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1、Growth,morphological and photosynthetic characteristics,antioxidant capacity,biomass yield and water use efficiencyof Gynura bicolor DC exposed to super-elevated CO2Minjuan Wanga,Chen Donga,Yuming Fua,b,Hong Liua,b,c,naInstitute of Environmental Biology and Life Support Technology,School of Biologic

2、al Science and Medical Engineering,Beihang University,Beijing 100191,ChinabInternational Joint Research Center of Aerospace Biotechnology&Medical Engineering,Beihang University,Beijing 100191,ChinacState Key Laboratory of Virtual Reality Technology and Systems,School of Computer Science and Engineer

3、ing,Beihang University,Beijing100191,Chinaa r t i c l e i n f oArticle history:Received 15 July 2014Received in revised form16 April 2015Accepted 4 May 2015Available online 13 May 2015Keywords:Antioxidant systemBioregenerative life support system(BLSS)Carbon dioxideG.bicolorPhotosynthesisa b s t r a

4、 c tAs a consequence of the increasing importance of leaf vegetables in Bioregenerative lifesupport system(BLSS),there is an interest in enhancing both the productivity and qualityof Gynura bicolor DC(G.bicolor).The effects of super-elevated carbon dioxide concentra-tion(3000 ppm)on G.bicolor in dif

5、ferent periods were investigated to reveal plausibleunderlying mechanisms for the growth,photosynthetic characteristics,antioxidantcapacity and biomass yield.Experimental results showed that not only the photosyntheticrate but also the antioxidant capacity were increased in short-term super-elevated

6、 CO2concentration.However,under long-term super-elevated CO2concentrations,photosyn-thetic rate and pigment contents of G.bicolor significantly decreased due to photoinhibition.Elevated CO2can be able to dwarf the G.bicolor plants.The leaves in high CO2concentration were curled,which may affect the

7、photosynthetic efficiency.At 3000 ppmlevel,leaf senescence accelerated but biomass yield had no significant difference.Furthermore,elevated CO2controlled transpiration rate and then enhanced water useefficiency.Superoxide dismutase(SOD)activity,peroxide activity(POD)and totalantioxidant capacity(T-A

8、OC)in 3000 ppm condition were significantly lower than thatin 400 ppm condition.On the contrary,catalase(CAT)activity,glutathione(GSH)contentand hydrogen peroxide(H2O2)were significantly enhanced under high CO2concentration.&2015 IAA.Published by Elsevier Ltd.All rights reserved.1.IntroductionThe gl

9、obal atmospheric carbon dioxide concentration(CO2),presently at about 385 mol mol?1CO2mol?1air,may surpass 700 mol mol?1before the end of thiscentury 1.A rise in atmospheric CO2 and othergreenhouse-effect gases is expected to cause changesin global climate,including increases in air temperaturesand

10、shifts in regional scale rainfall patterns,which couldresult in decreased soil water availability in some areasof the world 2,3.Growing plants under elevated CO2mayneutralizeormitigatethenegativeeffectsofthe stressful environmental conditions on production,maintaining or even improving the antioxida

11、nt system4,5.Contents lists available at ScienceDirectjournal homepage: Astronauticahttp:/dx.doi.org/10.1016/j.actaastro.2015.05.0100094-5765/&2015 IAA.Published by Elsevier Ltd.All rights reserved.nCorresponding author at:Institute of Environmental Biology and LifeSupport Technology,School of Biolo

12、gical Science and Medical Engineer-ing,Beihang University,Beijing 100191,China.Tel./fax:86 10 82339837.E-mail addresses:(M.Wang),wenjian_(C.Dong),(Y.Fu),LH(H.Liu).Acta Astronautica 114(2015)138146Establishment of Bioregenerative life support system(BLSS)is an ultimate way to provide food,oxygen andw

13、ater for astronauts carrying out long-term mannedspaceflight and deep space exploration 6,7.Plants willbe an integral part in BLSS for long-term,human spaceexploration 8,9.CO2enrichment is the sole source ofcarbon for plants,which will act as carbon fertilizer,resulting in far reaching consequences

14、for plant produc-tion generally,grain yield and quality specifically 10,11.More importantly,the CO2levels for advanced lifesupport systems may be at super elevated levels 12and CO2may be used as a pressurizing gas for growingplants on Moon or Mars.It is very important to selectplants which are resis

15、tant to high CO2concentration.The effects of carbon dioxide on plant growth have beenwidely studied,especially in conjunction with increasingconcerns about rising CO2concentrations in the Earthsatmosphere12,13.Ingeneral,thesestudieshaveshown that increases in CO2(e.g.,a doubling from 350to 700 mol m

16、ol?1)increase photosynthetic rates for C3species and decrease water use per unit area of vegeta-tion for C3and C4species 14,15.The present atmo-sphere CO2 limits the growth of C3crop plants,whichshow responses to elevated CO2 via reduced photo-respiration and enhanced photosynthetic rates,therebyinc

17、reasing their growth and yield.Gynura bicolor DC(G.bicolor),which originates in thetropical area of East Asia,is one of perennial vegetablesgrown for its edible young immature leaves and stems.Itcontains relatively high nutritional value of vitamin,ferrite,manganese and flavonoids with higher antiox

18、i-dant activity and serve as one of the candidate plants inthe life support system 5.Recent studies have shownthat a diet rich in antioxidants can mitigate the harmfuleffects of the stressful environmental conditions duringlong-term space flight 4,16.Therefore,planting thevegetables rich in antioxid

19、ants in the space environmentcannot only increase the dietary diversity of the astro-nauts,but also reduce the risk of space radiation to theastronauts 16.The responses of different species to highCO2were significantly different.Hence,it is necessary tounderstand the mechanisms underlying the differ

20、enteffects of high CO2concentration on the yield and anti-oxidant capability of G.bicolor.Moreover,as a conse-quence of the growing global demand for food,and theincreasing awareness of the importance of fruits andvegetables in the human diet,plant physiologists,agri-culturists,andplantbreedershaved

21、emonstratedincreasing interest not only in enhancing crop produc-tivity but also in improving the nutritional and health-promoting qualities of commonly consumed fruits andvegetables 17.In this study,we evaluated the response of growth,morphological and photosynthetic characteristics,antiox-idant ca

22、pacity,biomass yield and water use efficiency of G.bicolor plants under different CO2concentrations(400 and3000 ppm)in the closed environment growth chambers.The results provide some basic information on the optimalcultivation pattern for stable,good quality,high yields ofleaf vegetables in BLSS.2.M

23、aterials and methods2.1.Cultivation conditionsG.bicolor DC plants(16 seedlings per treatment)wereinitially maintained under low lighting for 3 days toacclimate the new growth circumstances after beingtransplanted in the pots,then subjected to different CO2treatments.Mixtures of red plus white LEDs(R

24、W,R:W1:1)were used 9.For all treatments lighting wascontinuous(24/0 h light/dark).Photosynthetic photon fluxdensity(PPFD)levels were measured at the top of plantcanopy with a quantum sensor(Li-250 A,Li-Cor,USA).PPFD was about 250 mol m?2s?1for all the treatments.Air temperature and relative humidity

25、 were maintained ingrowth chambers at 2171.3 1C and 7073.4%,respec-tively.The modified Hoagland nutrient solution(HoaglandandArnon,1950)included:NH49.0 mg/L;Ca280.1 mg/L;K 117.2 mg/L;Mg2 24.05 mg/L;NO?3?310.4 mg/L;H2PO?4?48.5 mg/L;SO?4?96.2 mg/L and thepH was 5.65.8.One chamber was set to maintain t

26、he CO2concentra-tion at 400 ppm(ambient conditions)and the other at3000 ppm(super-elevated CO2conditions).CO2monitor-ing was done with non-dispersive infrared CO2sensors(T6615,USA).Pure carbon dioxide(99.9%purity;Haipu gasco.ltd,Beijing)was supplied from a high concentrationcarbon dioxide cylinder a

27、nd injected through a pressureregulator into the closed chamber.Online infrared CO2analysis instrument(CICS)18 was used to measure theCO2concentration.Ambient CO2concentration served asthe control.2.2.Morphological and physiological analysesPlants were harvested at 15 days after planting(DAP),wherei

28、n 6 plants were selected randomly from eachtreatment to conduct morphological measurements.Plantheight(PH)and root height(RH)was measured from plantbase to tip of the main stem with straight scale.The freshweight(FWm1,g plant?1)of plants were determined,and then put it in the drying oven(85 1C)for 4

29、8 h.Thedried weight was expressed as m2.Biomass of the freshand dry weight were measured with an electronic balance.Water content(WC)was calculated according to thefollowing equation:WC m1?m2m1?100%12.3.Photosynthetic characteristics analyses2.3.1.Chlorophyll and carotenoid contentsChlorophyll was e

30、xtracted from the leaves of 6 plants at asimilar position within each treatment randomly whenplants were harvested at 15 days.The minced sample with0.1 g(fresh weight,FW)each,were placed in a mortar,and alittle of quartz sand and calcium carbonate powder,and 2 to3 ml of 95%ethanol were added,and the

31、n homogenized.M.Wang et al./Acta Astronautica 114(2015)13814613910 ml of 95%ethanol was added and grinded continuouslyuntil the plant tissues became white.The homogenizedsamples were filtered into a 25 ml volumetric flask afterkeeping for 3 to 5 min,and then 95%ethanol was added andmixed.The optical

32、 density was measured with a UV-1200spectrophotometer(SP-75,Shanghai spectrum instrumentsCo.,LTD,China)at 663 nm(OD663)for chlorophyll a(Chl a),and 645 nm(OD645)for chlorophyll b(Chl b)19.Total carotenoids were extracted with 100%acetone.Theirlevels were determined using the extinction coefficients

33、andequations predetermined by Wellburn in 1994 20.2.3.2.Photosynthetic efficiencyPortable photosynthesis instrument(Li-6400XT,Li-Cor,USA)was used for the determination of photosyntheticcharacteristics.Leaf gas-exchange parameters includedphotosynthetic rate(Photo),transpiration rate(Trmmol),intercel

34、lular CO2concentration(Ci)and stomatal conduc-tance(gs)of the second leaf at the G.bicolor terminal bud.These parameters were analyzed at 3,6,9,15 days.Wateruse efficiency(WUE)was calculated as the ratio of photo-synthetic capacity to transpiration.2.4.Antioxidant capacity analyses2.4.1.Superoxide d

35、ismutase(SOD)activitySOD activity was determined based on the method aspreviously described by Beauchamp and Fridovich 21.Briefly,0.5 g fresh weight of G.bicolor leaves were ice-bathed in homogenate and centrifuged at 4000 rpm for20 min.50 mL of supernate constant volume was added to10 mL reaction m

36、ixtures containing 5%phenols,5%H2O2and 87.5%distilled water.Then 0.25 mL enzyme solutionwas added into the reaction mixture and the reaction wascarried out at a constant temperature(25 1C)for 20 min.The optical density was determined at 470 nm.Oneunit(U)of SOD activity was defined as the amount ofpr

37、otein necessary to decrease the reference rate to 50%ofmaximum inhibition.2.4.2.Peroxidase(POD)activityPOD activity was analyzed spectrophotometrically at470 nm using guaiacol as a phenolic substrate with hydro-gen peroxide 22.The reaction mixture contained 0.15 mLof 4%(v/v)guaiacol,0.15 mL of 1%(v/

38、v)H2O2,2.66 mL of0.1 M phosphate buffer(pH7.0)and 40 L of enzymeextract.Blank sample contained the same mixture withoutenzyme extract.One unit of POD activity was defined as1 ml tissue proteins depleting 0.01 mol H2O2in 1 min.2.4.3.Catalase(CAT)activityCAT activity was determined according to the me

39、thoddescribed by Kumar and Knowles 23.CAT reactionsolution consisted of 100 mM Na2HPO4NaH2PO4buffersolution(pH7.0)and 0.1 M H2O2.The optical density wasdetermined every 1 min at 240 nm.One unit of CAT isdefined as the amount necessary to decompose 1 mol ofH2O2per minute at 25 1C.2.4.4.Glutathione(GS

40、H)contentThe low molecular weight thiols and their correspondingdisulfides were analyzed according to Airaki 24 to estimatethe combined contents of GSH and hydroxylmethyl GSH(hm-GSH).Approximately 200 mg of fresh shoot sampleswere homogenized in 5%(w/v)sulphosalicylic acid using achilled mortar and

41、pestle.The homogenate was centrifugedat 10,000g for 15 min at 4 1C.The supernatant obtained wasused to quantify the LMW(low molecular weight)thiols andtheir corresponding disulfides.2.4.5.Hydrogen peroxide(H2O2)contentSamples were lyophilized and ground to pass through a40-mesh screen.Freeze-dried s

42、amples(20 mg)were mixedwith 0.1 M phosphate buffer(pH7.2)in an ice bath made of10%homogenate,and then centrifuged(4000 rmp)at 4 1Cfor 15 min.The supernatant was removed and assayed forhydrogen peroxide(H2O2)content analysis using kits follow-ing the manufacturers instruction(Nanjing Jiancheng Bioen-

43、gineering Institute,Nanjing,China)25.The absorbance wasmeasured at 405 nm and the H2O2content as mmol g?1protein.2.4.6.Total antioxidant capacity(T-AOC)Total antioxidant capacity was determined using the FRAP(Ferric Reducing Antioxidant Power)assay 26,27.Themethod is based on the reduction of the Fe

44、3TPTZ complexto the ferrous form at low pH.A sample containing 900 L offreshly prepared FRAP solution(0.3 M acetate buffer pH 3.6containing10 mM2,4,6-tripyridyl-s-triazine(TPTZ)in40 mM HCl and 20 mM FeCl3?6H2O),90 L of deionizedwater and 30 L of extract was incubated at 37 1C for 30 min.The reductio

45、n is monitored by measuring absorbance(A)change at 595 nm.FRAP values were calculated using astandard curve obtained from the analysis of a standardmix containing increasing concentrations of Fe2and wereexpressed as mmol of Fe2equivalents per kg of edible partof the vegetables.One unit(U)of T-AOC wa

46、s defined as theamount that increased the absorbance by 0.01 at 37 1C.Datawere expressed as U/mg protein.2.5.Data statisticsAll experiments were performed in triplicate.Theaverage value of total 6 measurements7standard devia-tion was regarded as the final result.All statistical analyseswere conducte

47、d using Statistical Product and ServiceSolutionsforWindows,versionSPSS20(SPSSInc.,Chicago,Illinois).The data were analyzed using analysisof variance(ANOVA),and the differences between themeans were tested using Tukeys multiple range test(Pr0.05).3.Results3.1.The response of G.bicolor growth to diffe

48、rent treatmentsThere was no significant difference in shoots of G.bicolor plants as indicated in Table 1.However,in parti-cular,the plant height(PH)and roots height(RH)werelower under 3000 ppm condition,which means elevatedM.Wang et al./Acta Astronautica 114(2015)138146140CO2can be able to dwarf the

49、 G.bicolor plants and inhibitthe roots growth.The 400 ppm condition was beneficial toG.bicolor development and growth during the cycle.Asshown in Fig.1,the most obvious influence of different CO2concentration on morphological characteristics of G.bico-lor leaves was the leaf area.The leaf area in 30

50、00 ppmenvironment was smaller than that at 400 ppm level.Furthermore,G.bicolor leaves in high CO2concentrationwere curled,which may affect the photosynthetic effi-ciency and water use efficiency.3.2.The response of photosynthetic characteristics todifferent treatmentsElevated CO2did have a significa