represents the simplest acyclic sesquiterpene and is formed by action of phosphatases on the terminal phosphate moiety of FPP. Farnesol together with FPP provides the homeostatic control of carbon ux in the MVA pathway (Chappell and Coates 2010). In present study reduction in farnesol might be explained by involvement of farnesol in the synthesis of others sesquiterpenes metabolites, resulting in its reduced content under eUV-B. It was also observed that some metabolites (γ-gurjunene, β-selinene) decreased while others such as α-copaene, δ-cadinene, aromadendrene increased indicating differential responses of different enzymes or precursors involved in their synthesis against UV-B radiation. PCA results also showed that the metabolites which showed increasing trend or only detected under eUV-B, clustered together and form the cluster 1 whereas the metabolites which grouped together in cluster 2 showed reduction or not detected under eUV-B treatment.

Conclusions
Our study highlighted the negative effect of eUV-B on most of the growth and physiological parameters with a concomitant increase in UV-B absorbing compounds. The non-signi cant variations in Fv/Fm suggest, the reduction in photosynthesis was mainly associated with stomatal limitation and chlorophyll reduction, rather than the limitation of PSII. The negative effect of eUV-B on vegetative growth was also re ected by reproductive parameters (as showed by reduction in number of racemes and owers), as the branches provide maternal assurance for reproduction. Further reduction in number of seeds whereas non-signi cant variation in seeds mass and seeds size could be seen as an adaptive strategy of plant under stressful environment of eUV-B. The content of essential oil of seeds increased by 46.4 % due to eUV-B treatment. Under eUV-B exposure some metabolites such as α-copaene, δ-cadinene, aromadendrene, caryophylladienol, caryophyllene epoxide showed increment whereas farnesol, α-humulene, humulene oxide, β-linalool, β-geraniol and α-pinene oxide showed reduction; however the major metabolites like caryophyllene, caryophyllene oxide and bakuchiol which possess anticancerous and anti-in ammatory activities increased under eUV-B condition. Overall the study concludes that eUV-B enhanced the quality of seeds in terms of essential oil content and major active metabolites of essential oil obtained from seeds at the expense of growth, physiology and quantity of seeds. So if we aim to obtain a large quantity of seeds, UV-B may not be preferable but if the objective is to improve the medicinal quality of seeds of P. corylifolia, UV-B may be seen as a promising one. However for better understanding the detailed mechanism adopted by P. corylifolia under UV-B stress condition the study needs to be further explored. Visualization, Formal analysis. Shashi Bhushan Agrawal: Conceptualization, Validation, Supervision. All authors read and approved the nal manuscript Tables   Table 1 Two way ANOVA test to nd the effect of eUV-B treatment (T) plant growth stage (A) and their interaction (T×A) on different growth, physiological parameters and UV-B absorbing compounds of P. corylifolia (F ratios and level of signi cance; ns -non-signi cant, * p < 0.05, ** p < 0.01, *** p < 0.001) Phenols 9.6** 97.8*** 14.2*** Anthocyanins 3914.6*** 56.7*** 3.6*  Figure 1 Variation in shoot length, root length, number of leaves and leaf area of P. corylifolia at three growth stages under control and eUV-B treatment.

Figure 2
Variation in above and below ground biomass, number of branches and number of nodules of P. corylifolia at three growth stages under control and eUV-B treatment. Values of bars represent mean ± SE; Level of signi cance between control and eUV-B treated plants: ns, nonsigni cant, *p < 0.05, **p < 0.01, ***p< 0.001

Figure 5
Variation in Total chlorophyll, chlorophyll a/b, carotenoids, phenols, antocyanins and avonoids content of P. corylifolia at three growth stages under control and eUV-B treatment. Values of bars represent mean ± SE; Level of signi cance between control and eUV-B treated plants: ns, non-signi cant, *p < 0.05, **p < 0.01, ***p < 0.001