Exposure to Low UV-B Dose Induces DNA Double-Strand Breaks Mediated Onset of Endoreduplication in Vigna radiata (L.) R. Wilczek Seedlings

Abstract

Multiple lines of evidence indicate that solar UV-B light acts as an important environmental signal in plants, regulating various cellular and metabolic activities, gene expression, growth and development. Here, we show that low levels of UV-B (4.0 kJ m⁻²) significantly influence plant response during early seedling development in the tropical legume crop Vigna radiata (L.) R. Wilczek. Exposure to low doses of UV-B showed relatively less growth inhibition yet remarkably enhanced lateral root formation in seedlings. Both low and high (8.0 kJ m⁻²) doses of UV-B treatment induced DNA double-strand breaks and activated the SOG1-related ATM-ATR-mediated DNA damage response pathway. These effects led to G2-M-phase arrest with a compromised expression of the key cell cycle regulators, including CDKB1;1, CDKB2;1 and CYCB1;1, respectively. However, along with these effects, imbibitional exposure of seeds to a low UV-B dose resulted in enhanced accumulation of FZR1/CCS52A, E2Fa and WEE1 kinase and prominent induction of endoreduplication in 7-day-old seedlings. Low dose of UV-B mediated phenotypical responses, while the onset of endoreduplication appeared to be regulated at least in part via UV-B induced reactive oxygen species accumulation. Transcriptome analyses further revealed a network of co-regulated genes associated with DNA repair, cell cycle regulation and oxidative stress response pathways that are activated upon exposure to low doses of UV-B.

Introduction

Plants, being sessile in nature and with their obligatory dependence on sunlight for photosynthesis, cannot escape the damaging effects of solar UV-B light. Therefore, they have developed a sophisticated and highly regulated balance between optimal light capture and UV-B protection. Some plant species escape UV-B exposure by limiting their life span to the season or places where they perceive only low levels of UV-B, while others, including crops, grow during the summer months and experience a high incidence of solar UV-B light (Ulm and Jenkins 2015). In tropical climates, plants receive sunlight for longer duration, and the effects of UV-B radiation are greater under such conditions. Early studies in tropical crops, including Oryza sativa (Teramura and Sullivan 1994), Vigna mungo (Fukumoto and Mazza 2000), Vigna radiata (Amudha et al. 2005), Glycine max (Guruprasad et al. 2008) and Triticum aestivum (Kataria and Guruprasad 2014) have shown compromised growth and yield in response to exposure to ambient UV-B light. More recent studies in cucumber (Cucumis sativus L. cv. ‘Hi Jack’) have correlated the growth retardation effect with the regulatory mechanism associated with the acclimation processes of UV radiation (Qian et al. 2021). Other studies mainly in Arabidopsis and some other species indicated that two major mechanisms of UV-B mediated responses, including the accumulation of UV-B absorbing compounds and the DNA damage response, actually vary among various plant species. These factors eventually generate variations in UV-B response in plants (Hidema et al. 2007, Xu and Sullivan 2010). However, the variations in UV-B response in nonmodel crops and other field plant communities remain largely unexplored.

UV-B represents an energy-rich intrinsic component of solar radiation. It affects plant growth and development through diverse physiological and metabolic processes (Jenkins 2009). At high fluence rate, UV-B causes damage to the photosynthetic components (Correia et al. 1998), DNA (Schmitz-Hoerner and Weissenböck 2003), proteins and membranes (Bornman and Teramura 1993). The UV-B-induced photodimers (Taylor 2006), primarily cyclobutane pyrimidine dimers (CPDs) and pyrimidine-6,4-pyrimidinone dimers (6,4PPs) (Gill et al. 2015), create distortions in the DNA double-helical structure and eventually block transcription and replication (Britt 2004, Manova and Gruszka 2015). Furthermore, the inefficient repair of pyrimidine dimers before replication leads to the accumulation of double-strand breaks (DSBs) in DNA and oxidative damage (Britt 1999, Roy et al. 2011).

Oxidative stress plays a crucial role in modulating UV-B-related responses in plants (Lidon et al. 2012). The imbalance between the production of free radicals and the stimulation of reactive oxygen species (ROS) scavenging antioxidant activity results in the generation of oxidative pressure. This subsequently causes nonspecific oxidative damage to cellular biomolecules (Frohnmeyer and Staiger 2003, Pitzschke et al. 2006, Gill and Tuteja 2010). Furthermore, apart from the direct effect on DNA, high-energy photons of UV-B cause damage to plant cells through the formation of free radicals, mainly ROS, generated as typical byproduct of photo-excitation of certain intracellular components (Pristov et al. 2013). Excess intracellular ROS accumulation induces various forms of mutagenic oxidative DNA damage, including 8-oxo-G and 1,2-dihydro-2-oxoadenine (2-OH-A), respectively (Markkanen 2017). Oxidative lesions accumulate on both the DNA strands due to inefficient repair of these lesions via attempted base excision repair pathway under prolonged stress conditions. This situation activates the generation of potentially more harmful DNA lesions, including DNA single-strand breaks and DSBs (Zucca et al. 2013). Extensive accumulation and slower rates of repair of DSBs often cause loss of DNA segments, abnormalities in chromosome structure and, thus, generate significant genotoxic effects. Unrepaired DSBs inhibit DNA replication, generate replication stress and subsequently lead to cell cycle arrest. These factors eventually severely affect plant growth and development (Waterworth et al. 2011, Sedletska et al. 2013, Roy 2014, Graindorge et al. 2015).

Plant cells respond to genotoxic stress-induced DSBs, with mitotic escape and induction of endoreduplication cycle, suggesting a unique programmed mechanism for plants to survive under genotoxic stress (Barow and Meister 2003, Adachi et al. 2011). Compelling evidence indicates that endopolyploidy functions as an important cellular factor that correlates with organ size variation (Gegas et al. 2014). This also represents an important component of plant response to confer enhanced tolerance to UV-B radiation (280–320 nm) (Hase et al. 2006). The E2F transcription factor proteins act as an endocycle regulator and play key role in controlling cell proliferative activity in Arabidopsis (Vlieghe et al. 2005). Furthermore, involvement of CCS52A (Cell cycle Switch 52), a plant orthologue of yeast and animal cdh1/srw1/fzr genes, in the induction of endoreduplication has been demonstrated in plant cell. CCS52A causes altered degradation of mitotic cyclins, thus leading to the inhibition of M-phase and entry into the endoreduplication cycle from mitosis (Vinardell et al. 2003). However, comprehensive information on UV-B mediated response in plants, particularly in nonmodel tropical plant species, remains largely limited. Here, we demonstrate that exposure to low UV-B dose induces enhanced lateral root formation and DSB-mediated onset of endoreduplication in V. radiata seedlings. We further show that low UV-B-related phenotypical responses and induction of DSB-mediated endoreduplication are partly regulated through ROS generation. Moreover, transcriptome analysis reveals the network of co-regulated genes, including those involved in DNA damage response, cell cycle control, redox and phytohormone signaling in V. radiata following UVB exposure.

Results

High UV-B exposure negatively affects V. radiata germination and early seedling growth

To determine plant response to UV-B light during the early developmental stages, two different doses of UV-B, 4.0 and 8.0 kJ m⁻², were selected, representing low and relatively higher doses of UV-B, compared with the existing average daily natural dose (6.5 kJ m⁻², Panicker et al. 2014). To assess the growth response of V. radiata seeds to UV-B exposure, the germination frequency of water-imbibed seeds was initially determined at 24-h intervals up to 72 h (Supplementary Fig. S1A). The germination frequency decreased prominently during the course of UV-B exposure (both doses; Fig. 1A, B). Longer exposure time (90 min) to 4.0 kJ m⁻² of UV-B caused ∼42% inhibition in germination frequency (Fig. 1C) (P < 0.05). By contrast, 8.0 kJ m⁻² of UV-B caused significant inhibition (∼54% to 62%) of germination after 60- and 90-min exposure, respectively (Fig. 1C) (P < 0.01). UV-B exposure of seeds in the presence of supplemental white light reduced the inhibitory effect of UV-B on germination. This was more prominent with 4.0 kJ m⁻² of UV-B exposure (Supplementary Fig. S1B). Exposure of seeds to 8.0 kJ m⁻² of UV-B with supplemental white light for 60 and 90 min still caused ∼40% to 55% inhibition in seed germination (Supplementary Fig. S1C, D) (P < 0.01).

To assess the postgermination responses, seedlings germinated from untreated (control) and UV-B treated seeds were grown under long-day conditions (16-h light/8-h dark). Seven-day-old seedlings were then used for analysing the growth response. Hypocotyl length elongation was reduced to ∼2–2.3-fold when exposed to a 4.0 kJ m⁻² dose of UV-B for 60–90 min (Fig. 1F), while primary root growth was reduced by ∼1.6–2.1-fold under similar conditions (Fig. 1G). These responses were still detectable in the presence of supplemental white light (Supplementary Fig. S1E–H). Interestingly, lateral root formation enhanced conspicuously along with the increased time of exposure to low UV-B dose up to 60 min in comparison with the control condition (0 min exposure) (P < 0.01). This response subsequently declined with longer exposure periods (Fig. 1H). Lateral root formation was less prominent when a 4.0 kJ m⁻² dose of UV-B was applied in the presence of supplemental white light (Supplementary Fig. S1I). Nevertheless, 7-day-old seedlings grown from seeds exposed to 4.0 kJ m⁻² of UV-B showed no significant effect in fresh weights compared with control conditions (Fig. 1I). By contrast, significant inhibition of hypocotyl growth, primary root length and lateral root formation was detected in seedlings whose seeds were exposed to a 8.0-kJ m⁻² dose of UV-B. However, this effect was relatively diminished in the presence of supplemental white light (Supplementary Fig. S1G–I) (P < 0.01). Moreover, exposure to 8.0 kJ m⁻² of UV-B for longer duration caused prominent bending of the hypocotyl in seedlings. This response was reduced with supplemental white light (Supplementary Fig. S1F). Together, these results indicate a strong growth inhibitory effect of high UV-B dose. By contrast, moderate exposure to low UV-B dose caused less inhibitory effects but prominently enhanced lateral root formation.

Post-imbibitional exposure of V. radiata seeds to low doses of UV-B causes cell enlargement in the root apical region during early seedling growth

We next investigated the impact of UV-B exposure on growth response during the early stages of seedling growth. Although plant roots usually do not face direct UV-B exposure, considering the reduced primary root growth in seedlings raised from UV-B exposed seeds, we next analysed the possible effect of UV-B on primary root growth. Growth response of primary root epidermal cells close to the apical region in 7-day-old seedlings (grown from post-imbibitional UV-B light exposed seeds) was then studied. Roots of 7-day-old seedlings grown from 8.0 kJ m⁻² of UV-B exposed seeds showed increased disorganization at the root apical meristem region compared with the control and those exposed to low dose of UV-B (Supplementary Fig. S2A, I–III). These results indicate that exposure of seeds to high UV-B dose during early stages of germination after imbibition disrupts cellular integrity at the root apical meristem region. Consistent with this, compared with the control conditions and 4.0 kJ m⁻² of UV-B, root cell viability decreased significantly in 7-day-old seedlings grown from seeds exposed to 8.0 kJ m⁻² of UV-B for a longer duration (Supplementary Fig. S2B, I–IV, C, I–IV, D). On the other hand, we found a notable increase in cell area for cells positioned between 160 and 190 μm from the quiescent centre (QC) in seedlings grown from 4.0 kJ m⁻² of UV-B exposed seeds relative to the control condition (Fig. 2A, B). By contrast, this response was highly compromised of 8.0 kJ m² of UV-B (Fig. 2C, D). These results demonstrate that exposure to a low dose of UV-B mediates cell expansion in the smaller dividing cells at the root apical meristematic region. This response was also consistent with the cell expansion pattern observed in pregerminated (untreated) and UV-B-treated seedlings (at both 4.0 and 8.0 kJ m⁻² doses) (Supplementary Fig. S3A–D). Leaf epidermal cell area expansion was also detected in 7-day-old seedlings grown from seeds exposed to 4.0 kJ m⁻² of UV-B for 60–90 min (Supplementary Fig. S4B, F, I–IV). By contrast, exposure to high UV-B dose for 60 and 90 min appeared to disrupt the organization of leaf epidermal cell arrangement (Supplementary Fig. S4C, G, I–IV) without any noticeable expansion of leaf epidermal cell area. However, with both the UV-B doses, leaf epidermal cell number was decreased compared with the control (Supplementary Fig. S4D, E).

To further investigate the UV-B mediated response, 7-day-old pre-germinated V. radiata seedlings were exposed to 4.0 and 8.0 kJ m⁻² for 2 min or grown with supplemental UV-B light with either the daily dose 4.0 or 8.0 kJ m⁻² for 7 d. Compared with the control, low and high doses of UV-B treatment caused an increased accumulation of total UV-B absorbing compounds, particularly under longer exposure (Supplementary Fig. S5A, B). By contrast, total chlorophyll and carotenoid contents decreased in UV-B exposed or supplementary UV-B treated (both low and high UV-B doses) seedlings under similar treatment conditions (Supplementary Fig. S5C–F). Moreover, consistent with the previous observations (Tattini et al. 2000, Yan et al. 2012), UV-B exposure increased the trichome density on the leaf surface in 7-day-old seedlings but not in control (Supplementary Fig. S5G–I). This might be considered as the general protective morphological response. Interestingly, after longer exposure to low UV-B dose, trichomes showed enhanced ploidy levels compared with those exposed to the high UV-B dose (Supplementary Fig. S5J, K). Furthermore, V. radiata plants grown in the presence of supplemental low UV-B for 2 weeks showed only marginal difference in the fresh weights of aerial parts relative to the control. By contrast, the fresh weights of the aerial parts were compromised under supplemental high UV-B dose treatment (Supplementary Fig. S5L).

Longer exposure to UV-B induces DNA DSBs

To assess the direct genotoxic effects of UV-B exposure (Britt 2002), we next measured the level of induction and reduction of total CPDs in V. radiata seedlings formed due to UV-B exposure. CPD accumulation increased linearly with exposure time under both low and high UV-B doses compared with the control condition (Fig. 3A, B) (P < 0.01). However, CPDs increased prominently after 60 and 90 min of exposure to 8.0 kJ m⁻² compared with 4.0 kJ m⁻² of UV-B (Fig. 3B) (P < 0.01). Furthermore, the CPD recovery rate was notably slower after 8.0 kJ m⁻² UV-B treatment compared with the control and low UV-B dose (Supplementary Fig. S6A). These results indicate a strong genotoxic potential of high UV-B exposure and its possible impact on DNA repair activity.

Apart from oxidative damage to DNA (León-Chan et al. 2017), UV-B exposure also induces DNA strand breaks (Britt 1999, Roy et al. 2011, Biever and Gardner 2016), including DSBs. DSBs represent one of the most serious forms of DNA damage as it causes the loss of DNA fragments and significant genotoxic effects (Puchta 2005, Watanabe et al. 2018). We found a steady increase in DSB accumulation in 7-day-old V. radiata seedlings following exposure to both low and high UV-B doses. The effect was more prominent after 90 min of exposure to 8.0 kJ m⁻² of UV-B (Fig. 3C, D, H, I), with ∼1.4-fold higher DSBs compared to exposure with 4.0 kJ m⁻² of UV-B (Fig. 3H, I) (P < 0.01). Compared with the control, DSB accumulation was ∼2.6–4-fold and ∼4.1–5.4-fold higher after 60–90 min of exposure of seedlings to 4.0 kJ m⁻² (Fig. 3H, P < 0.01) and 8.0 kJ m⁻² (Fig. 3I) (P < 0.01) UV-B. In addition, seedlings exposed to 8.0 kJ m⁻² of UV-B showed a slower rate of DSB repair compared with either the low dose treatment or untreated control (Fig. 3J). Moreover, UV-B exposed (particularly with high UV-B dose) V. radiata seedlings showed significant accumulation levels of the phosphorylated form of H2AX (γH2AX, an immediate and highly conserved response for the induction of DSBs) (Friesner et al. 2005) (Fig. 3K, L), suggesting prominent genotoxic effect of high UV-B dose. These results could also be linked with the compromised growth response at 8.0 kJ m⁻² of UV-B (Fig. 1) due to induction of genome instability.

UV-B exposure activates DNA damage response through the SOG1-related ATM-ATR-mediated pathway

To investigate the UV-B mediated induction of DNA damage response (DDR), the accumulation levels of ataxia telangiectasia mutated (ATM) and ATM and Rad3-related (ATR) kinases (the two key regulators of the DNA damage response pathway) and the central DDR mediator, SUPPRESSOR OF GAMMA RESPONSE 1 (SOG1, a member of the NAC domain family transcription factor) (Bradbury and Jackson 2003, Yoshiyama 2016) was measured in UV-B exposed V. radiata seedlings. Immunoblot analyses showed enhanced accumulation of ATM (P < 0.01), ATR (P < 0.01) and SOG1 (P < 0.01) proteins with both low and high doses of UV-B treatment compared with control conditions (Fig. 4A–F, respectively). These observations (also confirmed in quantitative reverse transcription PCR, Supplementary Fig. S10) indicated UV-B mediated induction of DDR, involving the ‘classical’ SOG1 related ATM-ATR pathway. Following treatment with 4.0 kJ m⁻² of UV-B, ATM, ATR and SOG1 protein levels increased steadily along with exposure time. By contrast, 8.0 kJ m⁻² of UV-B caused an early induction of these proteins, reaching significantly high levels within 30 min of exposure, but then increased only marginally thereafter (Fig. 4D–F and Supplementary Fig. S9). Together, these results indicate an early induction of DDR after high UV-B exposure, which could be correlated to the enhanced sensitivity of V. radiata seedlings to high doses of UV-B (Supplementary Fig. S2A–D).

UV-B treatment compromises CYCB1 level and leads to cell cycle arrest

The cell cycle-specific cyclin-dependent protein kinases (CDKs) play a crucial role in regulating progression through the various phases of cell cycle in plants. The CYCB1;1/CDKB2;1 complex regulates cell cycle progression through the G2-M-phase and is essential for mitotic spindle formation. Like Arabidopsis, other genomes (of both monocotyledonous and dicotyledonous species) possess two family members of both CDKB1 (CDKB1;1 and CDKB1;2) and CDKB2 (CDKB2;1 and CDKB2;2), respectively, showing cell cycle phase-specific transcriptional regulation (Breyne et al. 2002, Menges et al. 2002). Molecular genetic studies using a dominant-negative allele have established that CDKB1 regulates progression through mitosis as reduced CDK1 activity leads to cell cycle arrest at the G2- to M-phase transition point (Porceddu et al. 2001). In Arabidopsis cell suspension culture, CDKB1;1 showed transcription peaks at the early G2 phase, indicating its function in the G2 phase of the cell cycle (Menges et al. 2002, Inzé and De Veylder 2006). Significant expression of CDKB1;1 in the progenitor cells of stomatal complexes also indicated its role in stomatal complex development (Boudolf et al. 2004). WEE1 protein kinase, another crucial and conserved component of the cell cycle machinery, also plays a key role in regulating the progression of cell division. WEE1 inhibits CDKB activity via phosphorylation and causes cell cycle arrest in response to DNA damage and replication stress (De Schutter et al. 2007, Dissmeyer et al. 2009, Waterworth et al. 2011). In this study, 7-day-old UV-B exposed (4.0 kJ m⁻² of UV-B) V. radiata seedlings showed decreased level of CDKB1;1 and CDKB2;1 protein accumulation with increasing time of UV-B exposure (Fig. 4G, H, Supplementary Fig. S9D, E) (p < 0.01). CYCB1;1 protein level also decreased under similar treatment condition compared with the control (Fig. 4I, Supplementary Fig. S9F) (p < 0.05). By contrast, WEE1 protein levels increased following exposure to 4.0 kJ m⁻² of UV-B compared with the control (Fig. 4J, Supplementary Fig. S9G) (P ≤ 0.01). These observations were consistent with the transcript abundance levels of these genes (Supplementary Fig S9). Taken together, the expression patterns of these key cell cycle regulators following low UV-B exposure suggest inhibition of cell division at the M-phase, leading to cell cycle arrest. However, the relative accumulation patterns of cell cycle regulators revealed early induction of cell cycle arrest following exposure to 8.0 kJ m⁻² of UV-B (Fig. 4K–N, Supplementary Fig. S9 A–G) (P ≤ 0.01).

UV-B exposure disrupts chromatin structure and induces histone modifications in V. radiata seedlings

We next analysed whether UV-B exposure affects the structural organization of chromatin in V. radiata seedlings. We performed micrococcal nuclease (MNase) assay to assess the degree of compaction of the chromatin following UV-B exposure (Moreno-Romero et al. 2012). In general, concentration kinetics and time kinetics of MNase activity on nuclear genomic DNA from UV-B exposed V. radiata seedlings indicated considerable DNA cleavage compared with the untreated control sample, and the effect was quite prominent after 8.0 kJ m⁻² of UV-B treatment (Fig. 5A, B). These results indicate that UV-B irradiation, apart from generating DSBs, alters chromatin structure.

In response to genotoxic stress and oxidative damage, altered histone methylation plays a crucial role in the regulation of DDR (Ayrapetov et al. 2014, Basenko et al. 2015, Questa et al. 2015). We found an increased signal of histone H3K9me3 (Fig. 5C–E) and H3K27me3 (Fig. 5F–H), in 7-day-old V. radiata seedlings exposed to low and high doses of UV-B, respectively (P ≤ 0.01) compared with control conditions. Accumulation of histone H3K9me3 and H3K27me3 was significantly higher with 8.0 kJ m⁻² dose of UV-B compared with the 4.0 kJ m⁻² dosage (Fig. 5C–H), suggesting the possibility of a transient repressive state of chromatin forming after longer exposure to high UV-B dose. In addition to methylation, acetylation of histone H3 and H4 also plays an important role in regulating DDR under genotoxic stress (Hunt et al. 2013). We found an increased H4K5 acetylation signal with 4.0 kJ m⁻² of UV-B, compared with the control, but this signal declined following longer exposure time (Fig. 5I, K). H4K5 acetylation levels decreased steadily along with exposure time under 8.0 kJ m⁻² of UV-B treatment (Fig. 5J, K). Increased H4K5 acetylation signal with 4.0 kJ m⁻² of UV-B indicated dynamic alteration in the chromatin structure, while reduced H4K5 acetylation levels with 8.0 kJ m⁻² of UV-B again suggests a repressive state of the chromatin structure, consistent with the strong genotoxic effect of high UV-B dose.

V. radiata response to UV-B exposure leads to endoreduplication

Previously, multiple studies in Arabidopsis have shown that genotoxic stress-mediated induction of endoreduplication represents an important component of plant response mechanisms (Barow and Meister 2003, Hase et al. 2006, Adachi et al. 2011). Based on this, we next investigated the possibility of whether UV-B mediated accumulation of DSBs along with the activation of DDR and cell cycle arrest (Figs. 3, 4) induces endoreduplication in V. radiata seedlings. Flowcytometric analysis of nuclear DNA content from leaves of 7-day-old seedlings grown from UV-B treated seeds showed a polypoid population of nuclei with prominent peaks at 4C, 8C, 16C and 32C with 60 min of 4.0 kJ m⁻² of UV-B exposure after imbibition, compared with the nonirradiated control (Fig. 6A, I–IV, B; Supplementary Table S1). Leaves of 14-day-old plants grown from seeds exposed to 4.0 kJ m⁻² of UV-B also showed polyploid nuclear populations with 8C and 16C DNA content, respectively (Fig. 6C, I–IV, D; Supplementary Table S1). Leaves from both 7- and 14-day-old seedlings (grown from seeds exposed to 4.0 kJ m⁻² UV-B dose for 60 min) showed prominent polyploid peaks at 4C and 8C level (Fig. 6A, C). Interestingly, for seeds exposed to 4.0 kJ m⁻² UV-B for 60 min, the pattern of polyploid nuclear population peaks found in 7-day-old seedlings was maintained at 14- and 21-day-old stages (Fig. 6C, Supplementary Fig. S11). Conversely, with 8.0 kJ m⁻² of UV-B dose, most of the nuclear populations were found in the 2C and 4C stages. Polyploid nuclear DNA peaks were less prominent in the leaves of both 7- and 14-day-old plants with high UV-B dose (Fig. 7A, I–IV, C, I-IV). Together, these results indicate effective induction of endoreduplication after longer exposure to low UV-B doses, while high UV-B dose compromises this response. Furthermore, it was also interesting to note that as an early response to low UV-B dose, the onset of the UV-B-mediated endoreduplication response was quite prominent and dramatic in the V. radiata model, which appeared to enter from the basal 2C–4C level in control conditions and then up to 32C after UV-B treatment (Fig. 6A, I–IV and B). By contrast, as indicated in previous studies, Arabidopsis leaves with 16C levels did not show the addition of any new ploidy levels (Gegas et al. 2014). Together, these results suggest that V. radiata appears to be more reactive compared with the Arabidopsis model in terms of induction of UV-B mediated endoreduplication.

To further validate the onset of endoreduplication following low doses of UV-B exposure, 7-day-old seedlings (germinated from nonirradiated control seeds) were exposed to 4.0 kJ m⁻² of UV-B for 2 min/day in the dark for consecutive 7 d and then grown under long-day conditions for another 7 d. Leaves of 21-day-old UV-B exposed plants showed clear polyploid nuclear population peaks at 4C, 8C and 16C positions compared with the control condition, while such response was significantly less in those exposed to 8.0 kJ m⁻² of UV-B (Supplementary Fig. S11B, C), again demonstrating prominent induction of endoreduplication following exposure to low UV-B dose.

To determine the link between UV-B mediated activation of DDR via SOG1 related ATM-ATR pathway and the onset of endoreduplication, we next utilized Arabidopsis thaliana atm-2, atr-2 and sog1-1 mutant lines due to the unavailability of well-characterized respective mutant lines in V. radiata. We found root epidermal cell area enlargement in wild-type, atm-2 and atr-2 seedlings upon UV-B exposure (seeds exposed to 4.0 kJ m⁻² of UV-B for 60 min) (Supplementary Fig. S14A, I–VIII). The response was less prominent in atm-2 and atr-2 mutant lines, while sog1-1 mutant seedlings showed only marginal enlargement in root epidermal cell area compared with the untreated control seedlings (Supplementary Fig. S14A, V–VIII). Flowcytometric analyses showed insignificant increases in polyploid nuclear populations in atm-2, atr-2 and sog1-1 mutant lines compared with the wild-type Arabidopsis exposed to 4.0 kJ m⁻² of UV-B (Supplementary Fig. S14D–K). Together, these results indicate that UV-B mediated activation of DDR involves the classical SOG1 related ATM-ATR pathway and is coupled with the onset of endoreduplication.

Since UV-B exposure caused increased accumulation of ATR (activated under replication stress) and cell cycle arrest in V. radiata seedlings (Fig. 4), we next investigated whether replication stress plays any role in UV-B mediated onset of endoreduplication. V. radiata seedlings, when treated with 2 mM HU (hydroxyurea, efficiently blocks replication) showed only marginal increase, if any, in root epidermal cell area and accumulation of DSBs compared with the control (Supplementary Fig. S15A–G). Furthermore, the relative proportion of polyploid nuclear population was less with 2 mM HU treatment compared with the UV-B treated seedlings (UV-B 4.0 kJ m⁻² for 60 min). Based on these results, we argued that the marginal level of cell area enlargement observed in HU-treated (2 mM) samples might be due to DSBs arising from HU-mediated replication stress. Together, these results demonstrate a crucial role of UV-B mediated accumulation of DSBs in the induction of endoreduplication, while replication stress possibly evokes an indirect effect through the accumulation of DSBs.

UV-B mediated accumulation of FZR1/CCS52A and E2Fa protein levels facilitates the onset of endoreduplication in V. radiata seedlings

In plants, the homologs of Fizzy-Related (FZR) family proteins, including Cell Cycle Switch 52A and B (CCS52A and CCS52B) play an important role in the induction of endoreduplication (Cebolla et al. 1999). We further detected an increased accumulation of FZR1/CCS52A protein level in the leaves of 7-day-old V. radiata seedlings exposed to 4.0 kJ m⁻² of UV-B light for different time points relative to the untreated control condition (Fig. 8A, B). CCS52A transcript accumulation level was also consistent with the protein level under similar conditions (Fig. 8C). Together, enhanced expression of CCS52A with low UV-B treatment was consistent with the onset of endoreduplication after exposure to a low dose of UV-B. However, at 8.0 kJ m⁻² of UV-B, both the protein and transcript levels of FZR1/CCS52A, after the initial increased accumulation, remained relatively unchanged along with the exposure time (Fig. 8A–C).

In plant genomes, apart from CCS52A, the E2F proteins also play an important role in the regulation of onset of endoreduplication by inducing the expression of S-phase related genes, thus facilitating additional DNA replication events after being released from RETINOBLASTOMA-RELATED (RBR) repression (De Veylder et al. 2002, Magyar et al. 2012). Among the three types of E2F/DP (dimerization partner) transcription factors present in the Arabidopsis thaliana genome (E2Fa, E2Fb, and E2Fc), E2Fa and E2Fb act as the positive regulator of cell division and related gene expression (Kosugi and Ohashi 2003). E2Fc acts as transcriptional repressor and inhibitor of cell division (Del Pozo et al. 2002). We found an early increase in E2Fa protein accumulation level following exposure to 4.0 kJ m⁻² dose of UV-B, and then it declined after longer exposure in 7-day-old V. radiata seedlings (Fig. 8D, E). By contrast, E2Fa protein, after the initial marginal decrease (30 min), did not show any significant change afterward with 8.0 kJ m⁻² of UV-B exposure (Fig. 8D, E). This was also consistent with the transcript abundance pattern (Fig. 8F). Taken together, these results elucidate that after exposure to low UV-B, increased accumulation of FZR1/CCS52A along with the initial increase in E2Fa protein facilitate effective increase in DNA replication. On the other hand, concomitant enhanced accumulation of WEE1 protein (Figs. 4, 8) causes cell cycle arrest, thus inducing endoreduplication.

Low dose of UV-B exposure mediated ROS accumulation enhances lateral root formation in V. radiata seedlings

Previous studies have shown the important effects of exposure to low UV-B dose and the associated ROS generation in the regulation of morphological responses in plants (Wan et al. 2018) through interaction with the phytohormone signaling cascade (Frohnmeyer and Staiger 2003, Orman-Ligeza et al. 2016). Within this context, 3-day-old V. radiata seedlings exposed to low and high UV-B doses showed a significant increase in UV-B mediated ROS accumulation (also H₂O₂ content in the roots) compared with the control conditions (Supplementary Fig. S16A–F) (p < 0.01) (Supplementary Fig. S16G–J). As expected, the response was more intense following exposure to a high dose of UV-B. We next investigated whether UV-B induced ROS generation plays any role in regulating the low dose of UV-B mediated phenotypic responses in V. radiata seedlings. Seeds treated with 10 µM solution of diphenyleneiodonium (DPI, a potential and specific inhibitor of NADPH oxidase and thus ROS generation) during imbibition, followed by a low dose of UV-B exposure showed ∼1.2-fold less germination frequency in comparison with the control condition (without DPI treatment) (Fig. 9A, C). Moreover, 7-day-old seedlings grown from seeds exposed to 4.0 kJ m⁻² of UV-B in the presence of DPI, apart from having shorter hypocotyl and primary root growth (Fig. 9B, E), showed significantly compromised lateral root formation compared with the control (UV-B exposure without DPI) (P < 0.01) (Fig. 9D). ROS accumulation levels in 3-day-old UV-B (4.0 kJ m⁻²) exposed V. radiata seedlings in the presence of 10 μM of DPI was significantly reduced (Fig. 9F, G), indicating that DPI treatment specifically inhibits UV-B mediated ROS accumulation in V. radiata seedlings. A similar effect was observed when we used ascorbic acid, which acts as a general ROS scavenger (Supplementary Fig. S17A–L) (Qian et al. 2014). Exogenously applied H₂O₂ (used as a positive control for ROS induction) also showed enhanced lateral root formation relative to the untreated control (Supplementary Fig. S18A, B). Taken together, these results indicate that ROS accumulation after exposure to low UV-B doses at least partly regulates enhanced lateral root formation in V. radiata seedlings.

Treatment with ROS inhibitor compromises V. radiata seedling response to low UV-B levels

To further investigate the role of UV-B induced ROS accumulation, other UV-B responses were then analysed following DPI treatment. DPI treatment appreciably reduced the cell area enlargement of root apical meristem epidermal cells near the QC region in roots of 7-day-old seedlings grown from seeds exposed to low doses of UV-B (Fig. 9L, M). Furthermore, DPI treatment notably reduced the DSBs and the cell population of polyploid nuclear DNA content in the roots and leaves of 7-day-old V. radiata seedlings grown from seeds exposed to low doses of UV-B in the presence of DPI (Fig. 9J, K, Supplementary Fig. S19). Taken together, these results provide an important clue to indicate that apart from the direct damaging effect on DNA, UV-B mediated ROS contribute appreciably in the induction of DSBs via oxidative damage and endoreduplication under a low dose of UV-B. By contrast, excess ROS accumulation and oxidative damage with longer exposure to low or high UV-B dose appears to reduce cell viability (Fig. 9F, G).

Transcriptome analysis reveals coordinated involvement of DNA damage repair, cell cycle regulation and redox signaling pathways in V. radiata upon UV-B exposure

Considering the enhanced lateral root formation and DSB-mediated induction of endoreduplication in V. radiata seedlings following exposure to low doses of UV-B, we next performed transcriptome analysis using RNA samples from roots of 3-day-old seedlings grown from control and 4.0 kJ m⁻² UV-B exposed seeds. Transcriptome and DEG analyses revealed 1,137 significantly differentially expressed genes, among which 671 were upregulated and 466 were downregulated. An overview of metabolic pathway, regulation, cell function and cellular responses exhibited that secondary metabolic pathway, DDRs, cell cycle regulation, phytohormones related responses and stress responses are the major processes triggered in response to UV-B treatment in roots of 3-day-old V. radiata seedlings (Supplementary Fig. S24A–E). Consistent with these observations, quantitative real-time PCR analyses confirmed upregulated expression of DNA repair protein RAD51 homolog (XR_002667491.1), DNA ligase 4 (XM_014646053.2), while DNA mismatch repair protein MutS2 (XM_014654405.2), Cyclin-dependent Kinase F-4 like (XM_014653758.2) and Cyclin D1-1 (XM_014668254.2) showed reduced expression. On the other hand, phospholipase D Z (XR_002667890.1), Auxin response factor 8 (XM_014663075.2), Ethylene-responsive transcription factor 13 (XM_022785891.1) showed upregulation. Among the transcription factors, probable WRKY transcription factor 53 (XM_014666894.2), NAC domain-containing protein 72 (XM_014635847.2) showed clear upregulation, while MADS-box transcription factor 23-like isoform X1 (XM_022787033.1) showed reduced expression with low UV-B dose treatment (Supplementary Fig. S24F).

To identify the protein interaction network among the selected DEGs, the major genes, with Arabidopsis homologs were screened using the STRING database and modified in Cystoscape. The association network of SOG1 (suppressor of gamma response 1) interacting proteins from the DEGs showed how SOG1 mediated repair pathway elements connected to other UV-B stress responses, such as cell cycle regulation and hormonal regulation related to stress responses. From the entire network, RRTF1 (redox responsive transcription factor 1) was identified as an interactor of multiple proteins related to UV-B mediated responses. In addition, EFS (early flowering in short days) was identified as interactors of FLD (flowering locus D), HUB2 (histone monoubiquitylation 2) and SDG2 (set domain group 2) (Fig. 10). Most of the interactors identified in this network have not been investigated in UV-B-mediated responses, to date, but given their comparable scores as bona fide interactors, these are expected to perform a vital role in UV-B-mediated responses in plant genomes.

Discussion

UV-B light, particularly low levels of UV-B, acts as an important environmental regulator and plays key role in regulating plant gene expression, cellular and metabolic processes, growth and development (Brosché and Strid 2000, Jenkins 2009, Rodríguez-Calzada et al. 2019). In this study, we further showed that exposure to a low dose of UV-B promotes endoreduplication in V. radiata seedlings by regulating the key components of DDR and cell cycle regulatory networks. In addition, low dose UV-B-mediated responses at the phenotypic and cellular levels appeared to be partly modulated through UV-B induced ROS generation. Conversely, exposure to high UV-B dose caused loss of cell viability (Fig. 11). Overall, this study unveils how exposure to low and high UV-B doses affect growth response in a nonmodel tropical plant species V. radiata, by integrating the UV-B mediated observed phenotypical responses at the cellular and molecular levels.

In Arabidopsis, WEE1 acts as a negative regulator of the cell cycle (De Schutter et al. 2007) and is one of the crucial targets of ATR-ATM signaling cascades, regulating cell cycle arrest upon induction of DNA damage (Eckardt 2007). Previous studies also indicated an increased abundance of endogenous WEE1 mRNA during the onset of endoreduplication in maize endosperm (Sun et al. 1999) and tomato fruit (Gonzalez et al. 2007). In developing tomato fruits, WEE1 regulates cell size and induces endoreduplication (Gonzalez et al. 2007). In Arabidopsis, TCP15 (CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF) transcription factor regulates endoreduplication via modulating the expression of positive regulators of endoreduplication, including CDT1A, CCS52A1 and WEE1, respectively (Li et al. 2012). We found an increased accumulation of WEE1 along with the induction of DDR, cell cycle arrest and the concomitant onset of endoreduplication in V. radiata seedlings following low UV-B exposure. Together, these results point to the function of WEE1 in cell cycle arrest and induction of endoreduplication via the SOG1 mediated ATM-ATR pathway (Figs. 4, 11). The pattern of root apical meristem epidermal cell area expansion and endoreduplication in UV-B treated wild-type, atm-2, atr-2 and sog1-1 mutant lines in Arabidopsis also supported the involvement of both ATM-SOG1 and ATR-SOG1 pathways in the induction of UV-B mediated DDR and endoreduplication (Supplementary Fig. S14).

Previous studies showed that M-phase CDK activity increases after the completion of S-phase, allowing further progression of the cell cycle through to G2 phase (Trovesi et al. 2013). CDKs directly activate the HR-mediated DSB repair, while CYCB1;1 (regulatory counterpart of mitotic CDKs) shows upregulated expression in response to genotoxic stress or due to disruption of chromosomal organization (Chen et al. 2003, Culligan et al. 2004, Liu et al. 2010). In Arabidopsis, SOG1 activates CYCB1;1 upon induction of DNA damage. This specifically activates the HR pathway by the plant-specific CDKB1-CYCB1 complex via phosphorylation of RAD51 (RADIATION SENSITIVE 51, a core component of HR-related DSB repair pathway). Interestingly, cycb1 and cdkb1 mutants did not display any strong induction of endoreduplication in the presence of the genotoxic agent cisplatin (Weimer et al. 2016). However, considering the previous observations of zeocin-induced DSBs and onset of endoreduplication in Arabidopsis root cells reported in multiple studies (De Veylder et al. 2002, Adachi et al. 2011, Edgar et al. 2014), the long-term effect of cisplatin in the induction of endoreduplication was not completely ruled in this study (Weimer et al. 2016). Within this context, in our study, we found a clear pattern of increased accumulation level of CCS52A and an initial increase in E2Fa protein level. This was associated with the steady-state increase of WEE1 in V. radiata seedlings after low UV-B exposure (Figs. 4, 6). By contrast, CCS52A, E2Fa and WEE1 protein levels were reduced following longer exposure to low UV-B (Fig. 4). Together, these results indicate the interplay in maintaining regulated expression and accumulation of the M-phase related key cell cycle regulators (CYCB1;1, CDKB2;1, CYCB1;1) and of positive modulators of endoreduplication (CCS52A, E2Fa and WEE1) for the induction of cell cycle arrest and endoreduplication after low UV-B exposure (Figs. 4, 7). On the other hand, higher levels of DSBs and ROS accumulation reduced cellular integrity and possibly promoted cell death (Supplementary Fig. S16).

Low levels of UV-B and the associated ROS generation play an important role in regulating plant growth responses (Jansen et al. 2012, Robson et al. 2015, Orman-Ligeza et al. 2016, Wan et al. 2018). We observed significantly reduced lateral root formation in V. radiata seedlings grown from UV-B (4.0 kJ m⁻²) treated seeds (Fig. 9) in the presence of DPI. Moreover, besides reduction in UV-B-mediated cell enlargement in root apical regions and leaf mesophyll cells, the induction of DSBs and the concomitant onset of endoreduplication were also appreciably compromised following DPI treatment (Fig. 9, Supplementary Fig. S19). Together, these observations suggest the involvement of ROS signaling at least in part in regulating the UV-B-mediated responses in V. radiata seedlings. This notion was also consistent with the results obtained from DEG analysis, which showed upregulation of RESPIRATORY BURST OXIDASE HOMOLOG (RBOH) protein B (XM_014663136.2) and phospholipase D Z, XM_014636502.2 (confirmed in Q-RT-PCR analysis), which are involved in ROS signaling (particularly RBOH in lateral root formation, Orman-Ligeza et al. 2016). Conversely, peroxidase A2 (XM_014662966.2) and folate receptor-like (XM_014658186.2) showed downregulated expression, probably for maintaining the UV-B induced ROS signal level (Supplementary Table S2 and Supplementary Fig. S19). Furthermore, DEG analyses revealed upregulation of the genes involved in auxin responses with a low dose of UV-B (Supplementary Table S2 and Supplementary Fig. S24). By contrast, some key cell cycle regulators showed downregulated expression (Supplementary Table S2). Together, these results suggest that the effects of UV-B mediated reduced expression of the core cell cycle regulators possibly override the auxin-related signaling, leading to cell cycle arrest and facilitated induction of endoreduplication.

Understanding the ecological and evolutionary roles of endoreduplication in the modulation of overall fitness and response to environmental stress in plants, in general, requires large-scale survey and analysis of endopolyploidy in natural populations of plants, including both crops and nonmodel plant species. This will help to further decipher the impact of environmentally triggered endoreduplication on the adaptive potential of natural plants populations (Neiman et al. 2017). The occurrence of endoreduplication is widespread among various plant species (Barow 2006) and has been connected in part to stress response with an important role in plant adaptation to environmental changes. However, its possible role in plant development and its adaptive significance remains largely unclear. In addition, information also remains limited regarding the possible role of endoreduplication in regulating plant growth following UV-B radiation in natural populations and nonmodel crops. In this context, our study has provided comprehensive information on low and high dose of UV-B mediated responses in the tropical legume crop V. radiata. Our results demonstrated that compared with the basal C levels of 2C or 4C nuclear DNA in the nonirradiated control, up to 32C content was observed in the leaves of V. radiata seedlings grown from UV-B irradiated seeds. By contrast, Arabidopsis leaves, already containing ploidy levels up to 16C, appeared to increase the proportion of 16C but without the addition of any new ploidy levels (Gegas et al. 2014). Therefore, our results demonstrate that UV-B mediated endoreduplication response is quite dramatic in the tropical legume crop V. radiata compared with the Arabidopsis model. Together, this information also encourages the potential utilization of V. radiata and other related nonmodel species for better understanding the mechanism of environmental factors mediated induction of DSB responses and endoreduplication to eventually decipher how endoreduplication affects the adaptive potential of crops and other natural populations of plants particularly in tropical climates.

Materials and Methods

Plant materials, growth conditions, UV-B treatment and quantitative plant growth measurement

Vigna radiata (L.) R. Wilczek cv. B1 seeds were obtained from IARI, Pusa, New Delhi, India. Surface sterilized seeds were imbibed in water for overnight. Next day, seeds were plated on sterile Petri plates with double layers of Whatman No.1 filter paper. UV-B treatment (post-imbibition) during germination was performed by following the method described previously (Hase et al. 2006, Roy et al. 2011, Singh et al. 2016). Seeds were exposed to UV-B light in a chamber equipped with a UV-B lamp source (UV-B bulb, Philips) radiating at a wavelength of >280 nm with a high peak at 312 nm. Seeds were exposed to either white light (control) or various doses of UV-B radiation (4.0 kJ m⁻² and 8.0 kJ m⁻² of UV-B without or with supplemental white light) for different time points (15, 30, 60 and 90 min, respectively). During UV-B treatment, the lid of Petri plate was removed, while the humidity was maintained between 70–75% levels. After UV-B exposure, seeds were kept in the dark at room temperature for 8 h and then transferred to light chamber for germination at 30°C under long-day conditions (16 h light/8 h dark period). Plates were irrigated daily with water and the temperature was maintained at 28°C along with the relative humidity of ∼70–75% (Singh and Padmakar 1991). Germination frequency was determined 2 d after germination and after 24 h interval for 3 d. Hypocotyl and primary root lengths, lateral root formation and plant fresh weight were measured in 7-day-old seedlings. UV-B treatment at seedling stage was performed following Hase et al. (2006).

Propidium iodide staining and cell viability assay

Roots of 7-day-old V. radiata seedlings grown from control or UV-B exposed seeds for different time points were stained with propidium iodide and stained samples were observed by using a confocal laser scanning microscope (Supplementary information). Root apical meristem cell area and distance from QC were measured using MBF ImageJ software by tracing the outline of the cells (Adachi et al. 2011). Cell viability in control and UV-B treated V. radiata root samples was carried out using the Trypan blue exclusion method with some modifications (Díaz-Tielas et al. 2012).

Measurement of leaf cell number and area

UV-B mediated changes in leaf cell area and density were measured by the method described previously (Gegas et al. 2014) (Supplementary information).

Flow cytometry

Flowcytometric analysis of nuclear DNA content was carried out by following the procedure described previously (Pal et al. 2004, Hase et al. 2006) with minor modifications. Nuclei were isolated from roots and the first and/or second true leaves of various aged (7–21 d) untreated control and UV-B exposed V. radiata seedlings and subsequently used for flowcytometry (Supplementary information).

ROS quantification and imaging

Quantitative estimation of total ROS was performed using root and leaf tissues from 7-day-old control and UV-B treated (both 4.0 and 8.0 kJ m⁻²) V. radiata seedlings. Details of imaging and DPI (diphenyleneiodonium chloride) treatment procedures are described under Supplementary information.

Analysis of H₂O₂ accumulation and localization

Total H₂O₂ content from control and UV-B treated 3-day-old V.  radiata seedlings were determined following the method described earlier with minor modifications (Velikova et al. 2000) (Supplementary information).

ELISA

The amount of total CPDs formed due to UV-B exposure was measured by ELISA with anti- CPD antibody (Roy et al. 2011). Seven-day-old seedlings grown under long-day conditions (16 h light/8 h dark) were exposed to 4 and 8.0 kJ m⁻² of UV-B doses for different time points in the dark condition and subsequently used for genomic DNA extraction for ELISA (Supplementary information).

Comet assay

The extent of UV-B-induced nuclear DNA damage in untreated control and UV-B exposed seedlings were detected by analysing the accumulation of DSBs using comet assay carried out under neutral condition (Roy et al. 2011) (Supplementary information).

Preparation of total protein extracts and immunoblotting

Total protein was extracted from ∼500 mg of control or UV-B-treated plant tissue samples by following the protocol described previously (Roy et al. 2007). Details of protein extraction, immunoblotting procedure and validation of specificity and cross-reactivity of the antibodies have been described under Supplementary information.

Histone protein extraction and detection of histone modifications

Extraction of histone protein was performed following the method described previously (Lang et al. 2012, Mahapatra et al. 2019). Immunoblotting was performed using anti-γH2AX (Sigma), anti-H3K9me2 (SCBT), anti-H3K27me2 (MERCK) and anti-H4K5ac (SIGMA) as the primary antibodies, respectively. Goat anti-rabbit IgG (alkaline phosphatase conjugated, Thermo Scientific) was used as the secondary antibody. The immune positive bands on the membrane were detected by using BCIP/NBT developer solution as substrate (Sambrook et al. 1989).

MNase assay

For the assessment of chromatin stability following exposure to UV-B, a micrococcal nuclease assay was performed following the procedure described previously (Moreno-Romero et al. 2012). Details of the procedure have been described under Supplementary information.

Extraction of chlorophyll, carotenoids and UV-B absorptive compounds

UV-B absorptive compounds were extracted from nonirradiated and UV-B treated V. radiata seedlings by following the method described previously (Hase et al. 2006) (Supplementary information).

RNA-Seq analysis

For RNA-seq analysis RNA was extracted from the root tissues of 3-day-old seedlings grown from untreated control and UV-B treated (4.0 kJ m⁻², 60 min exposure) seeds as three biological replicates. RNA-seq library was prepared using TruSeq RNA Library Prep Kits for Illumina® (NEB, USA). Details of the procedure and analyses have been described under Supplementary information.

Statistical analysis

The data were expressed as the mean of three independent replicates and were analyzed by using one-way ANOVA. P < 0.05 and P < 0.01 were considered significant. Error bars indicate ±SD. Statistical analyses were performed using Microsoft Excel 2016.

Supplementary Data

Supplementary data are available at PCP online.

Data Availability

All relevant data can be found within the manuscript and its supporting materials.

Funding

The authors gratefully acknowledge Council of Scientific and Industrial Research (CSIR), Govt. of India (Ref. No. 38(1417)/16/EMR-II, dated: 17/05/2016 to S.R.), SERB, DST, Govt. of India (Ref. No. ECR/2016/000539 to S.R.), SERB, DST, Govt. of India (Ref. No. SRG/2019/000901 to S.R.C.) and Ramalingaswami Re-entry Fellowship (Ref. No. BT/RLF/Re-entry/01/2018 to S.R.C.) for providing necessary financial supports. S.D. is the recipient of fellowship from the SERB, DST, Govt. of India (Ref. No. ECR/2016/000539) funded project. J.J. acknowledges the CSIR, Government of India for Ph.D. fellowship.

Acknowledgements

Authors acknowledge University Science Instrumentation Centre, University of Burdwan (Fluorescence microscope), West Bengal, India, Central Instrument facility, Bose Institute, Kolkata, India (Confocal microscope) and Centre for Research in Nanoscience and Nanotechnology, Calcutta University, Kolkata, India (BD- Flow cytometer) for providing necessary technical support. The authors also thank Prof. K.P. Das, former Professor, Department of Chemistry, Bose Institute Kolkata, for the language corrections and editing and Mr. Kalyan Mahapatra for supporting in the statistical analysis of the data.

Author Contributions

S.R. conceived the idea and designed the experiments. S.D. performed the experiments. S.R., A.P., S.R.C., S.D. and J.J. analysed the data; S.R., A.P., S.R.C. and S.D. wrote the manuscript.

Disclosures

The authors declare that they have no competing interest.

References