Seedling tretments nd phosphorus solution onentrtions ffet nodultion nd nodule funtions in soyen (Glyine mx L.) S.J. Mio 1, 2, 3, X.Z. Hn 1, X.B. Liu 1, Y.F. Qio 1 1 Northest Institute of Geogrphy nd Agrio-Eology, CAS, Hrin, Chin 2 College of Resoure nd Environment, Northest Agriulturl University, Hrin Heilongjing, Chin 3 Grdute Shool, CAS, Beijing, Chin ABSRAC he effet of three seedling tretments:, norml germintion; 1, otyledons removed; 2, otyledons removed 5 dys erlier thn in 1 ; nd two phosphorus levels ( nd P ) on nodultion nd nodule funtion in soyen [Glyine mx (L.) Merr.] were investigted in nutrient solution ulture. he numer of nodules formed t 3 ws in the order 2 > > 1, ut it ws > 2 > 1 t. Nodule dry weight per plnt hd the sme tendeny s the nodule numer. Nodule size (dry weight per nodule) in seedlings rnged from.61 to 1.89 mg in the order > 1 > 2, regrdless of P level. For exmple, nodule size in ws lrger y 86% nd 52% thn 2 t nd, respetively. Furthermore, regrdless of P level, speifi etylene redution tivity (ARA, µm C 2 /h/g nodule) inresed with P ontent in seedlings, ut no signifint differene ws found (P <.5). Leghemogloin (L) ontent ws not signifintly ffeted y P level; however, seedlings ( nd 1 ) signifintly ffeted the L ontent per unit plnt iomss (P <.5). All these results suggest tht seedling P ontent plys key role in nodultion nd nodule funtion of soyen. Keywords: soyen [Glyine mx (L.) Merr. ]; seedling; phosphorus nutrition; nitrogense tivity; leghemogloin onentrtion Phosphorus is one of the most importnt mroelements indispensle for plnt growth nd development (Mrshner 1986). Although soyen P 2 O 5 requirements re onsiderly lesser thn those of N or K, P is eqully importnt for plnt growth nd produtivity. Beuse of P funtions for the growth nd metolism of plnts, its defiieny retrded plnt growth, ell nd lef expnsion (Mrshner 1986). Some uthors reported tht P suffiieny signifintly inresed lef surfe of soyen (Wu 1999). It is known tht P regultion of photosynthesis nd rohydrte metolism in leves ws one of the mjor ftors limiting the plnt growth (Mrshner 1986). Moreover, P hs stimulting effets on nodule growth nd nitrogense tivity in nodules of legumes (Jkosen 1985, Isrel 1987, Hrt 1989, ng et l. 21); on the ontrry its defiieny deresed nodule mss (Singleton et l. 1985, Isrel 1987, Riet nd Drevon 1995, Drevon nd Hrtwig 1997), nodule numer (Jkosen 1985, ng et l. 21) nd nitrogense tivity (S nd Isrel 1991). All these reports foused on how exogenous P ffeted nodule formtion nd funtion. here is no report on the role of P reserved in seedlings in nodultion nd nodule funtion. Seed s P ws suffiient to estlish nodules; however in the se of the lk of P, plnts used the stored P in the seed to grow. Otherwise, nodules n e utoregulted without effet y exogenous P (Cludio et l. 22). Erly nodultion might e relted to the P onentrtion in seeds; high P in seeds my thus filitte the estlishment of symiosis (Cludio et l. 22). However, the reltionship etween seedling P onentrtion, nodultion nd nodule funtion is unler. In this work, we Supported y Ntionl Bsi Reserh Progrm of Chin, Projets No. 25CB12111 nd 23CCB1, y Heilongjing Provine, Projets No. GB5C21-1 nd CC5533, nd y Field Sttion Foundtion of CAS. PLAN SOIL ENVIRON., 53, 27 (2): 65 71 65
present the results on the role of seedling P in the nodultion nd nodule funtions of soyen [Glyine mx (L.) Merr.]. MAERIAL AND MEHODS Uniform-sized seeds of soyens [Glyine mx (L.) Merr., v. Heinong 35] were imied in wter overnight, nd then trnsferred onto mesh sitting ove n erted solution (ph ~ 6) of 1 mmol/l CCl 2 nd 5 µmol/l H 3 BO 3. hree P tretments were used on seedlings: (1), totl P onentrtion ws 1.316 mg/g, norml germintion; (2) 1, totl P onentrtion ws 1.232 mg/g, otyledons were removed when the first true lef prtly expnded, nd (3) 2, totl P onentrtion ws 1.67 mg/g, otyledons were removed 5 dys erlier thn in 1. Seeds were germinted for 4 5 dys when rdiles were out 4 m in length. Eighteen uniform seedlings were trnsferred into 5-litre pot with two P nutrient solution, i.e. [ ] nd 3 [ ] µmol/l. Phosphorus ws supplied s K PO 4 in nutrient solution. he omposition of the solution ws s follows (in µmol/l): K 2 SO 4, 6; MgSO 4.7 O, 2; CCl 2.2 O, 6; H 3 BO 3, 5; ZnSO 4.7 O,.75; MnSO 4. O, 1; CoSO 4.7 O,.2; CuSO 4.5 O,.2; N 2 MoO 4. O,.3; Fe-NEDA, 1. Brdyrhizoium jponium strin Hefeng 25 ws dded into pots s wter suspension to reh the finl onentrtion of out 1 5 ells/ml in the nutrient solution. his suspension ws dded gin 4 dys lter when solution ws renewed. Plnts were grown in glsshouse t 25/15 C dy nd night, respetively. Nutrient solutions were renewed twie week. Solution ph ws djusted to 5.5 6. one or twie dy, if neessry. retments were replited six times nd rndomized within replites. Plnts were hrvested in the 5 th week fter the tretments. Plnts were seprted into shoots, roots nd nodules. Biomss of ll prts ws mesured nd nodule numer ws lulted. Chlorophyll ws extrted with etone nd ethnol nd ws mesured with ultrviolet spetrometer (UV25 Jpn). he totl nd speifi etylene redution tivity (ARA) ws nlyzed with gs hromtogrph (GC21 Shimdzu Jpn) (ng et l. 21). Leghemogloin ontent ws mesured with ultrviolet spetrometer (UV25 Jpn). Plnt tissues were oven-dried t 8 C to the onstnt weight for P nd N nlysis. he totl P ontent fter digestion with SO 4 nd O 2 ws nlyzed with ultrviolet spetrometer (UV25 Jpn) nd N onentrtion ws nlyzed y uto-titrtion (Lu 2). he SAS softwre ws used to identify ny sttistilly signifint differenes. Dunn s multiple test t P <.5 ws used to ompre the mens of the tretments. RESULS Plnt growth From the 2 nd week, whole shoots ppered ple green, nd kept the olor until the lter stge; this indited slight nitrogen defiieny. By the 5 th week fter the tretments, hlorophyll onentrtion in the youngest expnded leves in ws the highest in this experiment, irrespetive of P level (Figure 1). For the sme seedlings, hlorophyll onentrtion in the youngest expnded leves deresed s P level inresed. Dry weight of shoots nd roots in t level ws y 41% nd 33% higher thn in 2, respetively; for level they were higher y 63% nd 33%. For oth shoots Plnt dry weight (g/plnt) Chlorophyll onentrtion.6.5.4.3.2.1. (mg/g fw) 2.5 2. 1.5 1..5. shoot root 1 1 2 2 1 1 2 2 retment 1 1 2 2 1 1 2 2 retment Figure 1. Dry weight of shoots nd roots nd hlorophyll onentrtion in the youngest expnded leves of soyen grown t, 1 nd 2 seedlings nd nd levels fter 5 weeks; letters on the top of the rs indite signifint differenes t 5% level d 66 PLAN SOIL ENVIRON., 53, 27 (2): 65 71
Nodule dry weight (g/plnt) Nodule numer per plnt 8 6 4 2.8.6.4.2 1 1 2 2 1 1 2 2 d retment 1 1 2 2 1 1 2 2 retment Figure 2. Numer of nodules nd nodule dry weight of soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week; letters on the top of the rs indite signifint differenes t 5% level nd roots, dry weight ws in the order: > 2 > 1 (Figure 1). Signifint differenes were reorded in the se of dry weight of shoots nd roots etween nd 2 ; however, no signifint differenes were oserved etween nd 1 or 1 nd 2. We found tht the effet of seedlings on dry weight of shoots nd roots ws lrger thn tht of P level. P level, seedlings signifintly ffeted nodule dry weight. Similrly, under the sme seedling tretment, the effet of P level on nodule dry weight ws signifintly different (Figure 2). Irrespetive of P level, nodule size (dry weight per nodule) in seedlings rnged from.61 to 1.89 mg in the order: > 1 > 2 (Figure 3). Dry weight per nodule in 1 ws y 86% nd 52% higher thn in 2 t nd levels, respetively. A sttistil nlysis showed tht the effet of seedlings on nodule size ws signifint t level; however, t level, this effet ws found only etween nd 1, or etween nd 2 (Figure 3). Nodule funtion At the sme P level, speifi ARA (µm C 2 /h/g nodule) in ws higher thn in other tretments, ut no sttistilly signifint differene ws found. Moreover, P level inresed speifi ARA in the sme seedlings. he effet of seedlings on totl ARA (µm C 2 /h/g plnt) ws minor t oth P levels, ut P level signifintly inresed totl ARA in 2 (Figure 4). he totl ARA in 2 seedlings ws the lrgest t level (8.21 µm C 2 /h/g nodule), followed y ( ), 1 ( ), 2 ( ), ( ) nd 1 ( ). C 2 -indued deline (C 2 -ID) of ARA ws oserved in ll the tretments (Figure 5). When 1% C 2 ws introdued into the gs mixture ottle with nodulted roots, the mount of C 2 produed inresed, nd then deresed with durtion of C 2 exposure time, ut the time until rehing the mximum vlue ws different. At level, the mount of C 2 from nodule tivity Nodule formtion Due to the low temperture, nodules firstly ppered during the 3 rd week fter the tretment. Nodule numer reorded in the 5 th week ws ffeted oth y seedlings nd P level (Figure 2). At level, seedlings did not signifintly ffet nodule numer; it rnged from 36 to 5 per plnt in the order: 2 > > 1 ; t level it rnged from 48 to 6 per plnt in the order: > 2 > 1. A signifint differene ws found etween nd 2 (Figure 2): level inresed the nodule numer per plnt; nd 2 tretments showed signifint differenes. For oth P levels, nodule dry weight per plnt rnged from.21 to.61 g in the order: > 2 > 1 (Figure 2); for tretment, nodule numer nd nodule dry weight per plnt in were y 11% nd 75% higher thn in 2. At the sme Dry weight per nodule (mg/g) 1.2 1..8.6.4.2. 1 1 2 2 1 1 2 2 retment Figure 3. Nodule size (dry weight per nodule) of soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week; letters on the top of the rs indite signifint differenes t 5% level PLAN SOIL ENVIRON., 53, 27 (2): 65 71 67
otl etylene redution (µm C 2 /h/g nodule) 6. 5. 4. 3. 2. 1.. 3. P 25. 2. 15. P 1P 1 2P 2 P3 P 1P3 1 2P3 2 P retment Speifi etylene redution (µm C 2 /h/g plnt) 1. 5.. 1 1 2 2 1 1 2 2 retment Figure 4. otl nd speifi etylene redution tivity (ARA) of soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week; ARA ws the verge ARA mesured etween 1 nd 6 min fter C 2 exposure; letters on the top of the rs indite signifint differenes t 5% level inresed, rehing the mximum vlue 4 min fter initil exposure to C 2. However, doule pek urve of C 2 -ID ws produed y the nodule tivity t level (Figure 5). he effet of seedlings on leghemogloin (L) onentrtion per nodule ws greter thn the effet of P level, espeilly in nd 1 seedlings; signifint differene ws found in these Reltive C 2 redution tivity (% of verge etween 1 6 min) 16 14 12 1 8 6 4 2 25 2 15 1 5 3 25 2 15 1 5 15 1 5 1 2 3 4 5 6 1 2 3 4 5 6 1 15 1 1 5 1 2 3 4 5 6 1 2 3 4 5 6 2 2 2 15 1 5 1 2 3 4 5 6 1 2 3 4 5 6 Durtion of C 2 exposure (min) Figure 5. ime ourse of etylene redution tivity (ARA) mesured on soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week 68 PLAN SOIL ENVIRON., 53, 27 (2): 65 71
L onentrtion (mg/g nodule fw) 1.9 1.85 1.8 1.75 1.7 1.65 1.6 1.55 1.5 1.45 1.4 1.35 P 3 ffeted P onentrtion, nd signifint differene ws oserved mong seedling tretments. For exmple, P onentrtion per plnt nd per unit weight in inresed from.74 to 2.62 mg/plnt nd from 1.16 to 4.4 mg/g, respetively. DISCUSSION Nodule formtion L onentrtion (mg/g plnt dw).35.3.25.2.15.1.5. 1 1 2 2 1 1 2 2 retment P 1 1 2 2 1 1 2 2 retment In the pre sent study, efore ino ulting Brdyrhizoium jponium strins, P onentrtion of seedlings ws 1.316 mg/g, for 1 it ws 1.232 mg/g, nd for 2 it ws 1.67 mg/g. Nodule numer ws signifintly ffeted y P levels in nd 2, not in 1 (Figure 2). his is euse 2 seedlings hd longer totl root length, whih provided more hnes for teri infetion thn 1. hus, when enough nodule teri were present, seedling P onentrtion eme key ftor for optiml nodultion if the P onentrtion ws low. P onentrtions in the seedlings Figure 6. Leghemogloin (L) onentrtion of soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week; letters on the top of the rs indite signifint differenes t 5% level seedlings t oth P levels (Figure 6). For the sme seedlings, P level hd no signifint effet on L onentrtion. he effet of P level on L onentrtion per plnt ws the sme s tht on speifi ARA, espeilly t. All these dt suggest tht L onentrtion in nodules n e onsidered s hrteristi of nitrogen fixtion effiieny (Shleev et l. 21). Chemil omposition Nitrogen onentrtion (per plnt nd per unit weight) in seedlings rnged from 14.75 to 26.18 mg/ plnt nd from 33.28 to 4.22 mg/g in the order: > 2 > 1, exept for nitrogen onentrtion per unit weight t level (Figure 7). Regrdless of P level, signifint differene of nitrogen onentrtion per plnt ws found etween nd 1. For nitrogen onentrtion per unit weight, signifint differene ws oserved etween nd 2 t oth P levels (Figure 7). Seedling effet on P onentrtion per plnt or per unit weight ws low t level. However, P level signifintly Conentrtion (mg/plnt) Conentrtion (mg/g) 45. 4. 35. 3. 25. 2. 15. 1. 5.. 5. 4. 3. 2. 1.. P P N 3 d d 1 1 2 2 1 1 2 2 retment P N d d d d 1 2 1 2 1 2 1 2 retment Figure 7. Nitrogen nd P onentrtions of soyen grown t, 1 nd 2 seedlings nd nd levels in the 5 th week; letters on the top of the rs indite signifint differenes t 5% level Note: P nd N onentrtions in plnts were sttistilly nlyzed seprtely PLAN SOIL ENVIRON., 53, 27 (2): 65 71 69
were higher thn required P level for rhizoi growth nd survivl (.5 µm P) (O Hr et l. 1988). his showed tht seedlings ontined enough P to estlish nodules, nd were thus omprle with the seeds tht reserved P s reported previously (Vlverde nd Wll 1999). Although the plnts of 2 seedlings hd the lowest onentrtion, nodule numer ws igger thn in 1, whih might result from the ft tht the plnts in 2 hd higher iomss thn 1, nd so more photossimiltes were trnsferred to nodule for nodule formtion nd growth (Voisin et l. 23). Chlorophyll onentrtion in the youngest expnded leves t level ws in the order: > 1 > 2 in the 5 th week, inditing tht photosynthesis ould not explin the vriility in growth of roots nd nodules (Voisin et l 23). Hene, in this experiment, plnt dry weight ws > 2 > 1, nd this trend ws the sme in the se of nodule numer nd nodule dry weight. Nevertheless, the effet of seedlings on plnt growth ws weker thn on nodule formtion nd development, suggesting thus tht P hd diret nd positive stimultion of nodultion in legumes (Jkosen 1985, Isrel 1987, 1993, Snging et l. 1989, Hellsten nd Huss-Dnell 21). However, n elevted P level ppered to inrese nodule development, whih is in greement with our previous reports (Mio et l. 27) nd other reports on soyen (Singleton et l. 1985, Drevon nd Hrtwig 1997). Nodule funtion his study suggested tht P ontent in seedlings plyed speifi role in nodule funtion of soyen; its effet on nitrogense tivity per unit nodule mss ws igger thn on totl nitrogense tivity (Figure 4). In soyen, P defiieny-deresed nitrogense tivity ws suggested to result from inhiited energy-dependent retions in nodules (ng et l. 21), deresed L onentrtion in nodules or deresed teroid iomss (S nd Isrel 1991). In our experiment, low P in seedlings deresed L onentrtion per plnt irrespetive of P level, whih my e used y the ft tht L is n oxygen-rrying heme protein in nodules, ple of inding oxygen to produe n oxygented form only in tive redued stte (Shleev et l. 21). hus, nodule ws kept under low free O 2 ondition to prevent nitrogense from irreversile intivtion y O 2 (kshi et l. 21). In mture nodules, L protein ws deteted t the onset of nitrogen fixtion (Kterin et l. 2), nd the higher nitrogense tivity plnts hd reltively higher L onentrtion per plnt in our experiment (Figure 6). L lso plyed n essentil role in nitrogen fixtion in nodules (Appley 1984, Ben nd Sprent 1989). Regrdless of P level, the C 2 -ID in nitrogense tivity under low P in seedlings ws lrger thn tht under high onentrtions, whih is onsistent with previous findings on soyen (Riet nd Drevon 1995, Drevon nd Hrtwig 1997) nd Medigo truntul L. (ng et l. 21). At µmol/l P level, the C 2 -ID rehed the mximum vlue 4 min fter C 2 introdution into the gs mixture; however, the two peks ppered fter 2 nd 5 min (Figure 5). he effet of seedlings nd P level on nitrogen onentrtion per plnt or per unit weight ws muh like tht on ARA. hese results show tht P-inresed ARA is ssoited with inresed N onentrtion nd N umultion (ng et l. 21). REFERENCES Appley C.A. (1984): Leghemogloin nd Rhizoium respirtion. Ann. Rev. Plnt Physiol., 35: 443 478. Ben M., Sprent J.I. (1989): Effet of nitrte on omponents of nodule leghemogloins. J. Exp. Bot., 4: 725 731. Cludio V., Alejndro F., Luis G.W. (22): Phosphorus nd the regultion of nodultion in the tinorhizl symiosis etween Disri trinervis (Rhmnee) nd Frnki BCU1151. New Phytol., 153: 43 51. Drevon J.J., Hrtwig U.A. (1997): Phosphorus defiieny inresed the rgon-indued deline of nodule nitrogense tivity in soyen nd lflf. Plnt, 21: 463 469. Hrt A.L. (1989): Distriution of phosphorus in nodulted white lover plnts. J. Plnt Nutr., 12: 159 171. Hellsten A., Huss-Dnell K. (21): Intertion effets of nitrogen nd phosphorus on nodultion in red lover (rifolium prtense L.). At Agr. Snd., 49: 135 142. Isrel D.W. (1987): Investigtion of the role of phosphorus in symioti dinitrogen fixtion. Plnt Physiol., 84: 835 84. Isrel D.W. (1993): Symioti dinitrogen fixtion nd host-plnt growth during development of nd reovery from phosphorus defiieny. Physiol. Plnt., 88: 294 3. Jkosen I. (1985): he role of phosphorus in nitrogen fixtion y young pe plnts (Pisum stivum). Physiol. Plnt., 64: 19 196. 7 PLAN SOIL ENVIRON., 53, 27 (2): 65 71
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