Ultraviolet B radiation-mediated stress ethylene emission from rice plants is regulated by 1-aminocyclopropane-1-carboxylate deaminase-producing bacteria

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Industrial emissions have played a major role in damaging the ozone layer,leading to increased atmospheric penetration of ultraviolet B(UV-B)(290—320 nm)radiation(Jordan,2002).The recovery rate of the damaged ozone layer in the mid-latitudes and tropics has been insignificant since 1979(Chipperfieldet al.,2017),even after the adoption of the Montreal Protocol(Protocol,1987),and the recovery process is expected to take many decades.In the meantime,UV-B radiation continues to penetrate the atmosphere(McKenzieet al.,2011;Baiset al.,2019).The characteristic short length and high energy of this radiation wavelength can directly affect major molecules,such as DNA,proteins,and lipids,which are important to living beings(Hideget al.,2013).Rice is one of the major global crops,which is generally cultivated at lower latitudes and can potentially be exposed to a higher flux of UV radiation due to greater solar angles and longer duration of sunlight(Kataria and Guruprasad,2012).

Exposure to UV-B radiation causes a decrease in plant height,total leaf area,and biomass(Newsham and Robinson,2009).The initial responses include elevated ethylene emission and reactive oxygen species(ROS)accumulation(Anet al.,2006;Czégényet al.,2016),which can eventually result in accelerated damage to DNA and cell membranes(Wanget al.,2010;Surjadinataet al.,2021).Additionally,prolonged exposure to UV-B radiation can also lead to destabilization of the cell membrane through enhanced lipid peroxidation(Berliet al.,2010;Liet al.,2014).In contrast,plants show a myriad of responses to coping with UV-B radiation-induced stress.For example,plants tend to enhance the deposition of polyphenols such as flavonoids in the epidermal tissues,acting as a potential antioxidant,like a sort of“sunscreen”to prevent the penetration of UV rays(Surjadinataet al.,2021).However,regulating ROS and ethylene emission can also be attained by inoculation with 1-aminocyclopropane-1-carboxylate(ACC)deaminase(ACCd)-producing plant growth-promoting bacteria(PGPB)to enhance stress tolerance,and the effectiveness of this method has been extensively studied in a plethora of abiotic and biotic stresses(Glick,2014).Additionally,designing effective synthetic communities(SynComs)for the alleviation of stresses(Trivediet al.,2021)is regarded as a sustainable and eco-friendly approach.Brevibacterium linensRS16 is an ACCd-producing PGPB that can alleviate salt and heat stresses in plants by ROS detoxification and regulation of ethylene and volatile organic compound(VOC)emissions(Siddikeeet al.,2010;Chatterjeeet al.,2020).The bacterium can also be regarded as a multifunctional PGPB that exhibits ACCd activity(4.13μmolα-ketobutyrate mg-1protein h-1),nitrogen fixation,indole-3-acetic acid production,ammonia production,and thiosulfate oxidation abilities(Siddikeeet al.,2010).Hence,this study was conducted to evaluate the efficiency ofB.linensRS16 in regulating UV-B stress-mediated ethylene emission,ROS accumulation,and cell damage to enhance the stress tolerance of rice(Or yza sativaL.)plants.

Rice seeds were soaked in water and germinated in a dark chamber at 30°C for 72 h.Germinated seedlings were transplanted in a seedling tray and transferred to a greenhouse at day/night temperature of 32°C/28°C and under natural illumination.Brevibacterium linensRS16 was cultured in nutrient broth until the optical density at 600 nm(OD600)reachedca.0.8.Subsequently,the inoculum was prepared by washing the bacterial cells in 0.03 mol L-1MgSO4three times and resuspended in the same manner,maintaining OD600atca.0.8.Five-day-old seedlings(30 rice seedlings per replicate)were transplanted into a 60-mL pot containing 50 g nursery soil(Doobaena,Nongkyung Co.,Republic of Korea).Precisely,10 mL bacterial inoculum was inoculated into the root zone and kept under greenhouse conditions for 2 d.The pots containing 7-d-old seedlings(2 d after inoculation)were transferred to two different growth chambers(DS 54 GLP,DASOL Scientific Co.Ltd.,Republic of Korea)equipped with UV-B lamps(306 nm,G20 T10E,Sankyo Ultraviolet,Co.,Ltd.,Japan)at two different intensities,viz.,0.5 and 1 W m-2.The control plants were treated with 0.03 mol L-1MgSO4and kept in a growth chamber without any UV lamps.The intensity of UV-B light was measured using a spectroradiometer(JAZEL 200,Ocean Optics,USA).The plants were kept at a day temperature of 32°C(16 h)and a night temperature of 28°C(8 h),withca.70%relative humidity.The UV-B stress was applied by turning on the lamps for 12 h daily,in addition to the illumination.Plants were harvested at days 2,3,and 4.Harvested plants were measured for the hydrogen peroxide(H2O2)concentration,malondialdehyde(MDA)concentration,electrolyte leakage,and polyphenol concentration.The H2O2concentration was measured by taking leaf samples(0.5 g)and grinding them with liquid nitrogen.Ground leaf samples were homogenized with 1 mL of cold acetone and centrifuged at 6 000×gfor 10 min at 4°C.The supernatant(1 mL)was carefully decanted into a test tube and 250μL of cold distilled water,0.1 mL of 5%(weight/volume)titanium sulfate,and 0.5 mL of 1 mol L-1ammonium hydroxide were added.The resultant mixture was centrifuged at 10 000×gfor 5 min at 4°C,and the supernatant was discarded.The pellet was dissolved in 2 mol L-1H2SO4,and the final volume was adjusted to 2 mL with cold distilled water.The absorbance was recorded at 415 nm using a UV-Vis spectrophotometer(UV-1601,Shimadzu,Japan),and the H2O2concentration was determined using a standard curve plotted with known concentrations of H2O2(Theochariset al.,2012).The MDA concentration was determined using ground leaf samples(0.5 g),homogenized with ice-cold phosphate buffer,and centrifuged at 6 000×gfor 10 min at 25°C.The supernatant(1 mL)was placed in a test tube,and 4 mL of 0.5%(weight/volume)thiobarbituric acid prepared in 20%trichloroacetic acid was added.The reaction mixture was incubated at 95°C for 30 min,and the reaction was terminated by transferring the tube into a cold-water bath.Absorption was recorded using a UV-Vis spectrophotometer at 532(A532)and 600(A600)nm.The resulting MDA concentration was calculated by subtractingA532fromA600and multiplying the result by the extinction coefficient of 155 mm-1cm-1(Samaddaret al.,2019).To determine electrolyte leakage,1 g leaf sample was taken and cut into small segments.The cut samples were placed in a conical tube containing 10 mL sterile deionized water and incubated for 24 h at 25°C.The electrical conductivity of each sample was measured,and the samples were incubated at 100°C for 30 min to determine the total electrical conductivity.The relative electrical conductivity was determined as a percentage(Subramanianet al.,2016).Polyphenol concentration was determined using fresh leaf samples(0.5 g)and homogenized with 80%ethanol,followed by agitation at 70°C for 20 min.The mixture was centrifuged at 9 000×gfor 10 min,and 2 mL supernatant was carefully placed into a test tube.The supernatant was mixed with 10 mL distilled water and 500μL Folin-Ciocalteu reagent and incubated in the dark for 30 min at 25°C.Absorption was recorded at 750 nm using a UV-Vis spectrophotometer,and the concentration was determined by plotting against a known concentration of phenol(Berliet al.,2009).

Ethylene emission was measured by transferring the plants into a customized 1-L gas chromatography bottle and closing the lid for 2 h,thereby collecting 1 mL air from the headspace and injecting it into a gas chromatograph(dsCHROM 6200,Donam Instruments Inc.,Republic of Korea)equipped with a Poropak-Q column.The study was carried out in a randomized block design,and three independent trials were conducted for data validity.The data obtained were subjected to one-way analysis of variance(ANOVA),and significant differences between the means within the treatments were determined by Tukey’s test atP＜0.05,using SAS package,Version 9.4.

These investigations,along with the genetic mapping ofB.linensRS16,assisted in addressing our objectives.Genetic mapping of the bacterium(NCBI accession no.NZ_CP030797.1)has been shown to alleviate UV-B radiation mediated stress as it contains three specific genes,viz.,phytoene desaturase(protein ID:WP_139908826.1),phytoene synthase(protein ID:WP_139908827.1),and ectoine synthase(protein ID:WP_135810511.1).These genes are important for protecting DNA from damage induced by ionizing radiation(Klassen,2010;Schröteret al.,2017).

Ethylene emission increased significantly for rice plants after 3 and 4 d of exposure to UV-B stress,compared to the control plants(Fig.1),indicating that the longer duration of exposure resulted in a significant increase in the ethylene emission.The two different levels of UV-B stress treatment showed similar trends in the induction of ethylene emission,with the plants exposed to 1 Wm-2UV-B treatment showing 26%higher ethylene emission rate compared to those exposed to 0.5 Wm-2UV-B treatment after 4 d of stress exposure.These observations are in line with a previous study in which elevated ethylene emission levels were observed under UVB exposure(Anet al.,2006).Moreover,inoculation withB.linensRS16 resulted in 12.2%and 16.4%decreases in ethylene emission,respectively,after 3 and 4 d of exposure to 0.5 Wm-2UV-B,compared to non-inoculated plants.On the other hand,bacterial inoculation resulted in 19.4%and 19.2%decreases in ethylene emission,respectively,after 3 and 4 d of 1 W m-2UV-B treatment.The inoculation ofB.linensRS16 reduced ethylene emission rate,as it has the ability to produce ACCd,which can scavenge the precursor of ethylene,ACC,and use it as a nitrogen source,for its growth and development(Siddikeeet al.,2010;Glick,2014).

Fig.1 Ethylene emission from rice plants inoculated with B.linens RS16 after 2,3,and 4 d of ultraviolet B(UV-B)exposure.Values are means with standard errors shown by vertical bars(n=3),and bars with different letters indicate significant differences(P＜0.05)among the treatments at each day.FW=fresh weight;NIc=non-inoculation;NIc+0.5W=noninoculation with 0.5 Wm-2 UV-B exposure;NIc+1W=non-inoculation with 1 Wm-2 UV-B exposure;Ic=inoculation;Ic+0.5W=inoculation with 0.5 Wm-2 UV-B exposure;Ic+1W=inoculation with 1 Wm-2 UV-B exposure.

Elevated ethylene emission level due to UV-B exposure can also lead to oxidative stress through higher accumulation of ROSin planta,by the upregulation of membrane-bound RBOHD protein(Yaoet al.,2017).In this study,UV-B induced significantly higher ROS accumulation in rice plants,measured in terms of the H2O2concentration,irrespective of bacterial inoculation throughout the stress exposure period(Fig.2a).Furthermore,B.linensRS16 inoculation significantly reduced the H2O2concentration compared to the non-inoculated plants(Fig 2a,b).After 4 d of UV-B stress exposure,bacterial inoculation resulted in 30%and 25%reductions in H2O2concentration for 0.5 and 1 Wm-2UVB exposure,respectively,compared to the non-inoculated plants(Fig.2a).Under stress conditions,B.linensRS16 enhances the activities of antioxidant enzymes such as catalase,superoxide dismutase,and ascorbate peroxidase,which might be a potential mechanism for scavenging ROS and decrease the detrimental effect of ROS on plant physiological processes(Chatterjeeet al.,2018).

Fig.2 Hydrogen peroxide(a)and malondialdehyde(b)concentrations of rice plants inoculated with B.linens RS16 after 2,3,and 4 d of ultraviolet B(UV-B)exposure.Values are means with standard errors shown by vertical bars(n=3),and bars with different letters indicate significant differences(P＜0.05)among the treatments at each day.FW=fresh weight.See Fig.1 for detailed description of each treatment.

The UV-B radiation can accelerate the destabilization of plant membrane integrity through enhanced lipid peroxidation and electrolyte leakage,due to ROS accumulation(Berliet al.,2010;Liet al.,2014;Maet al.,2019).This study also reported an increase in lipid peroxidation,measured in terms of MDAconcentration(Fig.2b).Furthermore,the decrease in H2O2concentration with inoculation ofB.linensRS16 significantly reduced the MDA concentration throughout the UV-B exposure period,with 15%and 14%reductions for 0.5 and 1 W m-2UV-B exposure,respectively,compared to the non-inoculated plants(Fig.2b).On the other hand,electrolyte leakage is a typical biomarker for monitoring damage to plant cell membranes from elevated H2O2concentration(Maet al.,2019).Electrolyte leakage was significantly enhanced upon UV-B exposure after 2,3,and 4 d of stress treatment,irrespective of bacterial inoculation.The plants exposed to 1 W m-2UV-B radiation showed higher electrolyte leakage compared to other treatments(Fig.3b).However,bacterial inoculation significantly decreased electrolyte leakage under UV-B exposure compared to non-inoculated plants.Inoculation withB.linensRS16 decreased electrolyte leakage by 13.6%,8.3%,and 13.4%after 2-,3-,and 4-d exposure to 1 Wm-2UV-B,respectively,whereas 15.6%,15.4%,and 13%reductions were observed for plants exposed to 0.5 Wm-2UV-B irradiation after 2,3,and 4 d,respectively,compared to non-inoculated plants.These results are in line with previous reports where inoculation with ACCd-producing bacteria resulted in lower MDA concentration and electrolyte leakage through the enhancement of antioxidant defense mechanisms under environmental stress conditions(Subramanianet al.,2016;Chatterjeeet al.,2018).

Fig.3 Polyphenol concentration(a)and electrolyte leakage(b)of leaves of rice plants inoculated with B.linens RS16 after 2,3,and 4 d of ultraviolet B(UV-B)exposure.Values are means with standard errors shown by vertical bars(n=3),and bars with different letters indicate significant differences(P＜0.05)among the treatments at each day.FW=fresh weight.See Fig.1 for detailed description of each treatment.

Polyphenols,including flavonoids,are regarded as potential antioxidants that restrict UV-B penetration through the epidermal tissues of plants(Surjadinataet al.,2021).In this study,polyphenol concentration was significantly higher in the leaves of UV-B-exposed rice plants than those of control plants throughout the exposure period(Fig.3a).The polyphenol concentration increased by 4.9%,18.7%,and 28%after 2,3,and 4 d of 0.5 Wm-2UV-B exposure,whereas 13%,35.8%,and 41.4%increases were observed after 2,3,and 4 d of 1 W m-2UV-B exposure,respectively,compared to the control plants(Fig.3a).Furthermore,bacterial inoculation significantly decreased polyphenol concentration after 2,3,and 4 d of UV-B exposure compared to the noninoculated plants.After 4 d of stress treatment,B.linensRS16 inoculation resulted in 11.6%and 11.8%decreases in polyphenol concentration compared to the non-inoculated plants for 0.5 and 1 Wm-2UV-B exposure,respectively.Our study corroborates a previous report where PGPR-inoculated cucumber plants exhibited lower polyphenol concentration under salinity and drought stress(Kanget al.,2014).The decreased polyphenol deposition in bacteria-inoculated plants can be attributed to enhanced tolerance to UV-B radiation.

This study showed that ethylene emission from rice plants increased with an increase in the intensity of UV-B exposure.The elevation in ethylene emission resulted in enhanced ROS accumulation,peroxidation of lipid residues in cell membranes,electrolyte leakage,and polyphenol deposition.Inoculation with ACCd-producing bacteria reduced ethylene emission from plants exposed to UV-B radiation and subsequently suppressed ROS accumulation,stabilized the cell membrane by decreasing lipid peroxidation and electrolyte leakage,and reduced polyphenol deposition in leaves.This study puts forward a new perspective in the characterization of UV-resistant ACCd-producing plant growth-promoting bacteria for the amelioration of ionizing radiation in stress agriculture.

ACKNOWLEDGEMENT

This study was supported by the Basic Research Program through the National Research Foundation(NRF)funded by the Ministry of Education,Science and Technology,Republic of Korea(No.2021R1A2C1006608).

CONTRIBUTION OFAUTHORS

Jeongyun Choi and Aritra Roy Choudhury contributed equally to this work.