The Effects of UV-B Radiation on Maize at the Seedling Stage

BeitaoXIE,QingmeiWANG,HaiyanZHANG,AixianLI,FuyunHOU,BaoqingWANG,Shunxu DONG,Liming ZHANG

1.Crop Science Research Institute,Shandong Academy of Agricultural Sciences,Jinan 250100,China;2.Shandong Academy of Agricultural Sciences,Jinan 250100,China

The ozone layer in the stratosphere has been thinning steadily,due to the increased emissions of CFCs and other nitrides such as N2O and so on,into the atmosphere since the last century.This has resulted in an intensity of ultraviolet-B(UV-B)radiation,which has been increasing steadily in recent decades[1].It has generally been reported that the O3layer has thinned by 1%,and the intensity of the UV-B radiation reaching the earth has increased by 2%[2].UV-B radiation is reported to alter plant growth,development,and morphology[3-5].The noxious effects of UVB radiation,such as causing damage to DNA,proteins,and membranes leading to altered metabolisms,are often a consequence of the reactive oxygen species(ROS)production[6].

Despite the potential impact of UV-B radiation on its growth and development,and therefore on the overall productivity of its species,maize remains the third most important worldwide crop,following wheat and rice.It has been reported that UV-B radiation decreased the biomass,plant height,and chlorophyll content of maize at the seedling stage[7].Maize is a crop with obvious hybrid vigor,and the current results were based on one or a few of the first-filial generation(F1).However,few studies regarding the response of maize parents and their F1to UV-B radiation stress have been conducted.In this research study,the response of maize parents and their F1to UV-B radiation has been characterized in terms of its effects on morphological development and photosynthesis,along with the activity of anti-oxidase and UV-B absorbing pigment content,in order to study the response of maize parents and their F1to UV-B radiation.

Materials and Methods

Plant growth and lighting conditions

The experiment was conducted in a culture room,and the hybrid maize F1 variety NONGDA108(ND108),its male parent HUANG C(HC),and female parent 178 were applied as the experimental material.The selected full seeds were soaked for 12 h,and were then placed in quartz sand.They were allowed to germinate in dark,with a controlled temperature and humidity for 3 d.The seedlings were grown at 25℃using a 12 h light/12 h dark photo-period,with 400 mmol/(m2·s)photosynthetically active radiation (PAR)from metal halide lamps(400 W,HPIT,Phillips Inc.,Belgium)until the second leaf was fully expanded.Then,the uniformly-developed seedlingswere hydroponically transplanted in a 1:2 diluted Hoagland nutrientsolution,which was aerated continuously by air compression for 3 days.At that point,the plants were grown in a full Hoagland nutrient solution.

Treatment of UV-B radiation

A UV-B treatment was applied to the plants 3 d after the transplanting of the seedlings.The control was generated with a combination of the metal halide lamps(400 W,HPI-T,Phillips Inc.,China),and fluorescent lamps(40 W,F72T12;Phillips Inc.,China),and filtered through clear polyester film(Folanorm 0.1 mm,Folex,Dreieich,Munich,Germany,cut-off 320 nm),in order to screen against UV-B and UVC radiation.The UV-B radiation,with a predominant irradiation of 308±10 nm,was imposed using UV-B emitting fluorescent lamps (TL12/40W/01,China Electricity-Optical Source Institute,Beijing,China)which were hung above the plants,and a cellulose acetate film(Ultraphan transparent;0.13 mm,Wettlinger Werkst of fe-Vienna,Austria)was used to screen against minor UV-C emissions (＜280 nm).The PAR fluence rate was measured using a calibrated spectroradiometer((Mod Li-1800,Li-Cor,Lincoln,USA).The UV-B radiation was measured with a broad-band UV-B spectroradiometer(peak wave-length:308 nm;Beijing Normal University,China).The UV-B radiation was applied during the central 6 h of the PAR photo-period,and the biologically-effective irradiance(UV-BBE)amounted to 5.4 kJ/(m2·d),using a Caldwell general plant damage action spectra normalized at 300 nm[1].

Activity measurement

The plants were harvested 6 d after the UV-B radiation treatment,and the fourth leaves of each block were sampled,weighed,snap frozen in liquid nitrogen,and stored at-80℃for the physiology determination.Six uniformly-developed plants were sampled for measuring the plant height,biomass,and area of the fourth expanded leaf.The chlorophyll content of the fourth expanded leaf was determined with a portable chlorophyll measurement(SPAD 502,Spectrum,USA).The chlorophyll fluorescence was determined with a portable PAM-2000 fluorometer (Heinz Wlaz,Effeltrich,Germany).The netphotosynthetic rate,stoma conductance,inner cellular CO2concentration,and transpiration rates,were measured using a Li-6400(Li-Cor,Lincoln,USA)portable photosynthesis measurement system.Eight plants per group were measured,taking the fourth fully expanded leaf.The applied light resource was a selfportable red-blue light,and during the measurements the PAR levels were maintained at 1 000 mmol/(m2·s).The gas flow through the ADC was 400 μmol/s,and the leaf chamber temperature varied between 25 and 27℃.

To measure SOD,CAT,POD,and MDA,0.5 g frozen samples were homogenized with 7 ml of 50 mmol/L phosphatebuffer solution(PH7.0),then ground in a mortar with an ice water mixture,and centrifuged at 16 000×g for 15 min in a refrigerated centrifuge.The supernatant was collected in a test tube,and then refrigerated for the determination of the enzyme activity and MDA content.The SOD activity was measured according to the methodintroducedbyZhangand Kirkham[8].At this point,20 μl of supernatant was added to a 3 mλ reaction solution containing 63 μmol/L of nitroblue tctrazolium (NBT;2’-di-p-nitrophenyl-5,5’-diphenyl [3,3’-dimcthoxy-4,4’-diphcnylcnc]);1.3 μmol/L riboflavin(7,8-dimethyl-10-ribitylisoalloxazine);13 μmol/L methionine;0.1 μmol/L thylenediaminelelraacctic acid(EDTA;thylenediamine-N,N,N’,N’-tetraacetic acid);and 50 mmol/L phosphate buffer(PH 7.8).The solutions were placed under light at 80 μmol photons m2·s for 10 min.The absorbance of the reacted solutions and non-reacted solutions at 560 nm was determined with a spectrophotometer(DU800,Beckman Coulter,Inc.,USA).One unit of SOD activity was defined as the amount of SOD required to cause a 50% inhibition of the rate of NBT reduction at 560 nm.The activity of CAT was determined as a decline in absorbance at 240 nm for 1 min[9].The 3-ml reaction solution contained 15 mmol/L H2O2,and a 50 mmol/L phosphate buffer(pH 7.0),as well as 0.1 to 0.2 ml enzyme extract.The activity of POD was measured as a decline in absorbance at 470 nm for 1 min.The 5-ml reaction mixture contained 20 mmol/L guaiacol (1-hydroxy-2-methoxybenzcnc),a 10 mmol/L phosphate buffer(pH 7.0),and 0.1 mmol/L enzyme extract.The reaction was started by adding 20 μl of 40 mmol/L H2O2.Then,the MDA content was measured using the method of Dhindsa[10].A 1-ml MDA extract was added to 4 ml of trichloroacetic acid containing 0.5% thiobarbittric acid [4,6(1H,5H)-pyrimidinedionc]. The solution was heated at 95℃for 30 min,and then quickly cooled in running water.The solution was centrifuged at 10 000×g for 10 min.The absorbance of the supernatant was measured at 532 and 600 nm.The concentration of MDA was calculated by the subtraction of A600from A532,and an extinction coefficient of 155 mm/cm for MDA[11].

The analysis of the UV-B absorbing compounds,and the UV-B absorbing compounds (mainly flavonoids),were measured using the procedure described by Mirecki and Teramura[12].A 0.5 g of fresh seedlings were excised and left overnight at 4℃with 25 ml acidified methanol(methanol/H2O/HCl=79/20/1,v/v/v).The extracts were diluted 1∶20 with the same solution,and their absorbance spectra were recorded in the range of 280 to 400 nm(UV/Vis DU800 spectrophotometer,Backman,USA)[13].

Results and Analysis

The effects on agronomy ideology

Fig.1 illustrates thatthe plant heights of 178 and ND108 undergoing UV-B radiation were decreased by 10.9% and 11.4%,respectively,compared with that of the control.Also,the plant height of HC was increased by 15.5%,which indicated that the effects of the UV-B radiation on the plant height of ND108 fell in between the effects on that of the 178 and HC.The leaf area of the ND108 exposed to the UV-B radiation was decreased by 20.6% ;the leaf area of the 178 was decreased by 25.3% ;and the leaf area of the HC was decreased by 10.2% compared with the control.The decrease of the ND108 and 178 were significant.The decrease of ND108 was found to be between that of the 178 and HC.The inhibition of the UV-B radiation on the ND108 was stronger than in the male parent HC,and weaker than in the female parent.The UV-B radiation evidently inhibited the biomass of the ND108 and its parents.The biomass of 178,ND108,and HC was decreased by 28.1%,25.0%,and 15.4%,respectively.The fall of the biomass in the ND108 and 178 was found to be remarkable,while that of the HC was insignificant.

The chlorophyll content of the fourth leaf in the ND108 was higher than that in 178 and HC under the controlled conditions.The chlorophyll content in the ND108 was decreased significantly(12.1% )under the UV-B radiation conditions,while that of 178 was hardly decreased,and the HC was decreased minimally.These results indicated that the F1ND108 genetically surmounted its male parent in regard to chlorophyll content.

The effects on photosynthesis

The photosynthesis as the base of the biomass,corresponded to the reduction of the biomass under the UV-B treatment.The photosynthetic rate of the ND108 dropped observably by 26.6% as shown in Fig.2A,and the photosynthetic rate of the 178 and HC decreased slightly.

In addition to the photosynthetic rate,the stoma conductance,inner cellular CO2concentration,and transpiration rate,were the important indexes ofthe photosynthesis.The stoma conductance indicated the amount of H2O and CO2which entered the leaves.The more H2O and CO2entering the leaves,the higher the stoma conductance.Also,the photosynthetic rate was correspondingly higher.Fig.2B shows that the UV-B treatment reduced the stoma conductance of the ND108 and its female parent 178 significantly,which dropped 24.0% and 47.6%,respectively.However,the stoma conductance of the HC increased slightly with the UV-B radiation treatment.

While corresponding to the reduction of the stoma conductance,the transpiration rate of the ND108 and 178 decreased with the UV-B radiation,especially the transpiration rate of the ND108,which dropped significantly.However,the transpiration rate of the HC increased while undergoing the UV-B radiation treatment.

CO2is one of the most important raw materials of photosynthesis,and the inner cellular CO2concentration in the leaves increased due to the decrease of the photosynthetic rate.Fig.2D illustrates that the inner cellular CO2concentration of the ND108 exposed to UV-B radiation rose by 8.1%,when compared to that of the control.These results corresponded to the reduction of the photosynthetic rate.The inner cellular CO2concentration of the 178 and HC samples changed only slightly.

The effects on the chlorophyll fluorescence(Fv/Fm)

The chlorophyll fluorescence(Fv/Fm)of the leaves was also a factor impacting the photosynthesis,and theFv/Fmvalue presented a positive relation with the photosynthetic rate.Fig.3 shows that theFv/Fmvalue of the ND108 dropped by 2% after being slightly treated with UV-B radiation.The 178 showed a similar trend to the ND108,and at the same time,theFv/Fmvalue of the HC increased by 2% while undergoing the UV-B radiation.

The effects on anti-oxidase activity

The anti-oxidase are the important enzymes which eliminate the active oxygen in plants,the activity of which changes with the plants’development and the variations of the external environment.It can be seen from Fig.4A that the SOD activity of the ND108 decreased with the UV-B radiation exposure,and that of the 178 was nearly unchanged.Meanwhile,that of the HC increased significantly(34.4% ).With the UV-B radiation,the POD activity of the ND108 decreased by 13.3% as shown in Fig.4B,while that of the 178 increased significantly(35.3% ),and that of the HC changed minimally.The treatment of the UV-B radiation increased the CAT activity by 8.6% in the ND108.In Fig.4C,it can be seen that the effects of the UV-B radiation on the CAT activity of the 178 was similar to that of ND108,but the CAT activity of the HC decreased by 11.5%,which was significant.The UVB radiation increased the MDA content of the leaves in the 178,ND108,and HC by 131.3%,39.2%,and 202%,respectively.These difference were all significant(P＜0.01),which indicated that the resistance capability of the ND108 to UV-B radiation stress was stronger than the parent plants,and the hybrid vigor was obvious.

The effects on flavonoid content

The absorbance range of 290 to 350nm represent the flavonoid contents of the leaves.The higher the absorbance,the higher the flavonoid content.Fig.5 shows that the UV-B radiation increased the flavonoid of the leaves in the 178,ND108,and HC.The difference was that the flavonoid of the leaves in the 178 was lower than that in the ND108 and HC on the matter undergoing the UV-B radiation,as well as in the control condition.

Discussion

The effects of the UV-B radiation on the plants were diverse.The variant trier,experimental material,and experimental method may achieve different results[14].Generally speaking,the plants which grew to higher dimensions,or a high elevation zone,presented a higher resistivity to the UV-B radiation than the plants which grew in lower dimensions or a lower elevation zone.Themonocotyledonshada higher resistivity to the UV-B radiation than dicotyledons[15].Maze is a worldwide field crop with a higher resistivity to UV-B radiation when compared with soybean and cotton.Maze presented a different resistivity due to its breeding from different genetic backgrounds[16].ND108 is a widely planted variety with large acreage in China,and the male parent HC and female parent 178 were grown in different environments.For this reason,the resistivity to the UV-B radiation of the ND108 and its parents was not exactly the same.Some of the effects of the UV-B radiation to the ND108 were similar to that of 178;some were similar to that of the HC;some were the same as 178 and HC;and still others were different from 178 and HC.

It was found that a certain dose of the UV-B radiation was necessary for maize to proceed with normal physiological and metabolic activity[17].When the external UV-B radiation was lower than the optimum dose required,then the growth of the maize was promoted with the increase of the UV-B radiation.However,when the external UV-B radiation exceeded the optimum dose,then the growth of the maize was inhibited,while different maize varieties required different optimum doses of UV-B radiation.For this reason,in this experimentsomemorphologicalor physiological data of the ND108 or its parentswerepromotedunderthe stress of the UV-B radiation.

In this study,the UV-B radiation was stronger than the optimum dose of UV-B radiation required for the ND108,and the maize was suppressed and damaged.This resulted in the reduction of plant height and biomass,and the narrowing of the leaf area,as well as visible inhibition being a direct reason for the inhibition of the photosynthesis in the ND108 exposed to the UV-B radiation.The reason that caused the decrease of photosynthesis in ND108 and itsparentsare twofold.One factor was that the stoma resistance increased with the UV-B radiation exposure,and the amounts of H2O2and CO2photosynthesis raw material entering into the leaves were decreased.This represented a falling of the stoma conductance and transpiration rate,which was consistent with most of the previous reports[18].The other factor was that the photosynthetic ability decreased as it was irradiated by UV-B radiation,which was mainly exhibited in the increase of the inner cellular CO2(Ci)concentration in the ND108 and its parents,and also the decreased Fv/Fm value.The main cause was that the activity of the enzyme(PEP and Rubiscase)involved in photosynthetic reaction decreased or was devitalized[19-20].The stoma conductance and transpiration rate of the HC rose slightly when irradiated by the UV-B radiation,while the Ci and photosynthetic rate were nearly unchanged.These results meant that the resistance of the HC to the UV-B radiation was higher than that of the ND108 and 178.

The inhibition and damage to maizecausedbyUV-B radiation,such as damage to the DNA,and proteins and membranes, which leads to an altered metabolism,are often a consequence of the reactive oxygen species(ROS)production in the plant cells[6].The ROS led peroxidation in the membranes resulted in a leakage of the inramembranous component production of malondialdehyde(MDA)[21].The defense mechanism of the maize to stress was to generate large numbers of inoxidizable substances,including antioxidase and antioxidant.The antioxidase contained SOD,CAT,and POD,and the antioxidase and ROS in the maize was kept in equilibrium in the control condition.When the maize was exposed to UV-B radiation,the ROS was largely produced,and the antioxidase was subsequently activated in order to reach a new equilibrium in the stress condition.The further the stress was imposed,the more ROS was produced,and then the antioxidase activity reduced sharply,after increasing to some extent[22].The time period of the antioxidase initiated change in the plants with different resistances to stress was found to be different.As shown in Fig.4D,HC suffered the greatest degree during the UV-B radiation exposure,and ND108 also was greatly affected,followed by the 178 with the weakest degree of damage.Fig.4A shows that with UV-B radiation,the SOD activity of the 178 and ND108 changed invariably,and that of HC and increased signally,which indicated that the activation of the SOD activity in HC was the largest.The inconsistency of the MDA content,and the activity of the antioxidase indicated that there were other antioxidants besides antioxidase to scavenge the ROS in the maize.The main antioxidants were secondary metabolic matter including flavonoid,cinnamic acid,and vitamin E,and so on[23].Among these,the flavonoid was one type ofimportantsecondary metabolic matter.Fig.5 shows that the flavonoid content of the ND108 and its parents increased significantly after being irradiated by UV-B radiation,and it seemed that the UV-B radiation was involved in the biosynthesis of the flavonoid[24].

As shown in Fig.1,2 and 3,maize is a field crop with visible hybrid vigor in its yield,which is apparent in the plants’height,leaf area,and biomass.However,the maize barely displayed any hybrid vigor in its resistance to UV-B radiation,which was represented by the inhibition rate[Inhibition rate(% )=(CC-CU)/CC×100].The lower the inhibition rate,the higher the resistance of the maize to the UV-B radiation.In regard to the biomass,the inhibition rate of UV-B radiation in 178,ND108,and HC was found to be 28.1%,25.0%,and 15.4%,respectively.No hybrid vigor was presented,but a mid-parent effect in the biomass was evident.Meanwhile,the inhibition rate in the chlorophyll content was 0%,10%,and 4%,respectively,which displayed a sub-parent effect.It was possible that the yield was one of the important goals in the breeding of the ND108,and the resistance to UV-B radiation was not taken into account.With the continuing increases of UV-B radiation,it is suggested that the resistance to UV-B radiation should be one of the goals in the future breeding of maize or other field crops.

Conclusions

Plants at the seedling stage were grown in soilless cultures,with and without UV-B radiation at a dose of 5.4 kJ/(m2·d).Three days later,it was found that the treatment of UV-B radiation significantly reduced such characteristics as biomass,plant height,leaf area,and so on.Also,the photosynthesis of the maize was inhabited,the anti-oxidase activity decreased,and the MDA concentration increased with the UV-B radiation exposure.The maize presented visible hybrid vigor under control condition,while the hybrid vigor under UV-B radiation barely emerged in the biomass,plant height,leaf area,and physiological metabolic index.

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