JOURNAL OF ENVIRONMENTAL SCIENCES 34 (2015) Available online at ScienceDirect

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1 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) 9 99 Aville online t SieneDiret Effets of nitrogen dioxide nd its id mist on retive oxygen speies prodution nd ntioxidnt enzyme tivity in Aridopsis plnts Xiofng Liu,, Fen Hou,, Gungke Li,, Nn Sng,. College of Environment nd Resoure, Reserh Center of Environment nd Helth, Shnxi University, Tiyun 6, Chin. College of Environmentl Siene nd Engineering, Nnki University, Tinjin 7, Chin ARTICLE INFO ABSTRACT Artile history: Reeived 7 Otoer 4 Revised 7 Ferury 5 Aepted Mrh 5 Aville online June 5 Keywords: Nitrogen dioxide Nitri id mist Aridopsis thlin Retive oxygen speies Antioxidnt system Nitrogen dioxide (NO ) is one of the most ommon nd hrmful ir pollutnts. To nlyze the response of plnts to NO stress, we investigted the morphologil hnge, retive oxygen speies (ROS) prodution nd ntioxidnt enzyme tivity in Aridopsis thlin (Col-) exposed to.7, 4, 8.5, nd 8.8 mg/m NO. The results indite tht NO exposure ffeted plnt growth nd hlorophyll (Chl) ontent, nd inresed oxygen free rdil (O ) prodution rte in Aridopsis shoots. Furthermore, NO elevted the levels of lipid peroxidtion nd protein oxidtion, ompnied y the indution of ntioxidnt enzyme tivities nd hnge of sorte (AsA) nd glutthione (GSH) ontents. Following this, we mimiked nitri id mist under experimentl onditions, nd onfirmed the ntioxidnt mehnism of the plnt to the stress. Our results imply tht NO nd its id mist used pollution risk to plnt systems. During the proess, inresed ROS ted s signl to indue defense response, nd ntioxidnt sttus plyed n importnt role in plnt protetion ginst NO /nitri id mist-used oxidtive dmge. 5 The Reserh Center for Eo-Environmentl Sienes, Chinese Ademy of Sienes. Pulished y Elsevier B.V. Introdution Nitrogen dioxide (NO ) is one of the most ommon nd hrmful ir pollutnts. Due to its mssive dishrge from motor vehile exhust nd sttionry soures suh s eletri utilities nd industril oilers, the onentrtion of NO in the tmosphere hs grdully inresed in mny res of the world during the pst few dedes. Therefore, NO hs een strong inditor in tmospheri environment monitoring nd potentil risk ftor in dverse effet explortion. As reported, pek levels of up to.8 8 mg/m hve een enountered in the outdoors, prtiulrly long ursides in downtown res with hevy motor vehiulr trffi (Pthmnthn et l., ). However, due to the development of industrilized prodution nd the ontinuous rise of utomoile exhust emissions, NO onentrtion will e likely to further inrese in the future. Following the inrese of NO environmentl onentrtion, nother serious issue is the ourrene of nitri id preipittion (nitri id mist), whih results from its omining with tmospheri moisture nd uses dverse eologil effets. Corresponding uthors. E-mil: ligungke@sxu.edu.n (Gungke Li), sngnn@sxu.edu.n (Nn Sng). These uthors ontriuted eqully to this work / 5 The Reserh Center for Eo-Environmentl Sienes, Chinese Ademy of Sienes. Pulished y Elsevier B.V.

2 94 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) 9 99 Reently, epidemiologi dt nd experimentl results hve linked NO exposure with wide rnge of effets on humn helth, inluding inresed mortlity risk, inresed rtes of hospitl dmissions nd emergeny deprtment visits, exertion of hroni respirtory onditions (e.g., sthm), nd deresed lung funtion (Smet nd Krewski, 7). Therefore, it is expeted tht plnts might e ffeted fter exposure to tmospheri NO nd its id mist pollution. Some studies hve demonstrted tht NO ould enter lef mesophyll tissue through stomt, omine with wter nd then onvert to nitri id, whih urns plnt tissue. The symptom ppers s lesions visile on oth sides of the leves, whih initilly ours etween lef veins or long lef edges (Li et l., 7; Mio et l., 8). A deeper level of toxiity ws lso studied for some plnts suh s Brssi mpestris seedlings, Cinnmomum mphor seedlings nd tomto plnts (M et l., 7,; Pndey nd Agrwl, 994; Teng et l., ). NO ould indue hnge to growth nd photosyntheti tivity, using oxidtive dmge ompnied y hnges in the ntioxidnt defense system (Chen et l., ; M et l., 7; Tkhshil et l., ). However, whether these toxi effets of NO pply to other plnts or whether similr symptoms will our under the stress of nitri id mist is not yet ler. Hene, Aridopsis thlin, n interntionlly reognized model plnt, ws hosen in this study to evlute the speifi plnt response to NO nd nitri id mist exposure, nd the reserh results will e extended to other plnts. There is mple evidene tht retive oxygen speies (ROS) re ruil seond messengers involved in the response to diverse ioti nd ioti stresses, nd n onfer degree of ross-tolerne ginst distint stresses (Apel nd Hirt, 4; Foyer nd Notor, 5). NO is n oxidnt pollutnt, nd indues oxidtive dmge to ell memrnes, resulting in the genertion of ROS (Mustf nd Tierney, 978; Pthmnthn et l., ). Inresed ROS n ttk iomromoleules nd result in oxidtive dmge to nulei ids, proteins nd lipids (Foyer nd Notor, 5; Mittler et l., 4; Yi et l., 5). However, plnts n svenge exess ROS y invoking the ntioxidnt defense system to void oxidtive dmge (Inzé nd Vn-Montgu, 995; Mittler, ). The indution of ntioxidnt enzymes hs een thought to e protetive retion of plnts ginst NO stress, ut the ext defense mehnism ws not ler. In the present study, we hrterized the hnges in morphologil fetures nd physiologil indexes in response to NO nd nitri id mist in Aridopsis shoots, nd determined ROS prodution nd ntioxidnt enzyme tivities. Our reserh provides prtiulr insight into the pity of NO / nitri id mist to indue ellulr ROS nd ntioxidnt response in plnt ells, nd ontriutes to understnding the underlying mehnisms of toxiologil retion nd plnt dpttion to NO /nitri id mist stress.. Mterils nd methods.. Plnt mterils nd NO /nitri id mist tretment Plnts of A. thlin (L.) eotype Columi (Col-) were purhsed from the Chinese Ademy of Agriulturl Sienes. The seeds were disinfeted with sodium hypohlorite t % (V/V, ontining Triton- t %), wshed three times with running wter, ndsokedinthewter.aftervernliztionfordyst4 C, they were seeded in seedling trys out m, 5 plnts per try, with pet imported from Germny s sustrte ( kind of pure moss pet, with proportions of N, P nd K of 4/6/8, nd ph ). Aridopsis plnts were grown in ontrolled growth hmer t ± C with 6 hr photoperiod per dy, 7% reltive humidity nd photosyntheti photon flux density of 4 μmol/(m se). Four-week-old plnts were exposed to.7, 4, 8.5, nd 8.8 mg/m NO t 6 hr/dy for 7 dys, respetively, while ontrol group ws pled in nother identil hmer, whih ws ontinully flushed with filtered ir for the sme period of time. The NO gs ws diluted with fresh ir t the intke port of the hmer to yield the desired onentrtions; then delivered to the plnts through tue positioned in the upper prt of eh hmer nd distriuted homogeneously vi fn. The NO onentrtion within the hmers ws mesured every min y the Sltzmn olorimetri method using spetrometer lirted t 545 nm (Kumie et l., 9). The desired NO onentrtions were ontrolled y the opening of flow vlve. For nitri id mist tretment, four-week-old A. thlin ws exposed to nitri id fog (ph ), whih ws produed y misting HNO solution for 4, 6 nd 8 hr/dy for 7 dys, respetively. To otin the misting HNO solution, HNO solution ws diluted y distilled wter to the proper ph vlue, nd then spryed on the lef surfe with spryer in mist flow. Menwhile, ontrol group ws pled in nother identil hmer, whih ws ontinully exposed to wter fog for the sme period of time.. Mesurement of hlorophyll (Chl) ontent Chl ontent ws determined y the method reported y Bo (5). Briefly, fresh shoots were pulverized with distilled wter, nd the homogente ws extrted y 8% etone. Asorne of the superntnt ws mesured t 66 nd 645 nm using spetrophotometer nd Chl ontent ws expressed s mg/g fresh weight (fw)... Mesurement of oxygen free rdil (O ) prodution rte prodution rte ws determined y the hydroxylmine method (Zhng nd Qu, ). Briefly, fresh lef smple ws homogenized in phosphte uffer (5 mol/l, ph 7.8), nd the homogente ws entrifuged t, g for min. The superntnt ws mixed with hydroxylmine hydrohloride, nd then kept t 5 C for hr. α-nphthylmine nd minoenzenesulfoni id were used s hromogeni gents. Asorne of the superntnt ws mesured t 5 nm using spetrophotometer. O.4. Estimtion of sorte (AsA) nd glutthione (GSH) pool The ontent of redued AsA ws determined spetrophotometrilly ording to the method of Kmpfenkel et l (995). The mount of redued GSH ws exmined ording to the method of Sgherri nd Nvri-Izzo (995).

3 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) Mesurement of lipid peroxidtion level The level of lipid peroxidtion ws estimted y mesuring the onentrtion of mlondildehyde (MDA). MDA is ommon produt of lipid peroxidtion nd is sensitive dignosti index of oxidtive injury (Jnero, 99; El-Moshty et l, 99). Fresh lef smple ws ground in 5% trihloroeti id (W/V) nd the homogente ws entrifuged t 4 g for min. The superntnt frtion ws dded to n equl volume of.6% thiorituri id (W/V). The mixture ws heted t C for min nd then entrifuged t g for min fter it ws ooled. The sorne of the superntnt ws mesured t 5 nm nd lipid peroxidtion level ws expressed s μmol MDA/g fw. Protein ronyl (PCO) ontent is the most generl inditor nd mrker of protein oxidtive dmge. In the present study, PCO level ws tested y,4-dinitrophenylhydrzine (DNPH) spetrophotometry (Levine et l., 99). Briefly, fresh lef smple (.5 g) ws ground in phosphte uffer (5 mol/l, ph 7.) nd the homogente ws entrifuged t, g for 5 min t 4 C. The superntnt ws used for DNPH retions. PCO ontent ws lulted y the sorne t 7 nm, nd the result ws expressed s nmol ronyl/mg protein..6. Assy of ntioxidnt enzyme tivities Superoxide dismutse (SOD) tivity ws mesured y Nitro Blue tetrzolium (NBT) spetrophotometry (Zhu et l., 99). Fresh lef smple (.5 g) ws homogenized in phosphte uffer (5 M, ph 7.8) nd the homogente ws entrifuged t g for min t 4 C. The superntnt ws dded to retion mixture ontining mmol/l DL-methionine (Met), 75 μmol/l NBT, μmol/l EDTA-N,ndμmol/L ltoflvin, in order to determine the sorne t 56 nm. Inhiition of 5% of the retion ws defined s one unit of enzyme nd the enzyme tivity ws expressed s U/g fw. Ctlse (CAT) tivity ws ssyed ording to the method of Zhng (99), with some modifitions. Fresh lef smple (.5 g) ws homogenized in phosphte uffer (. mol/l, ph 7.8) ontining % polyvinylpyrrolidone K. The homogente ws entrifuged t 4 g for 5 min t 4 C nd the superntnt ws used for the enzyme ssy. CAT tivity ws exmined y mesuring the derese of sorne t 4 nm in retion mixture ontining. ml H O (. mol/l) nd. ml extrt. Results were expressed s U/(min g) fw. Peroxidse (POD) tivity ws determined using the guiol method (Mehly, 955). Briefly, fresh lef smple (. g) ws homogenized in phosphte uffer (. mol/l, ph 6.) nd the homogente ws entrifuged t 4 g for 5 min t 4 C. The superntnt ws dded to the retion mixture, inluding 5 mmol/l guiol s the donor nd 6.7 mmol/l H O s the sustrte. The rte of hnge in sorne t 47 nm ws mesured nd the tivity ws expressed s DA47/min/g fw..7. Determintion of dissolved protein The mount of dissolved protein ws determined y the Brdford method (Brdford, 976). We used ovine serum lumin s the stndrd nd results were expressed s mg/g fw..8. Sttistil nlysis of dt Experiments were repeted three times, nd ll vlues were expressed s men ± SE. In the study, ll experiment groups nd ontrol group were pled in severl identil hmers seprtely, whih were kept t the sme temperture, photoperiod, reltive humidity, et. The exposure to NO nd nitri id mist were onsidered to e the single ftor for NO tretment nd nitri id mist tretment, respetively, whih were dopted to perform single ftor nlysis. Therefore, the sttistil differenes (5, or ) mong the negtive ontrol nd series of treted groups were nlyzed y one-wy nlysis of vrine (ANOVA), using the Origin 7. softwre pkge.. Results.. Effet of NO exposure on the morphology nd Chl ontent in Aridopsis shoots To find the generl toxi response, we first exposed Aridopsis to NO t different onentrtions (.7, 4, 8.5 nd 8.8 mg/m ) nd oserved their morphologil hnges. As shown in Fig., NO t lower onentrtions ffeted the plnt growth, nd indued the ourrene of yellowing lef fter 4 nd 8.5 mg/m exposure. However, when the NO onentrtion inresed to 8.8 mg/m, the plnt ppered to hve visily ute injuries on the lef nd eventully died fter tretment. Beuse of this, the 8.8 mg/m exposure ws not onsidered in follow-up experiments. Following this morphologil hnge, we further determined Chl ontent in Aridopsis shoots, whih in vivo ws n importnt kind of pigment involved in the proesses of photosynthesis. Fig. indites tht Chl ontent ws the lowest fter 4 mg/m exposure, ut there ws no signifint differene mong the treted plnts ompred to the ontrol... Effet of NO on ROS genertion nd ntioxidnts (AsA nd GSH) in Aridopsis shoots Exposure to NO used n inrese of ROS genertion in Aridopsis shoot ells. The O genertion rte enhned with inresing NO onentrtion, nd sttistil differene ws oserved t ll exposure onentrtions (Fig. ). AsA ontent tended to derese with the enhnement of NO onentrtion, nd sttistil differene ourred t the 8.5 mg/m group (Fig. ). On the ontrry, GSH ontent showed n inresing tendeny with inresing NO onentrtion, nd ws signifintly indued fter.7 nd 8.5 mg/m exposure (Fig. )... Effet of NO on levels of lipid peroxidtion nd protein oxidtion in Aridopsis shoots The levels of lipid peroxidtion nd protein oxidtion in NO -treted Aridopsis plnts were mesured from the ontents of MDA nd PCO. As presented in Fig., MDA ontent nd PCO level vried in NO onentrtion-dependent mnner nd sttistil inreses were oserved t 4 nd 8.5 mg/m,

4 96 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) 9 99 the NO onentrtion rehed 4 nd 8.5 mg/m, despite slight deline t 8.5 mg/m. For CAT, it tended to e indued fter the 8.5 mg/m tretment, ut no sttistil differene ws oserved t ll exposure onentrtions. Control.5. Effet of nitri id mist exposure on Chl ontent, lipid peroxidtion nd protein oxidtion, nd ntioxidnt enzyme tivities in Aridopsis shoots.5.7 mg/m 4 mg/m 8.5 mg/m 8.8 mg/m To mimi the tully environmentl exposure, we treted Aridopsis with nitri id mist for different exposure durtions. Fig. 5 shows tht with exposure to nitri id mist, the Chl ontent signifintly deresed with the inrese of tretment time, nd sttistil differene ws oserved fter 6 nd 8 hr/dy exposure. Following the result, we oserved the indution of MDA nd PCO ontents fter nitri id mist exposure for different durtions (Fig. 5). The MDA level tended to inrese, ut no sttistil differene ourred, while the PCO ontent inresed with the enhnement of tretment time, nd showed signifint differene when the dily exposure time rehed 8 hr/dy. Also, we further tested the hnge of SOD, POD nd CAT tivities (Fig. 5). SOD tivity hnged slightly in ll groups of A. thlin, ut no sttistil differene ourred. POD tivity signifintly inresed fter exposure to nitri id mist. CAT tivity remined unhnged fter 4 nd 6 hr/dy tretments for 7 dys, nd ws slightly ut sttistilly enhned fter the dily exposure time rehed 8 hr/dy. Chl ontent inresed fold NO onentrtion (mg/m ) Fig. Effet of Nitrogen dioxide (NO ) exposure on the morphology () nd hlorophyll (Chl) ontent () in Aridopsis shoots. Dt represent the men ± SE of three different experiments. inditing oxidtive dmge in plnt ells fter NO exposure t higher onentrtions..4. Effet of NO on ntioxidnt enzyme tivities in Aridopsis shoots Fig. 4 indites the hnges of ntioxidnt enzyme tivities in response to NO exposure. SOD tivity inresed s funtion of NO onentrtion, nd signifint differene ourred t 4 nd 8.5 mg/m. POD tivity remined unhnged fter.7 mg/m tretment, nd sttistilly inresed when. Disussion NO ffeted the growth nd Chl ontent in Aridopsis shoots, nd the effets vried s funtions of exposure onentrtion. Inhiited growth nd Chl ontent in the present study might e ttriuted to the dmged defense system nd onsequently unlned metolism. In plnt ells, ROS re unvoidle y-produts of eroi metolism. Under norml growth onditions, mounts of ROS re modest nd ells experiene only mild oxidtive stress, wheres mny stresses enhne ROS prodution (Inzé nd Vn-Montgu, 995; Mittler et l., 4). The results of our present study lerly show tht NO triggered rpid inrese in O genertion rte in Aridopsis shoots. The enhned prodution of ROS under stress n pose thret to ells, ut it is lso thought tht ROS serve s signl moleules to tivte the stress responses nd defense pthwys (Apel nd Hirt, 4; Knight nd Knight, ; Mittler, ). Thus, NO -indued ROS n e viewed s ellulr inditors of stress nd s seondry messengers involved in the stress-response signl trnsdution pthwy. Sine memrne lipids nd proteins re the preferred trgets of ROS in plnts under environmentl stress (Prssd, 996), they re onsidered to e relile inditors of ontrolled modultion of ROS levels nd oxidtive stress (Hlliwell nd Gutteridge, 99). Therefore, we further investigted the levels of lipid peroxidtion nd protein oxidtion y mesuring MDA nd PCO ontents. In Aridopsis shoots, the MDA level nd PCO ontent inresed, nd oxidtive stress ourred fter

5 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) O - genertion rte inresed fold ASA ontent inresed fold NO onentrtion (mg/m ) N O onentrtion (mg/m ) NO onentrtion (mg/m ) GSH ontent inresed fold Fig. Effet of Nitrogen dioxide (NO ) on retive oxygen speies (ROS) genertion () nd ntioxidnt ontents (sorte (AsA) () nd glutthione (GSH) ()) in Aridopsis shoots. Dt represent the men ± SE of three different experiments. p < 5, p <, p < vs. ontrol. 4 mg/m NO exposure. This finding suggests tht NO t lower onentrtions stimulted defense retions, nd these defense retions might ountert ROS generted vi elevted ntioxidnt defense systems in the tissue; wheres high onentrtion NO exposure indued the prodution of ROS, nd the pity of the ntioxidnt system ws exeeded, or inhiited. As result, the exessive ROS ttked lipids nd proteins nd the plnt experiened sustntil oxidtive stress. Additionlly, the tivities of ntioxidnt enzymes (SOD, CAT nd POD) were tested under the sme tretment onditions. In this study, NO enhned the tivities of severl ntioxidnt enzymes inluding SOD, POD nd CAT ssoited with inresed ROS genertion, suggesting n indution of ntioxidnt defenses regulted y ROS-medited signling pthwy. SOD is protein lss tht ontins metls nd tlyzes the dismuttion of superoxide rdil nions into H O nd moleulr oxygen (Sndlios, 99). The elevted SOD tivity in Aridopsis plnts suggests tht the hemil indued SOD to inrese the genertion of H O. CAT nd POD re the primry H O -svenging enzymes in plnt ells. In this study, NO pronounedly inresed POD tivity, wheres it hd little effet on CAT tivity. CAT hs high pity ut low ffinity enzyme for H O, wheres POD hs high ffinity for H O (Mittler, ). After NO exposure nd shorter dily exposure to nitri id mist, POD tivity ws signifintly enhned nd eliminted prt of the generted H O. As result, the remining H O might not e enough to indue CAT tivity. This ws not the se for the longest dily exposure, whih umulted more H O nd indued CAT tivity. Thus, POD, ut not CAT, ws the most effiient svenging enzyme to derese the ellulr levels of H O in plnt ells under NO stress. Moreover, the inrese of POD tivity n ontriute to the overll ell resistne to NO stress sine POD prtiiptes in mny other ell proesses involved in the plnt defense retion (Vlério et l., 4). POD n initite ell-wll toughening events suh s phenoli ross-linking nd lignifition, whih n strengthen lef nd stem tissues ginst potentil dmge (Polle et l., 994). The roles tht POD n ply in ell wll toughening nd in the prodution of seondry metolites, nd its simultneous oxidnt nd ntioxidnt pilities, mke it n importnt ftor in the integrted defense response of plnts to NO stress. Additionlly the AsA GSH redox system in hloroplsts is lso n effetive svenger of O nd H O in plnt ells (Foyer nd Hlliwell, 976; Jing nd Chen, 8). Thus s NO onentrtions inresed, GSH ontent inresed, ut AsA ontent deresed, suggesting tht MDA ontent inresed fold.6 PCO ontent inresed fold NO onentrtion (mg/m ) NO onentrtion (mg/m ) Fig. Effet of Nitrogen dioxide (NO ) on levels of lipid peroxidtion () nd protein oxidtion () in Aridopsis shoots. Dt represent the men ± SE of three different experiments.p < 5, p <, p < vs. ontrol.

6 98 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) 9 99 SOD tivity inresed fold POD tivity inresed fold NO onentrtion (mg/m ) NO onentrtion (mg/m ) NO onentrtion (mg/m ) CAT tivity inresed fold.6 Fig. 4 Effet of Nitrogen dioxide (NO ) on ntioxidnt enzyme tivities (inluding: Superoxide dismutses (SOD) (), Peroxidse (POD) (), Ctlse (CAT) ()) in Aridopsis shoots. Dt represent the men ± SE of three different experiments. p < 5, p <, p < vs. ontrol. the ntioxidnts lso plyed n importnt role in plnt defenses ginst NO stress. 4. Conlusions NO exposure ffeted plnt growth nd hlorophyll level, nd inresed O prodution rte in Aridopsis shoots. Furthermore, NO elevted the levels of lipid peroxidtion nd protein oxidtion, ompnied y the indution of SOD, POD nd CAT tivities nd the hnge of ntioxidnt ontents. Following this, we mimiked nitri id mist under experimentl onditions, nd onfirmed ntioxidnt mehnisms of the plnt in response to the stress. Our results imply tht NO nd its id mist used pollution risk to plnt systems, inresed ROS ted s signl to indue defense PCO ontent inresed fold MDA level inresed fold Chl ontent inresed fold SOD tivity inresed fold POD tivity inresed fold CAT tivity inresed fold Fig. 5 Effet of nitri id mist exposure on hlorophyll (Chl) ontent (), oxidtive stress () nd ntioxidnt enzyme tivities () in Aridopsis shoots. Dt represent the men ± SE of three different experiments.p < 5, p <,p <vs. ontrol.

7 JOURNAL OF ENVIRONMENTAL SCIENCES 4 (5) response, nd ntioxidnt sttus plyed n importnt role in plnt protetion ginst NO /nitri id mist-used oxidtive dmges. Aknowledgments This study ws supported y the Ntionl Nturl Siene Foundtion of Chin (Nos. 4777, 7776), the Speilized Reserh Fund for the Dotorl Progrm of Higher Edution (SRFDP) (Nos. 4, 45), the Projet for Siene nd Tehnology Development of Shnxi Provine (No. 9-), the Reserh Projet supported y the Shnxi Sholrship Counil of Chin (No. -9), nd the Progrm for the Top Young nd Middle ged Innovtive Tlents of Higher Lerning Institutions of Shnxi (TYMIT, No. ). REFERENCES Apel, K., Hirt, H., 4. Retive oxygen speies: metolism, oxidtive stress, nd signl trnsdution. Annu. Rev. Plnt Biol. 55, Bo, S.D., 5. Agriulturl nd Chemil Anlysis of Soil. Agriulture Press of Chin, Beijing, pp. 4. Brdford, M.M., 976. A rpid nd sensitive method for the quntittion of mirogrm quntity of protein utilizing the priniple of protein dye inding. Anl. 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