This is the published version of a paper published in Physiologia Plantarum: An
International Journal for Plant Biology.
Citation for the original published paper (version of record):
Sáenz-de la, O D., Morales, L O., Strid, Å., Torres-Pacheco, I., Guevara-Gonzáles, R G.
(2021)
UV#B exposure and exogenous hydrogen peroxide application lead to cross#tolerance
toward drought in Nicotiana tabacum L.
Physiologia Plantarum: An International Journal for Plant Biology
https://doi.org/10.1111/ppl.13448
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S P E C I A L I S S U E A R T I C L E
Ultraviolet-B exposure and exogenous hydrogen peroxide
application lead to cross-tolerance toward drought in
Nicotiana tabacum L.
Diana Saenz-de la O
1|
Luis O. Morales
2|
Åke Strid
2|
Irineo Torres-Pacheco
1|
Ramon G. Guevara-Gonzalez
11
Biosystems Engineering, School of Engineering, Autonomous University of Queretaro-Campus Amazcala, Querétaro, Mexico 2
School of Science and Technology, Örebro University, Örebro, Sweden
Correspondence
Åke Strid, School of Science and Technology, Örebro University, Örebro, Sweden. Email: ake.strid@oru.se;
Ramon G. Guevara-Gonzalez, Biosystems Engineering, School of Engineering, Autonomous University of Queretaro-Campus Amazcala, Querétaro, Mexico.
Email: ramon.guevara@uaq.mx Funding information
Consejo Nacional de Ciencia y Tecnología, Grant/Award Numbers: 283259, 707895; Faculty for Business, Science and Technology at Örebro University; Stiftelsen för Kunskaps-och Kompetensutveckling, Grant/Award Number: 20130164; Svenska Forskningsrådet Formas, Grant/Award Number: 942-2015-516 Edited by: F. Pescheck
Abstract
Acclimation of plants to water deficit involves biochemical and physiological
adjust-ments. Here, we studied how ultraviolet (UV)-B exposure and exogenously applied
hydrogen peroxide (H
2O
2) potentiates drought tolerance in tobacco (Nicotiana
tabacum L. cv. xanthi nc). Separate and combined applications for 14 days of
1.75 kJ m
2day
1UV-B radiation and 0.2 mM H
2O
2were assessed. Both factors,
individually and combined, resulted in inhibition of growth. Furthermore, the
com-bined treatment led to the most compacted plants. UV-B- and UV-B
+ H
2O
2-treated
plants increased total antioxidant capacity and foliar epidermal flavonol index. H
2O
2-and UV-B
+ H
2O
2-pre-treated plants showed cross-tolerance to a subsequent 7-day
moderate drought treatment, which was assessed as the absence of negative impact
on growth, leaf wilting, and leaf relative water content. Plant responses to the
pre-treatment were notably different: (1) H
2O
2increased the activity of catalase
(EC 1.11.1.6), phenylalanine ammonia lyase (EC 4.3.1.5), and peroxidase activities
(EC 1.11.1.7), and (2) the combined treatment induced epidermal flavonols which
were key to drought tolerance. We report synergistic effects of UV-B and H
2O
2on
transcription accumulation of UV RESISTANCE LOCUS 8, NAC DOMAIN PROTEIN 13
(NAC13), and BRI1-EMS-SUPPRESSOR 1 (BES1). Our data demonstrate a
pre-treat-ment-dependent response to drought for NAC13, BES1, and CHALCONE SYNTHASE
transcript accumulation. This study highlights the potential of combining UV-B and
H
2O
2to improve drought tolerance which could become a useful tool to reduce
water use.
1
|
I N T R O D U C T I O N
Extreme weather events limit plant production, which, in turn, may affect agricultural important plant species resulting in reduced food production. In addition, the frequency of such extreme events is likely to increase as climate change worsens (Lesk et al., 2016). Therefore,
plant drought tolerance is an important trait for which we need to develop a more complete understanding at the physiological and molecular level (Godfray et al., 2010). Drought stress diminishes crop growth, disturbs plant water and plant nutrient relations, reduces pho-tosynthesis, and causes oxidative damage due to the generation of reactive oxygen species (ROS) (Salehi-Lisar and
Bakhshayeshan-Physiologia Plantarum
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Agdam, 2016). Thus, plant drought tolerance is a complex process that involves osmotic adjustment, osmoprotection, and antioxidant activity in the form of an ROS scavenging defense system (Farooq et al., 2009). In addition, some chemical and physical stressors that at high concentra-tions cause toxicity may at low or moderate concentraconcentra-tions play a posi-tive role by pre-conditioning plants against the negaposi-tive influence of other abiotic factors, such as drought (Vazquez-Hernandez et al., 2019). Promoting tolerance to a strong stress by a different type of mild stress, applied either simultaneously or sequentially, is referred to as cross-tolerance (Dhanya Thomas et al., 2020; Hossain et al., 2018). Cross-tolerance against stress may involve an intricate pattern of activation of stress-responsive pathways leading to changes in morphology, anatomy, physiology, and biochemistry (Hossain et al., 2013).
The ultraviolet (UV) region of the electromagnetic spectrum has been conventionally defined as UV-C (<280 nm), UV-B (280–315 nm), and UV-A (315–400 nm) (Björn 2015). However, more recent studies argue that divisions of the UV waveband should rather consider absorption and physiological functions of UV photoreceptors when assessing plant responses to UV radiation (Rai et al., 2020; Rai et al., 2021), emphasizing UV wavelengths below (UVsw) and above (UVlw) 350 nm. Notwithstanding, approximately 95% of UV radiation reaching the Earth's surface is UV-A, whereas UV-C and UV-B radia-tion below 290 nm are absorbed by ozone in the stratosphere. UV-B, despite being only a small proportion of the UV spectrum, has the potential of altering plant morphology and metabolism (Jenkins 2009). Previous studies have reported that UV-B stress has negative effects on photosynthesis, plant growth, and biomass accumulation (Albert et al., 2011; Frohnmeyer and Staiger, 2003; Jansen et al., 1998; Jor-dan 2002). However, research of the last decades has shown that ambient levels of UV-B, perceived through the UV-B photoreceptor UV RESISTANCE LOCUS 8 (UVR8), regulate photomorphogenesis and defense responses, including photo repair processes, antioxidant activities, and UV-screening (Jansen and Bornman, 2012; Jenkins 2009; Morales et al., 2013; Rizzini et al., 2011). In this sense, UV-B radiation has been suggested to act as a signal that can promote toler-ance to biotic and abiotic stress conditions. The potential use of UV-B radiation to alleviate the effects of drought stress has been reported in different plant species such as silver birch (Robson et al., 2015a), rice (Dhanya Thomas et al., 2020), and wheat (Kovacs et al., 2014). Plant responses to the combination of drought and UV-B depend on whether the treatments are simultaneous or sequential (Escobar-Bravo et al., 2021). The sensitivity of the plant species also determines whether the interaction between drought and UV-B is additive, syner-gistic, or antagonistic (Bandurska and Cieslak, 2013). Cross-tolerance to drought may be activated by defense-related molecules, such as hydrogen peroxide (H2O2), nitric oxide (NO), abscisic acid, ethylene, jasmonic acid, or salicylic acid, which are produced as responses to UV-B (Bandurska and Cieslak, 2013; Mackerness et al., 2001; Man-nucci et al., 2020; Tossi et al., 2014). However, how UV-B mechanisti-cally interacts with these molecules while inducing cross-tolerance to drought remains poorly understood.
Plant responses to H2O2have received much attention because of its role as a signaling molecule, being considered an elicitor that,
when exogenously applied, can induce defense in plants prior to stress exposure (Parola-Contreras et al., 2020; Vazquez-Hernandez et al., 2019). Among H2O2-triggered responses, cross-tolerance to abiotic and biotic stresses is often observed (Hossain et al., 2015). Alleviation of drought using H2O2-controlled elicitation has been reported in cucumber (Sun et al., 2016), rice (Sohag et al., 2020), and soybean (Ishibashi et al., 2011). Drought stress increases the levels of ROS, such as H2O2and singlet oxygen (O2), in chloroplast, peroxisome, and mitochondria, which in turn can lead to oxidative damage (Cruz De Carvalho 2008). Despite this, priming with H2O2 can protect plants against damage by triggering antioxidant enzymes, such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase, and ascorbic acid peroxidase (APX), thus reducing the accumulation of different ROS (Hossain et al., 2015). This means that oxidative damage may be effectively alleviated by the antioxidant machinery after activation through exogenous H2O2application. Application of either UV-B in barley or H2O2in cucumber provided drought protection (Bandurska et al., 2013; Sun et al., 2016). However, in what way UV-B and H2O2 together can prime drought tolerance remains unknown.
The aim of this study was to evaluate cross-tolerance responses under moderate drought conditions in Nicotiana tabacum plants. A factorial experiment was implemented to assess the interaction of simultaneous exposure to UV-B exposure and exogenous application of H2O2.
2
|
M A T E R I A L S A N D M E T H O D S
2.1
|
Plant material, growth conditions
Tobacco seeds (N. tabacum L. cv. xanthi nc) were surface sterilized, sown in vitro on Murashige and Skoog medium and kept in a germina-tion chamber at a temperature of 25 ± 1C, 60 ± 5% of relative humidity (RH) and 70–85 μmol m 2s 1of photosynthetically active radiation (PAR). When plants were 1 week old (counting from radicle emergence) they were transplanted into individual 8 8 cm plastic pots containing fertilized peat moss-based substrate (Grön Torvmull, SW Horto, Hasselfors Garden, Örebro, Sweden) and transferred to a controlled environment greenhouse chamber at 25/20 ± 1C day/night and 75 ± 5% RH. Pots were arranged in trays containing 24 pots each. Plants were watered daily by adding 2 L of water to the trays. To compensate for the loss of nutrients in the substrate, plants were irrigated every third day with a 25% diluted Hoagland solution (Table S1) instead of using only water. Seedlings were exposed to PAR from high-pressure sodium lamps (Vialox NAV-T Super 4Y; Osram) corresponding to 200–240 μmol m 2 s 1 for 16 h per day (from 06:00 to 22:00 h) daily.
2.2
|
Experimental treatment conditions
The experiment started when plants were 3 weeks old (counting from radicle emergence) by applying UV-B- and H2O2-pre-treatments for
14 days, resulting in four plant treatment groups: control, UV-B-, H2O2-, and UV-B+ H2O2-pre-treated plants. The number of plants that were grown under each pre-treatment condition was 36.
The pre-treatment conditions were as follows:
1. Supplementary UV-B was applied for 4 h per day (from 10:00 to 14:00 h) and provided using fluorescent lamps (Philips TL40/12 UV, Eindhoven, The Netherlands). Then, 122 mW m 2of plant-weighted UV-B irradiation normalized to 300 nm (Kalbina et al., 2008; Thimijan et al., 1978; Yu and Björn, 1997) was applied (including some UV-A; cf. Qian et al. (2019) for the spectral infor-mation for this light source and the shape of the plant-weighted UV-B curve). The 4 h exposure gave a total dose of 1.75 kJ m 2 day 1of plant-weighted UV-B. As described in Qian et al. (2019), trays with plants were put inside three open top, front, and back-side Perspex boxes (two trays per box), covered with 0.13 mm cel-lulose acetate sheets (Nordbergs Tekniska AB, Vallentuna, Sweden), which function as filters to remove any UV-C radiation. The UV-B levels were selected based on previous UV-B-induced photomorphogenic responses in economically important plant spe-cies (Cucumis sativus, Qian et al., 2019, 2020, 2021; Capsicum annuum, Rodríguez-Calzada et al., 2019; Anethum graveolens, Castro-Alves et al., 2021). Such specific regulatory events have been reported to be triggered by low UV-B doses (Brosché and Strid, 2003; Jansen and Bornman, 2012; Robson et al., 2015b). UV radiation was measured using an Optronic Laboratories OL756 (Orlando, FL) double monochromator spectroradiometer. Control plants were exposed to white light only in the same chamber as the UV-B plants. Trays with control plants were also put inside three boxes (also two trays per box) covered on all sides, as well as the top, with UV-blocking Perspex (Plastbearbetning AB).
2. A solution of 0.2 mM H2O2 (H2O2 30% Perhydrol for analysis EMSURE ISO) was foliar sprayed (using 2.5 ml per plant) and applied to irrigation (using 10 ml per plant). H2O2 was applied every third day at the end of the UV-B treatment (at 14:00 h), giv-ing a total of five applications. All plants were randomly arranged on the trays, but those that received the H2O2treatment (H2O2 -and UV-B+ H2O2-pre-treated plants) were each placed in an indi-vidual plastic cup to avoid contaminating the irrigation water for the other pre-treated and control plants in the trays. The H2O2 solution and irrigation were applied to these plants by adding to each plastic cup 10 ml 0.2 mM H2O2plus the proportional amount of water or Hoagland's solution that corresponded to each plant in the trays. To perform foliar spraying, plants were taken to a sepa-rate chamber, having the same climatic conditions, and were ret-urned to their place when no traces of the solution remained on the leaves and tissue. The H2O2concentration used was deter-mined considering the levels reported in a number of studies where H2O2was used as a pre-treatment to enhance abiotic stress tolerance (Hossain et al., 2015). H2O2was applied to the roots fol-lowing our preliminary study that showed no differences in pheno-type or stress resistance in tobacco plants after foliar spray only (data not shown). Therefore, we chose to also elicit the roots
following the logic that drought stress is related to this organ of the plant.
3. Plants were exposed to a combined treatment of UV-B and H2O2 under the aforementioned conditions for each factor.
On Day 15 of the experiment (1 day after the completion of the 14-day pre-treatment and thus 36 days after radicle emergence), 12 plants per pre-treatment were used to carry out a 7-day drought experiment. Six of them were subjected to drought treatment, and six were used as well-watered controls. To achieve moderate drought con-ditions, plants were watered and allowed to reach maximum pot capac-ity (when drainage stopped). Thus, on Day 15 of the experiment, each pot was weighed, and this value was established as 100%. After 3 days of water withdrawal, potting capacity reached 40–45%. The drought treatment was achieved by weighing the pots daily and watering, when necessary, by placing the pots in a tray with water only for the neces-sary period of time so that the pot capacity remained at 40–45% until the end of the experiment. Viability was determined by visual assess-ment and measuring leaf relative water content (RWC). The RWC (%) was measured in the second fully developed leaf from the apex. Leaves were collected, the fresh weight (Fw) was recorded, and the leaves incu-bated in distilled water for 4 h at 4C in the dark. The leaves were blot-ted, and the turgid weight (Tw) measured. Finally, leaves were dried at 80C overnight and the dry weight (Dw) measured. RWC was calculated as RWC= (Fw - Dw)/(Tw - Dw) (Jones 2007).
For further analysis, the first, second, and third fully developed leaves from the apex of three plants per treatment were collected during the 21 days of the experiment at 0, 4, and 28 h and at Days 7, 14, and 21 after the start of the pre-treatment exposures. Root samples of three plants per treatment were collected at the end of the drought treatment (Day 21). Samples were frozen in liquid nitro-gen and stored at 80C. For the analysis of total antioxidant capac-ity, fresh tissue was used. For enzymatic assays, gene expression, and root proline analyses, the samples had been lyophilized by freeze-drying for 3 days (Lyolab 3000, ThermoFisher Scientific). Three plants per treatment were collected at each sampling time for performance of the analyses. Over the course of the 21 days of the experiment, the positions of the plants in the trays and the positions of the trays in the Perspex boxes were rotated daily.
2.3
|
Morphological measurements and
noninvasive leaf epidermal flavonol index
Morphological measurements were performed on randomized plants, using six plants per treatment, at the beginning (Day 1), middle (Day 7), and end of the pre-treatment (Day 14). Stem length was measured using a ruler, the number of leaves was counted, and the stem diameter was measured using a digital caliper. Measurements of the adaxial side epidermal flavonol index were also performed in six plants per treat-ment in the second fully developed leaf at Hours 0, 4, and 28, and every third day thereafter (at 14:00 h) until the end of the experiment using a DUALEX SCIENTIFIC optical sensor (ForceA, France).
2.4
|
Trolox equivalent antioxidant capacity
Total antioxidant capacity was measured using a commercially avail-able kit (Total Antioxidant Capacity Assay kit, Sigma). A total of 100 mg of fresh leaf tissue from a pool of leaves was frozen at 80C and homogenized using liquid nitrogen and a mortar and pestle. Sam-ples were extracted in 1 ml of 4C 1 phosphate buffered saline, and the supernatant was diluted 1:30 to bring values within the range of the kit standards. Samples were assayed according to the manufac-turer's protocol by comparing the absorbances of diluted extracts at 570 nm with Trolox standards. Values were then normalized to tissue Fw as equivalents of mg Trolox using the standard reference curve.
2.5
|
Preparation of leaf extracts and enzymatic
assays
Lyophilized leaf samples (50 mg) were ground with 2 ml ice-cold potassium-phosphate buffer (0.05 M, pH 7.8) using a mortar and pes-tle. Samples were centrifuged at 16 100 g for 15 min at 4C. The supernatants were stored at 20C until further used for assays of CAT, phenylalanine ammonia lyase (PAL), SOD, and peroxidase (POD) activities. Total soluble protein content was determined spectropho-tometrically (λ595 nm) according to the standard Bradford assay (Bradford 1976), using bovine serum albumin as standard.
The catalytic activity of CAT (EC 1.11.1.6) was measured spectro-photometrically (λ240 nm) by monitoring the rate of H2O2decrease for 6 min at room temperature as described by Afiyanti and Chen (2014). The reactions were started by adding 1.4 ml 50 mM potassium phosphate buffer (pH 8.0), 140μl 100 mM H2O2, and 70μl sample extract. CAT activity was expressed in units of mmol of H2O2 decomposed (mg protein min) 1.
PAL (EC 4.3.1.5) activity was determined by measuring spectro-photometrically (λ290 nm) the increases in the formation of cinnamic acid according to Toscano et al. (2018) by incubating 0.1 ml of sample extract in 1.5 ml of 0.1 M borate buffer (pH 8.8) containing 10 mM
L-phenylalanine for 1 h at 40C. The reaction was stopped by the addition of 0.25 ml 1 N HCl. One unit (U) of PAL released 1μmol of cinnamic acid (min) 1 at pH 8.8 and 40C. The PAL activity was expressed as U (mg protein) 1.
The SOD activity (EC 1.15.1.1) was assayed spectrophotometri-cally (λ560 nm) using the method of Hayat et al. (2018). Total volume of 50μl of the sample extract was added to 2.95 ml of the reaction mixture. The mixture contained 1.5 ml 0.05 M phosphate buffer (pH 7.8), 0.3 ml 0.1 mM EDTA, 0.3 ml 0.13 M methionine, 0.3 ml 0.75 mM nitroblue tetrazolium (NBT), 0.3 ml 0.02 mM riboflavin, and 0.25 ml distilled water. One unit (U) of SOD activity was defined as the amount of enzyme required for 50% inhibition of photochemical reduction of NBT, expressed as U (mg protein) 1.
POD activity was recorded using a commercially available kit (Amplex Red Hydrogen Peroxide/Peroxidase Assay Kit, ThermoFisher Scientific). Samples were diluted 1:5 to bring values within the range of the kit standards. Samples were assayed according to the
manufacturer's protocol by detecting the fluorescence signal in a plate reader with emission at 590 nm.
2.6
|
Determination of proline content in the roots
Proline was measured in roots by the ninhydrin-based colorimetric method as described by Lee et al. (2018). Lyophilized root tissue (0.05 g) was extracted by homogenization in 1 ml of 1% (w/v) sulfosalicylic acid. Then, 1 ml of plant extract was reacted with 2 ml acidic ninhydrin (1.25% [w/v] ninhydrin in 80% [v/v] acetic acid) and incubated at 100C for 60 min. The reaction was stopped by putting the samples in an ice bath for 10 min. The absorbance was measured using a spectrophotometer atλ510 nm, and the proline concentration was calculated using the standard curve.
2.7
|
Gene expression analysis
RNA extraction was performed using Direct-zol RNA miniprep kit (BioAdvanced Systems Co.) according to the manufacturer's instructions. cDNA synthesis was performed using the Maxima First Strand cDNA Synthesis (ThermoFisher Scientific) for RT-qPCR according to the instructions of the provider (10 min at 25C followed by 15 min at 50C). Accumulation of mRNA transcripts for marker genes involved in UV-B signaling or responding to UV-B (UVR8; BRI1-EMS-SUPPRESSOR 1, BES1; CHALCONE SYNTHASE, CHS; NAC DOMAIN PROTEIN 13, NAC13) was measured. The forward (F) and reverse (R) primers, designed by using the Primer-Blast tool from the National Center for Biotechnology Information, and used in the experiments, are given in Table S2.
Quantitative PCR was performed using the Maxima SYBR Green/ ROX qPCR Master Mix (Thermo Scientific) kit following the manufac-turer's instructions. The PCR conditions were as follows: 5 min at 94C, 40 cycles of 1 min at 94C, and 1 min at 56C for UVR8, 64C for NAC13, 62C for UVR8 and CHS. Bio-Rad CFX manager software was used to automatically calculate the cycle threshold (Ct) value for each reaction. Each reaction was performed at least in duplicate in two inde-pendent experiments. The expression of all genes was normalized to the mean of the housekeeping gene ELONGATION FACTOR 1 ALPHA (EF-1A). The following primers were used for EF-1A: EF-1AF—TGAGA TGCACCACGAAGCTC and EF-1AR—CCAACATTGTCACCAGGAAGTG. The quantification of mRNA levels was based on the relative quantifica-tion method (2 ΔΔCt) (Livak and Schmittgen, 2001).
2.8
|
Statistical analysis
Statistical analyses were carried out using the GraphPad prism 9.0 program (GraphPad Software). Data were collected from two inde-pendent experiments. One-way ANOVA was used to analyze data obtained during the first part of the experiment when pre-treatments were applied (Day 14). These data included stem length; basal diame-ter; number of leaves; Trolox equivalent antioxidant capacity (TEAC);
enzymatic activities (PAL, POD, SOD, and CAT); and epidermal UV absorbance. TEAC and epidermal UV absorbance at the end of the second part of the experiment (Day 21) was analyzed using two-way ANOVA with drought treatments and pre-treatments as factors. Two-way ANOVA was also used to analyze gene expression data using day of measurement and pre-treatments as factors. Treatment mean values were compared with Tukey's test at P≤ 0.05. Data are pres-ented as the mean ± standard deviation of six plants per treatment for morphological parameters and epidermal flavonol index estimated with Dualex, and of three plants per treatment for TEAC, enzymatic activities, RWC, proline content, and gene expression.
3
|
R E S U L T S
3.1
|
Effects of pre-treatments on morphological
and biochemical features
Among the morphological parameters studied, UV-B and H2O2 treat-ments individually reduced stem length, resulting in plants shorter than those in the control group (Figure 1A). The UV-B+ H2O2 treat-ment led to a reduced stem length compared to both control and the individual effect of the H2O2treatment (Figure 1A). The basal stem diameter was only decreased by the combined UV-B+ H2O2 treat-ment (Figure 1B). In addition, the number of leaves of UV-B+ H2O2 -treated plants was lower compared to the control and to the effect of either of the individual pre-treatments (Figure 1C). Thus, the com-bined treatment effect on plant morphology was shorter and more compact plants (Figure 2A).
The total antioxidant capacity increased in plants subjected to UV-B by 33% compared to the control, whereas no change was
observed in H2O2-treated plants (Figure 1D). The UV-B+ H2O2 treat-ment led to increased total antioxidant capacity by 31% compared to the control (Figure 1D).
The foliar adaxial epidermal flavonol index followed a similar trend, where the UV-B treatment as a single factor increased this parameter compared to the control from Day 7 of the experiment and until the end of the pre-treatment exposure. As with the total antioxidative capacity, the H2O2 treatment did not lead to any changes in the flavonol index throughout the 14 days of exposure (Table 1). For the combined treatment, the difference was significant from Hour 28 after the start of the exposure and was maintained throughout the 14 days (Table 1). The combined treatment induced a significantly higher leaf epidermal flavonol index between Days 5 and 12 when compared to the UV-B treatment. On the last day of expo-sure, the foliar epidermal index of flavonols was similar between these treatments (Table 1).
3.2
|
Leaf RWC and root proline content induced
by drought treatment
The first signs of drought stress were visible on Day 6 of drought treatment (i.e. on Day 20 after the start of the experiment) in control plants, which exhibited wilting of their older leaves (as shown for plants on Day 7 of drought treatment in Figure 2B). On Day 7 of drought treatment (i.e. on Day 21 from the start of the experiment), plants exposed to UV-B also showed signs of wilting (Figure 2C), whereas plants treated with H2O2and UV-B+ H2O2did not exhibit any visible signs of stress during the experiment (Figure 2D,E, respec-tively). On Day 7 of drought treatment, the RWC decreased significantly in the control plants by 21.9% (from 95.2% RWC in
non-F I G U R E 1 (A) Effects of ultraviolet (UV)-B, H2O2, and UV-B + H2O2on the (A) length and (B) basal diameter of the stem, (C) number of leaves, and (D) antioxidant capacity in Trolox equivalent antioxidant capacity (TEAC) in Nicotiana tabacum plants after 14 days of pre-treatment. The number of plants per treatment was n= 6 for morphological data and n= 3 for TEAC data. Different letters indicate significant
differences between the treatments using Tukey's test (P≤ 0.05). The standard deviation of the measurements is shown
drought-treated plants to 74.3% RWC in drought-treated plants). Cor-respondingly, the RWC decreased significantly in UV-B-pre-treated plants by 25.5% (from 96.0% RWC in non-drought-treated plants to 71.5% RWC in drought-treated plants) (Table 2). In plants pre-treated with H2O2 only, the RWC decreased significantly by 6.8% (from 93.3% RWC in non-treated plants to 87.0% RWC in drought-treated plants). The combined UV-B+ H2O2-pre-treatment led to a significant decrease in RWC from 93.5 to 86.2% (i.e. by 7.8%). The proline content in roots did not show any significant changes between treatments in this experiment (Table 2).
3.3
|
CAT, PAL, POD, and SOD activities induced
by the drought treatment
PAL activity significantly increased under non-drought conditions in plants of all three pre-treatments compared with pre-treatment con-trols. Whereas the increase was similar in the UV-B-pre-treated and UV-B+ H2O2-pre-treated plants, H2O2-pre-treatment led to a sig-nificantly higher PAL activity still (Figure 3A). Drought did not fur-ther affect PAL activity in the H2O2-pre-treatment plants, whereas PAL activity in plants pre-treated with either UV-B or UV-B+ H2O2 was significantly higher under drought conditions. The UV-B-pre-treated plants showed the highest PAL activity during drought con-ditions, tightly followed by plants that had received the combined pre-treatment. Interestingly, non-pre-treated control plants had a
higher PAL activity after drought than the H2O2-pre-treated plants (Figure 3A).
The H2O2-pre-treatment induced significantly higher CAT activity under non-drought conditions than in control plants or UV-B-pre-treated plants (Figure 3B). Drought again did not alter CAT activity in H2O2-pre-treated plants, whereas all three other pre-treatments did. Control, UV-B-pre-treated and plants pre-treated with both UV-B and H2O2all had a similar CAT activity after drought exposure. This activ-ity was significantly higher than that of the H2O2-pre-treated plants (Figure 3B).
Similarly to CAT, H2O2-pre-treated plants had significantly higher POD activity under non-drought conditions than plants that had expe-rienced any of the other pre-treatments (Figure 3C). Yet again, drought treatment did not alter POD activity in H2O2-pre-treated plants. The low POD activity under non-drought conditions in UV-B-treated plants did not change as a result of drought. However, plants exposed to the double pre-treatment exhibited an approximately four-fold increase in POD activity after drought. Even more pronounced was the increase in control plants of the POD activity when drought was applied, induction being two-fold larger still (Figure 3C).
Finally, the SOD activity of non-drought exposed plants was low independently of what pre-treatment the plants had been given. Any differences between the pre-treatments were too small to be statisti-cally different (Figure 3D). In this case, drought did lead to increased SOD activities in plants after all four pre-treatments, the increase being considerably larger in control plants and in H2O2-pre-treated plants.
F I G U R E 2 (A) Representative images of plants exposed to 14 days of the respective pre-treatments. Also, representative images of wilting in plants exposed to 14 days of the respective pre-treatments and additional 7 days of drought treatment (Days 15–21 of the experiment), (B) no pre-treatment, (C) UV-B pre-treatment, (D) H2O2 pre-treatment, and (E) UV-B+ H2O2 pre-treatment
3.4
|
TEAC and leaf flavonol index induced by the
drought treatment
ANOVA showed a significant main effect (P < 0.05) of the pre-treatment and drought, and of the interaction pre-treatment drought on TEAC (Supporting Table S3). TEAC was similar in all pre-treatment groups under non-drought conditions (Figure 4A). On Day 21, drought had led to a significant increase in the control group and in the UV-B-pre-treated plants. On the other hand, the H2O2and UV-B+ H2O2 -pre-treated plants did not show any increase between drought and non-drought conditions (Figure 4A).
ANOVA showed a significant main effect (P < 0.01) of the pre-treatment and drought, and of the interaction pre-pre-treatment drought on the leaf flavonol index (Table S3). UV-B+ H2O2-pre-treated plants exhibited significantly higher leaf epidermal flavonol index under non-drought conditions than H2O2-pre-treated and control plants on Day 21 (Figure 4B). Drought treatment increased the content even more in UV-B+ H2O2-pre-treated plants, which showed the highest levels, followed by the UV-B-treated plants and, finally the non-pre-treated plants (Figure 4B). Drought treatment did not increase the index of leaf epidermal flavonols in H2O2-pre-treated plants (Figure 4B).
3.5
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Effects of UV-B, H
2O
2, and drought on
transcript accumulation of
UVR8, NAC13, BES1,
and
CHS
ANOVA showed a significant main effect (P < 0.01) of the pre-treatment and day of measurement on transcript accumulation of all genes analyzed (Table S4). The interaction pre-treatment day of measurement was also significant for all genes. Except for UVR8, the expression of all other genes was affected by the interaction pre-trea-tment drought (Table S4).
Increased transcript accumulation of UVR8 was only triggered in UV-B- and UV-B+ H2O2-pre-treated plants on Day 14 (Figure 5A). At this time point, UV-B-pre-treatment led to a 27-fold increase in the UVR8 mRNA levels when compared with the corresponding control. Plants pre-treated with UV-B+ H2O2 showed a 63-fold increase in UVR8 transcript levels (Figure 5A). On Day 21, the UVR8 transcript abundance had returned to control levels, independently of pre-treatment and whether the plants had been exposed to drought or not.
For NAC13, on Day 14, the expression pattern was very similar to that for UVR8. UV-B and UV-B+ H2O2pre-treatments led to tran-script accumulation by 13- and 97-fold, respectively (Figure 5B). In contrast to UVR8, NAC13 transcript levels were still enhanced (by 41-fold) on Day 21 in well-watered UV-B+ H2O2-pre-treated plants. In drought-treated plants on Day 21, both UV-B- and UV-B + H2O2-pre-treated plants had a similar NAC13 expression about fivefold above the control level (Figure 5B).
BES1 transcript levels increased on Day 14 by 267-, 772-, and 1956-fold, in plants that had received UV-B-, H2O2-, and UV-B + H2O2-pre-treatments, respectively (Figure 5C). On Day 21, i.e. after 7 days of drought, BES1 levels were higher in plants pre-treated with
TAB L E 1 Leaf ada xial epi derm al flavon ol inde x estim ated with Duale x a t 0 , 4 , and 28 h and at Days 3, 5, 7, 9, 12, and 14 in Nicotian a tabacu m leave s culti vated unde r pre-tre atme nt wi th UV-B, H2 O2 , o r UV-B + H2 O2 , o r in the absence of pre-tr eatment . Data a re mean s o f measu rement s from si x plan ts per treatm ent ± stan dard devi ation. Significa nt dif ferences w ere de termine d a mong the mean s using Tu key's test at P < 0 .05, 0.0 1, 0. 001, a n d 0.0001 whic h are indicated with *, **, ***, ****, respe ctively Hour (H)/day (D) Control H2 O2 treated Increase in H2 O2 treated compared to control (%) UV-B treated Increase in UV-B treated compared to control (%) UV-B + H2 O2 treated Increase in H2 O2 + UV-B treated compared to control (%) Increase in H2 O2 + UV-B treated compared to UV-B treated (%) H 0 0.32 ± 0.008 0.34 ± 0.008 5 0.32 ± 0.01 0 0.34 ± 0.008 5 5 H 4 0.33 ± 0.006 0.33 ± 0.008 1 0.35 ± 0.01 5 0.35 ± 0.012 6 1 H 2 8 0.32 ± 0.006 0.33 ± 0.010 4 0.36 ± 0.01 12 0.37 ± 0.013 15* 3 D 3 0.32 ± 0.004 0.32 ± 0.005 1 0.35 ± 0.01 8 0.38 ± 0.014 17** 8 D 5 0.34 ± 0.005 0.34 ± 0.012 1 0.38 ± 0.02 12 0.42 ± 0.027 24**** 11* D 7 0.34 ± 0.010 0.36 ± 0.004 7 0.40 ± 0.02 19*** 0.44 ± 0.048 32**** 11* D 9 0.35 ± 0.009 0.36 ± 0.012 4 0.48 ± 0.05 38**** 0.53 ± 0.045 52**** 10* D 1 2 0.43 ± 0.025 0.45 ± 0.044 4 0.63 ± 0.07 48**** 0.71 ± 0.050 65**** 12**** D 1 4 0.44 ± 0.026 0.48 ± 0.020 9 0.71 ± 0.06 59**** 0.72 ± 0.060 62**** 2
UV-B (100-fold), H2O2 (12-fold), and UV-B+ H2O2 (7-fold) and exposed to drought than the non-pre-treated control plants (Figure 5C). On Day 21 under no-drought conditions, only the H2O2 -pre-treatment led to increased BES1 transcript levels compared to the corresponding control (by fivefold) (Figure 5C).
On Day 14, tobacco plants displayed a clear and significant CHS mRNA induction by UV-B-, H2O2-, and UV-B+ H2O2-pre-treatments by approximately 34 000-, 55-, and 37,000-fold, respectively, compared to the non-pre-treated control plants. A similar trend was maintained on Day 21 after 7 days of subsequent drought treatment for the plants that had been pre-exposed to UV-B- (40-fold) and UV-B+ H2O2 -(65-fold) pre-treatments (Figure 5D). In contrast, on Day 21, in non-drought treated plants, the CHS transcript levels had almost returned to the basal level, only displaying a twofold to fourfold increase compared to the corresponding control in all cases (Figure 5D).
4
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D I S C U S S I O N
The increase of stress cross-tolerance through the application of physical and chemical factors is accompanied by morphological,
biochemical, and molecular changes, which are important to study since their interaction determine the desired response. In this study, cross-tolerance in tobacco plants, induced by UV-B exposure and exogenously applied H2O2, toward a subsequent drought treatment was studied independently and in combination by assessing several morphological, physiological, and biochemical parameters.
With regard to the morphological parameters, the shorter stem length exhibited by the UV-B-pre-treated plants (Figure 1A) agrees with previous studies reporting significant decreases in stem length in different plant species under UV light regimens (Qian et al., 2020; Robson et al., 2015b; Rodríguez-Calzada et al., 2019). In contrast, with regard to H2O2-pre-treated plants, our results differ from other reports that have shown increased biomass and plant height in response to exogenously applied H2O2(Ashfaque et al., 2014; Basal and Szabo, 2020; Sun et al., 2016). In these previous papers, and in most studies where exogenous H2O2 have been applied, foliar spraying has been used as the sole application method. We, on the other hand, in our H2O2-pre-treatment also applied H2O2 to roots through irrigation, a difference that might have influenced the results. The fact that more compact plants were obtained under all the pre-treatments must, for economically important plants, also be
F I G U R E 3 Activities in Nicotiana tabacum leaves after 7 days of drought treatment of important defense enzymes. (A) Phenylalanine ammonia lyase (PAL); (B) catalase (CAT); (C) total peroxidase (POD); and
(D) superoxide dismutase (SOD). Different letters indicate significant difference between treatments using Tukey's test (P≤ 0.05). The standard deviation of the measurements is shown. The number of plants per treatment was n= 3
T A B L E 2 Leaf RWC and root proline content at the end of drought treatment (Day 21). Different letters indicate significant differences between the treatments using Tukey's test (P≤ 0.05). Data represent means of n= 3 measurements ± standard deviation Treatment
Leaf RWC (%) Root proline content (μM/mg)
Control Drought Control Drought
Control 95.17 ± 1.33a 74.33 ± 1.86b 2.67 ± 0.74a 2.94 ± 0.18a UV-B 96 ± 0.63a 71.50 ± 1.76b 2.55 ± 0.13a 3.26 ± 0.54a H2O2 93.33 ± 2.73a 87 ± 1.26c 3.06 ± 0.98a 2.87 ± 0.07a UV-B+ H2O2 93.50 ± 2.59a 86.17 ± 0.75c 2.83 ± 0.40a 2.94 ± 0.05a Abbreviation: RWC, relative water content.
considered in a horticultural production scenario, since this might not be a desired property in commercial production if larger biomass is expected.
The observed increase in the total antioxidant capacity of UV-B-and UV-B+ H2O2-treated plants at the end of the pre-treatment period (Figure 1D) could be explained by an increase in non-enzymatic antioxidants, since the foliar epidermal flavonol index (Table 1) was significantly higher in these groups of plants (Qian et al., 2021). Fur-thermore, our data indicate that regarding these two biochemical parameters, plants treated with UV-B+ H2O2 were primarily influenced by the effect of the UV-B treatment rather than by H2O2. This novel finding highlights the need to study the dominant conver-gence of UV-B and ROS signaling pathways against other possible pathways triggered by additional factors for conferring defense responses in plants. The UV-B-induced foliar flavonol index in this experiment (Table 1) is consistent with the fact that a primary mecha-nism by which plants acclimate to UV exposure is the accumulation of UV-absorbing compounds, such as flavonoids and anthocyanins in leaf epidermal tissue, acting as UV shielding components, but which also are efficient ROS scavengers (Agati et al., 2013; Agati and Tattini, 2010; Neugart and Schreiner, 2018).
The leaf RWC and root proline content were measured on Day 21 to evaluate differences between those pre-treatments that led to drought cross-tolerance (H2O2 and UV-B+ H2O2) and those that induced foliar wilting caused by the drought treatment (Control and UV-B). The wilting observations (Figure 2) were corroborated by RWC measurements (Table 2), the values of which were lower in plants that presented this trait. H2O2and UV-B+ H2O2treatments significantly reduced losses in leaf water content compared to control and UV-B treatments, but the fact that there was no change in root proline con-tent suggests that drought stress tolerance was not due to osmotic
F I G U R E 5 Transcript accumulation for (A) UV RESISTANCE LOCUS 8 (UVR8); (B) NAC DOMAIN CONTAINING PROTEIN 13 (NAC13); (C) BRI1-EMS-SUPPRESSOR 1 (BES1); and (D) CHALCONE SYNTHASE (CHS) genes on Days 0, 14, and 21 after
commencement of the experiment. Pre-treatment was performed for the first 14 days and drought treatment was performed on Days 15–21. Relative expression levels were measured in Nicotiana tabacum leaves using qPCR. The number of plants per treatments was n= 3. Error bars represent standard errors. Different letters indicate a significant difference between the treatments using the Tukey's test (P≤ 0.05)
F I G U R E 4 Effects of UV-B-, H2O2-, and UV-B+ H2O2 -pre-treatments on Nicotiana tabacum responses to 7 days of drought treatment (Day 21 of the experiment). (A) Trolox equivalent antioxidant capacity (TEAC) and (B) leaf adaxial epidermal flavonol index estimated using a Dualex instrument. Different letters indicate significant difference between treatments using Tukey's test (P≤ 0.05). The standard deviation of the measurements is shown. The number of plants per treatment was n= 3 for TEAC data and n = 6 for FLAV index data
adjustment related to this osmolyte (Table 2). Neither H2O2treatment of roots nor leaves promoted root proline accumulation in our studies, in contrast to other studies where H2O2priming led to osmotic stress tolerance by modulating proline accumulation (Ashfaque et al., 2014; Hossain et al., 2015). Similar results in which RWC was increased due to the effect of exogenous H2O2have been reported under different drought stress regimens (Basal and Szabo, 2020; Ishibashi et al., 2011; Sun et al., 2016). Thus, our data, together with results from previous studies, indicate that a strong defense response under drought in plants pre-treated with H2O2is the maintenance of high levels of leaf RWC, an effect that was also found in the combined pre-treatment, and which probably plays a key role in the cross-tolerance shown by the plants.
CAT is considered a major H2O2-scavenging enzyme that is mainly active at relatively high H2O2 concentrations (Anjum et al., 2016). Our UV-B treatment did not affect CAT activity in non-drought-treated plants (Figure 3B), which indicates that the low UV-B doses used did not induce significant changes in the produc-tion of H2O2. Since POD and SOD activities also did not increase in the UV-B-pre-treated group (Figure 3C,D), this confirms that the treatment did not induce oxidative stress. Increases in CAT, SOD, and POD activities by UV-B have previously been reported in experiments using higher daily doses. Priming with 6 kJ m 2 of UV-B radiation in rice seedlings induced CAT as well as SOD and APX activities (Thomas and Puthur, 2019). In another study, expo-sure of N. tabacum plants to high (13.6 kJ m 2 day 1) biologically effective UV-B radiation led to increases in POD defense, specifi-cally APX, and of the chloroplast-localized Fe-SOD (Majer et al., 2014). Racz et al., (2018) also reported that leaf acclimation to UV-B, corresponding to a 7.7 kJ m 2 day 1 biologically effective dose, is conferred by selective activation of POD isoforms. Our data showed that UV-B exposure specifically increased PAL activity (Figure 3A) and stimulated the downstream synthesis of foliar epi-dermal flavonols (Table 1) in the absence of oxidative stress.
The high level of CAT activity in H2O2-pre-treated tobacco plants under non-drought conditions (Figure 3B) indicates that the application of this elicitor either led to uptake of this uncharged molecule by the plant, or induced ROS production. Moreover, the CAT activity remained at the same level in drought-treated plants, which shows that a durable post-application effect was induced by the exogenous H2O2. The PAL and POD activities of H2O2-pre-treated tobacco plants showed the same behavior (Figure 3A,C). These results suggest that activation of the enzymatic antioxidant machinery due to H2O2 pre-treatment induced the observed drought cross-tolerance in these plants.
Other studies have also concluded that the capacity for osmotic adjustment via up-regulating antioxidant enzymes influences drought tolerance due to elicitation by H2O2. Sun et al. (2016) found that drought tolerance of cucumber seedlings was improved by foliar appli-cation of 1.5 mM H2O2. In their study, the CAT activity was signifi-cantly increased under severe drought conditions but not under moderate drought, contrary to the SOD and POD activities, which were significantly increased by the elicitor under medium drought-stress conditions. Similarly, exogenous soil application of 5 mmol L 1
H2O2improved water stress tolerance of rice seedlings at the same time as CAT activity remained unaltered compared with the control. APX and guaiacol peroxidase activities were, however, significantly increased (Sohag et al., 2020).
With regard to UV-B+ H2O2-pre-treated plants, the enzymatic activities of PAL, CAT, and SOD (Figure 3A,B,D) followed a similar trend as those of the UV-B-pre-treated plants, again confirming the strong influence of UV-B for these plants. Interestingly, with regard to POD (Figure 3C), neither UV-B-pre-treatment nor H2O2 -pre-treatment led to any increases in activities as the result of drought treatment. However, the combined UV-B+ H2O2-pre-treatment led to a synergistic increase in POD activity requiring both pre-treatment factors to be present for induction of the response.
To further study the influence of UV-B under the different treat-ments, we measured changes in transcript accumulation of a set of UV-B marker genes. The UV-B photoreceptor UVR8 (Jenkins 2014) mediates responses to UV-B through interactions with specific signal-ing components and transcription factors which ultimately lead to the biosynthesis of phenolic compounds, such as flavonoids (Hideg et al., 2013; Hideg and Strid, 2017; Jenkins 2014; Liang et al., 2019; Rai et al., 2020). Transcript accumulation of UVR8 was strongly increased in UV-B-treated tobacco plants on Day 14. Interestingly, UVR8 in Ara-bidopsis does not appear to be regulated neither at the transcript nor at the protein level (Jenkins 2014; Morales et al., 2013). Thus, tran-scriptional regulation of UVR8 by UV-B appears to be species-depen-dent. Our data further indicate that ROS and UV-B signaling interact to regulate UVR8 expression in tobacco (Figure 5A). A similar synergis-tic effect on the mRNA abundance was also observed on Day 14 for the NAC13 and BES1 genes, given the higher transcript accumulation after UV-B+ H2O2pre-treatment as compared to plants pre-treated with UV-B or H2O2alone (Figure 5B,C). Thus, the convergence of UV-B and ROS signaling impacts on the expression of genes involved in multiple pathways.
The increase of NAC13 transcript levels in UV-B- and UV-B + H2O2-pre-treated plants on Day 14 (Figure 5B) indicates that NAC13 in tobacco is induced by low UV-B doses. Previous research showed that NAC13 expression is induced by UV-B in Arabidopsis independently of the UVR8 photoreceptor (O'Hara et al., 2019) and is also induced by salt stress in poplar (Zhang et al., 2019), as well as by drought in broomcorn millet (Shan et al., 2020). Although NAC13 tran-script levels decreased between Days 14 and 21, particularly in UV-B-pre-treated plants, our results suggest that drought helped to maintain an elevated NAC13 expression in these plants. Thus, our data together with earlier reports indicate that NAC13 mediates diverse stress responses in plants. However, drought on its own did not induce NAC13 expression in tobacco (Figure 5B). Transcriptional regulation of NAC members appears to be complex since different NAC genes play different roles in response to drought stress. Overexpression studies have shown that apple NAC1, rice NAC066, and tomato NAC2 increased drought tolerance (Borgohain et al., 2019; Jia et al., 2019; Yuan et al., 2019), whereas rice NAC095 is a negative regulator of drought stress resistance (Huang et al., 2015). In fact, heterologous expression of Lilium pumilum NAC13 in tobacco decreased drought
tolerance but increased salt tolerance (Wang et al., 2020). Thus, the role of NAC genes in drought tolerance needs further elucidation, especially when considering this large gene family with more than 100 members in different plant species (Wang et al., 2020).
BES1 is a transcription factor involved in brassinosteroid (BR) signaling that, when activated, regulates expression of thousands of BR-responsive genes (Clouse 2011). In response to UV-B, the expression of BES1 in Arabidopsis appears to be dependent on the nature of the UV-B treatment. While UV-B levels in natural sun-light (1.5μmol m 2s 1for 6 h) did not affect BES1 transcript accumu-lation in wild type plants (Rai et al., 2020), exposure to UV-B stress provided by broad-band UV (5 μmol m 2 s 1 for 8 h per day for 10 days) lowers BES1 transcript levels (Liang et al., 2020). Here we show that low level UV-B-pre-treatment led to increased BES1 tran-script levels in tobacco plants (Figure 5C). Furthermore, plants exposed to H2O2- or combined UV-B+ H2O2-pre-treatments showed an even greater increase in BES1 mRNA, revealing an important role for H2O2in induction of BES1 expression. These data correlate with recent studies that have shown a positive H2O2regulation of BR sig-naling. Tian et al. (2018) uncovered a critical role of H2O2in BR signal-ing in Arabidopsis plants through the oxidation of the BES1 homologous transcription factor BRASSINAZOLE-RESISTANT1 (BZR1), leading to its interaction with key regulators in cell growth and the auxin-signaling pathway. In our study, BES1 transcript abun-dance decreased from Day 14 to 21 in well-watered plants exposed to all three pre-treatments. However, significant amounts of BES1 mRNA were maintained on Day 21 under drought conditions in plants subjected to the three different pre-treatments. These findings indi-cate that BES1 in tobacco is transcriptionally regulated by low UV-B doses and exogenous H2O2. Drought obviously also influences BES1 expression induced by UV-B and H2O2 pre-treatment, although drought itself could not regulate BES1 transcription.
Flavonoids have been reported to have functional roles in absorb-ing UV radiation and to function as antioxidants (Agati et al., 2013; Hideg and Strid, 2017). Furthermore, many research groups have reported that flavonoids have a function in defense against drought, when acting as free radical scavengers (Kumar et al., 2020; Nakabayashi et al., 2014; Ramakrishna and Ravishankar, 2011). Our
data are in agreement with previous research which showed that UV-B enhances accumulation of CHS transcripts, representing the first committed step of the flavonoid biosynthesis pathway (Brown and Jenkins, 2008; Rai et al., 2020; Rodríguez-Calzada et al., 2019; Santin et al., 2020). It is well known that H2O2plays a role in regulation of genes associated with phenolic compound biosynthesis, of which CHS is one (Nyathi and Baker, 2006). Our data also show that drought amplifies the expression pattern induced particularly by UV-B-pre-treatment in tobacco, a result that coincides with increased levels of epidermal flavonoids (Figure 4B). Thus, this strengthens the notion that flavonoids have an important role in inducing drought tolerance and mitigating oxidative stress.
Our data also demonstrate a pre-treatment-dependent response to drought for NAC13, BES1, and CHS transcript accumulation. Our data also demonstrate a pre-treatment-dependent response to drought for antioxidant capacity (TEAC) and leaf epidermal flavonol index (see interaction information of drought and pre-treatments fac-tors in the Table S3), which highlight the potential of using UV-B and H2O2as pre-treatments to help maintain induced antioxidant defense to improve subsequent tolerance to adverse conditions.
In Figure 6, we summarize the effects of the three environmental cues UV-B radiation, oxidative pressure (H2O2-pre-treatment) and drought and their mechanistic interplay with regards to gene expres-sion. The UV-B-pre-treatment increased the expression of all four molecular markers in tobacco, whereas drought alone did not alter gene expression (Figures 5 and 6). H2O2-pre-treatment does not lead to expression of UVR8 or NAC13 (Figures 5A,B and 6), but induces transcription of BES1 and CHS (Figures 5C,D and 6). Furthermore, H2O2 potentiates the UV-B-induced expression of NAC13 (Figures 5C,D and 6). Drought also prolongs the effect of UV-B-pre-treatment of all genes, except UVR8 (see interaction information on drought and pre-treatments factors in the Table S4).
5
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C O N C L U S I O N S
To conclude, this study highlights the potential of using a novel combination of factors to provide cross-tolerance to drought which
F I G U R E 6 Schematic
representation of the effects of UV-B- and H2O2-pre-treatments and subsequent drought treatment on the expression of UVR8, NAC13, BES1, and CHS in tobacco leaves. Upward arrows indicate induced expression by a given treatment (gray—UV-B; white—H2O2). Black filled arrows show interactive effects between UV-B and H2O2 -pre-treatments and UV-B-pre-treatment and drought conditions
could become a useful tool, for instance, to reduce water use in agriculture in a current challenging environmental scenario. In order to strengthen these findings, it would be of importance to perform experiments with plants both in a more advanced vegetative state and under longer periods of pre-treatment and drought exposure, either in tobacco or in other species of commercial value. Moreover, a key aspect of controlled elicitation would be to carry out further studies of the biochemical and molecular responses toward these factors in order to find specific conditions under which plants show desired responses.
A C K N O W L E D G M E N T S
This research was supported by research grants to Å. S. from the Knowledge Foundation (kks.se; grant #20130164), and the Swedish Research Council Formas (formas.se/en; grant #942-2015-516). The project was also supported by the Faculty for Business, Science and Technology at Örebro University. Moreover, this research was par-tially supported by SEP-CONACyT Mexico (Ciencia basica 2016, grant 283259). D. S.-d acknowledges Dr Irina Kalbina for the technical assis-tance and the doctorate scholarship 707895 and travel grant given by Consejo Nacional de Ciencia y Tecnología (CONACyT, Mexico). A U T H O R C O N T R I B U T I O N S
Ramon G. Guevara-Gonzalez and Diana Saenz-de la O: Conceived the research. Ramon G. Guevara-Gonzalez, Åke Strid, and Luis O. Morales: Coordinated the research. Diana Saenz-de la O: Per-formed the experiments. Diana Saenz-de la O, Luis O. Morales, Åke Strid, Irineo Torres-Pacheco, and Ramon G. Guevara-Gonzalez: Ana-lyzed the data and wrote the manuscript.
D A T A A V A I L A B I L I T Y S T A T E M E N T
The data that support the findings of this study are available from the corresponding authors upon reasonable request.
O R C I D
Diana Saenz-de la O https://orcid.org/0000-0001-5115-6513
Luis O. Morales https://orcid.org/0000-0002-9233-7254
Åke Strid https://orcid.org/0000-0003-3315-8835
Ramon G. Guevara-Gonzalez https://orcid.org/0000-0002-5748-7097
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S U P P O R T I N G I N F O R M A T I O N
Additional supporting information may be found online in the Supporting Information section at the end of this article.
How to cite this article: Saenz-de la O, D., Morales, L.O., Strid, Å., Torres-Pacheco, I., Guevara-Gonzalez, R.G. (2021) Ultraviolet-B exposure and exogenous hydrogen peroxide application lead to cross-tolerance toward drought in Nicotiana tabacum L. Physiologia Plantarum, 1–14. Available from:https://doi.org/10.1111/ppl.13448
