氮素與豆科作物固氮關(guān)系研究進(jìn)展

夏玄，龔振平

（東北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，哈爾濱 150030）

氮素與豆科作物固氮關(guān)系研究進(jìn)展

夏玄，龔振平

（東北農(nóng)業(yè)大學(xué)農(nóng)學(xué)院，哈爾濱 150030）

氮素與根瘤固氮結(jié)合可達(dá)高產(chǎn)，兩者矛盾對(duì)根瘤固氮產(chǎn)生不利影響。氮素影響根瘤固氮作用機(jī)制仍不明確。文章在現(xiàn)有研究成果基礎(chǔ)上，總結(jié)氮素與大豆根瘤固氮關(guān)系研究發(fā)展進(jìn)程；綜述不同氮素形態(tài)和氮素濃度對(duì)根瘤形成和生長(zhǎng)、根瘤固氮酶活性影響及相關(guān)機(jī)制研究動(dòng)態(tài)；闡述氮素抑制根瘤固氮碳水化合物爭(zhēng)奪、抑制根瘤氧供給、硝酸鹽毒害、反饋調(diào)節(jié)及其他可能抑制機(jī)制；提出氮素抑制結(jié)瘤氮濃度界限模糊化，不同氮素形式、豆科種類氮素抑制機(jī)制差異化及詳細(xì)抑制機(jī)制不明確等問(wèn)題。

氮素；根瘤；抑制；機(jī)制

豆科作物與根瘤菌通過(guò)復(fù)雜相互作用，形成豆科作物獨(dú)特高效生物固氮體系。但豆科作物尤其是大豆，單純依靠根瘤固氮無(wú)法達(dá)到高產(chǎn)目標(biāo)。氮肥施入尤為重要。氮素與根瘤固氮存在正負(fù)相關(guān)并存的復(fù)雜關(guān)系。土壤中某種鹽類阻礙根瘤正常生長(zhǎng)，如硝酸鹽類[1]。探索氮素與根瘤固氮關(guān)系可提高共生固氮能力，優(yōu)化作物管理措施，克服化合態(tài)氮對(duì)根瘤固氮不利影響，促使兩者可持續(xù)協(xié)調(diào)發(fā)展。目前氮素影響根瘤作用機(jī)制仍無(wú)明確結(jié)論，國(guó)外取得成果較多，國(guó)內(nèi)發(fā)展相對(duì)滯后。本文總結(jié)氮素與大豆根瘤固氮關(guān)系成果，旨在探究?jī)烧咦饔脵C(jī)制提供理論基礎(chǔ)。

1 氮素對(duì)大豆根瘤固氮影響

1.1 氮素對(duì)大豆根瘤形成和生長(zhǎng)影響

1.1.1 氮素形態(tài)對(duì)根瘤形成和生長(zhǎng)影響

氮素形態(tài)主要為硝態(tài)氮、銨態(tài)氮和尿素。劉莉等對(duì)不同品種大豆接種根瘤菌與根毛侵染試驗(yàn)研究認(rèn)為，高濃度化合態(tài)氮通過(guò)早期阻礙根瘤菌對(duì)根毛侵染而抑制根瘤菌與大豆共生關(guān)系建立，抑制影響程度排序?yàn)椋海∟H4）2SO4＞NH4NO3＞KNO3，抑制結(jié)瘤作用NO3-濃度高于NH4+濃度[2]。Guo等以尿素、硝酸鹽、銨鹽及硝酸銨為氮源，在兩種濃度（5和10 mmol·L-1）下研究氮素對(duì)蠶豆（Fababean）、白羽扇豆（White lupin）、苜蓿根瘤形成、生長(zhǎng)、固氮酶活性影響，高濃度較低濃度抑制效果更明顯，而NH4Cl抑制根瘤最嚴(yán)重，5 mmol·L-1濃度尿素抑制根瘤效果最輕[3]。嚴(yán)君等施用不同形態(tài)氮肥研究表明，不同形態(tài)氮源對(duì)根瘤干重、數(shù)量有促進(jìn)作用，增加根瘤中含氮量和氮積累量，根瘤干重及數(shù)量變化表現(xiàn)為銨態(tài)氮＞蛋白氮（豆粉）＞硝態(tài)氮＞氨基酸態(tài)氮（甘氨酸）＞酰胺態(tài)氮（尿素）＞不施氮肥[4]。Gan等研究認(rèn)為，在低濃度下與硝酸銨、硝態(tài)氮相比，單獨(dú)施用銨態(tài)氮使大豆具有更高生物積累量、根瘤干重、總氮積累量[5]。董守坤等采用15N標(biāo)記法以硫酸銨為氮源研究表明，隨氮濃度增加,根瘤干重呈先升后降變化趨勢(shì)，當(dāng)營(yíng)養(yǎng)液氮濃度為3.57 mmol·L-1（50 mg·L-1）時(shí)，有利于根瘤生長(zhǎng)[6]。宋海星等研究表明，銨態(tài)氮較硝態(tài)氮有利于大豆生長(zhǎng)，前者對(duì)根瘤固氮抑制作用明顯低于后者[7]。Svenning等利用水培法研究氮素對(duì)白三葉草結(jié)瘤影響認(rèn)為，NH4+與NO3-在20 mmol·L-1濃度恒定不變條件下，以NH4+為氮源處理較以NO3-為氮源處理固氮量大[8]。Rys和Phung以NH4Cl、Na-NO3和NH4NO3為氮源研究表明，NH4NO3對(duì)三葉草根瘤重量、固氮酶活性影響抑制最嚴(yán)重，而NH4+降低溶液pH，未對(duì)根瘤數(shù)量、重量和固氮酶活性及植株生長(zhǎng)產(chǎn)生較大影響[9]。Wahab和Abd-Alla對(duì)兩種大豆Clark和Crauford作KNO3和NH4Cl不同施氮量試驗(yàn)（0、16、32、64、128 kg N·hm-2），在較低施氮水平下兩個(gè)大豆品種生長(zhǎng)和結(jié)瘤均明顯增加，NH4+更適合作為臨時(shí)氮源提高根瘤數(shù)量和重量[10]。Gulden和Vessey研究認(rèn)為，低濃度NH4+促進(jìn)豌豆根瘤形成及生長(zhǎng)，一旦將NH4+移除，相關(guān)植株生長(zhǎng)率，結(jié)瘤數(shù)量、重量及固氮酶活性均與未施加NH4+處理相似[11]。Imsande將大豆經(jīng)不同濃度NO3-和NH4+連續(xù)培養(yǎng)，相同濃度下NO3-抑制根瘤重量及固氮酶活性程度均高于NH4+，試驗(yàn)控制pH，說(shuō)明NH4+抑制結(jié)瘤并非酸毒害結(jié)果[12]。

由氮素形態(tài)對(duì)結(jié)瘤抑制試驗(yàn)可見(jiàn)，不同形態(tài)氮對(duì)結(jié)瘤和固氮酶活性抑制效果不同，硝態(tài)氮對(duì)根瘤生長(zhǎng)發(fā)育及固氮酶活性抑制作用較銨態(tài)氮更嚴(yán)重。暗示硝態(tài)氮、銨態(tài)氮抑制大豆根瘤形成至少有兩種機(jī)制作用。以往研究中NO3-對(duì)結(jié)瘤和固氮影響研究較多，對(duì)NH4+研究相對(duì)較少[13-14]，兩者對(duì)豆類作物結(jié)瘤固氮影響差異性比較研究較少[15-16]。

1.1.2 氮素濃度對(duì)根瘤形成和生長(zhǎng)影響

氮素抑制結(jié)瘤固氮濃度問(wèn)題是研究熱點(diǎn)，尤其是較高濃度氮對(duì)結(jié)瘤及根瘤固氮抑制作用。

Daimon等研究指出，氮濃度在14 mmol·L-1以上，根瘤數(shù)量和重量均受到顯著抑制[17]。Guo等指出，5和10 mmol·L-1硝酸鹽、銨鹽混合物和尿素均抑制豆科作物根瘤生長(zhǎng)，濃度越高抑制效果越明顯[3]。Gan等研究表明，10 mmol·L-1氮濃度無(wú)論是硝態(tài)氮、銨態(tài)氮還是硝酸銨，均顯著降低大豆根瘤數(shù)量、根瘤重量以及總固氮量[5]。Macduff等通過(guò)0、10、100和1 000 mmol·L-1不同濃度NO3-對(duì)固氮抑制試驗(yàn)表明，隨NO3-濃度增加固氮抑制越嚴(yán)重，即使在提供最低氮濃度10 mmol·L-1水平下，增加NO3-固氮無(wú)提高[18]。Carroll等認(rèn)為在5.5和5 mmol·L-1氮濃度條件下，大豆根生長(zhǎng)及結(jié)瘤受到抑制[19-20]。Serraj等提出3 mmol·L-1KNO3可完全抑制大豆結(jié)瘤[21]。較高濃度氮抑制結(jié)瘤及固氮，低濃度氮促進(jìn)或降低結(jié)瘤及固氮研究已有報(bào)道。Gulden和Vessey通過(guò)砂培試驗(yàn)報(bào)道，銨鹽在大豆結(jié)瘤過(guò)程起負(fù)面影響，但與0和2 mmol·L-1銨鹽處理相比，0.5和1 mmol·L-1銨鹽使大豆植株結(jié)瘤和固氮效果更佳[13]。Daimon等利用溶液培養(yǎng)試驗(yàn)發(fā)現(xiàn)，3.5和7 mmol·L-1對(duì)根瘤形成起促進(jìn)作用，而在0.7 mmol·L-1下根瘤數(shù)量下降，但根瘤鮮重提高[17]。通過(guò)溶液培養(yǎng)大豆植株結(jié)瘤在4 mmol·L-1KNO3和2 mmol·L-1（NH4）2SO4中受抑制，其中4 mmol·L-1KNO3根瘤干重顯著受抑制，顯著抑制早晚兩階段根瘤發(fā)展[12]。張榮銑等在水培條件下研究不同NO3-濃度對(duì)大豆結(jié)瘤固氮影響發(fā)現(xiàn)，0.71 mmol·L-1（10 mg·kg-1）NO3-使根瘤干重增加43%；而≥2.14 mmol·L-1（30 mg·kg-1）時(shí)，根瘤干重受抑制[22]。Gan等將大于5 mmol·L-1氮設(shè)為高濃度氮，認(rèn)為1和3.75 mmol·L-1為低濃度氮，試驗(yàn)表明低濃度氮顯著提高大豆根瘤數(shù)量、根瘤重量及每株大豆總固氮量[5]。Streeter等指出大豆適于生長(zhǎng)在復(fù)合氮量較小環(huán)境中，建議砂培培養(yǎng)氮濃度為1～2 mmol·L-1[23]。Gibson和Harper提出，當(dāng)NO3-濃度1 mmol·L-1時(shí)，大豆根瘤形成及根瘤固氮酶活性均受到延緩和阻礙[24]。

1.2 氮素對(duì)大豆根瘤固氮影響的局部性和系統(tǒng)性

1.2.1 氮素對(duì)根瘤固氮酶活性影響

根瘤固氮酶是衡量根瘤固氮活性重要指標(biāo)。乙炔還原法推進(jìn)了氮與抑制根瘤關(guān)系研究。Minchin等認(rèn)為乙炔還原法存在誤導(dǎo)結(jié)果問(wèn)題，對(duì)多種處理比較無(wú)效[25]。但Schuller等指出，利用乙炔還原法和15N標(biāo)記法研究硝酸鹽對(duì)大豆根瘤固氮影響結(jié)果一致[26]。乙炔還原法測(cè)定根瘤固氮酶活性已廣泛應(yīng)用，但氮素抑制固氮酶活性機(jī)制仍不明確。

研究指出，在硝酸鹽存在條件下，固氮效率明顯降低[27-29]。Streeter利用砂培供給大豆15 mmol·L-1硝態(tài)氮7 d，在供氮1 d后固氮酶活性受抑制，與對(duì)照相比，隨時(shí)間推移固氮酶活性抑制程度達(dá)80%[30]。Skrdleta等用珍珠巖培養(yǎng)豌豆，供給20 mmol·L-1硝態(tài)氮處理，固氮酶活性同樣在1 d受顯著抑制，而根瘤生長(zhǎng)（根瘤干重）則在開(kāi)始處理后3 d受抑制[31]。硝酸鹽施入豆科作物生長(zhǎng)介質(zhì)后，通過(guò)NO3-還原酶還原成NO2-，NO2-直接抑制固氮酶活性[32]，或形成NO混合物阻礙固氮進(jìn)程[33]。

1.2.2 氮素對(duì)大豆根瘤形成和固氮酶活性影響的局部性與系統(tǒng)性

Hinson利用土壤盆栽，將大豆根系分為兩份，其中一側(cè)大豆根系施氮，抑制另一側(cè)不施氮根瘤重量，但并未抑制根瘤數(shù)量[34]。Carroll和Gresshoff利用相似方法研究硝酸鹽對(duì)白三葉草結(jié)瘤及固氮影響，根部直接施氮產(chǎn)生局部性抑制根瘤形成現(xiàn)象[35]。Silsbury等供給小型草地三葉草（Small swards of subterranean clover）1和5 mmol·L-1NO3-與0 mmol·L-1對(duì)照相比，三葉草固氮酶活性明顯降低，而植株和根部NO3-含量明顯增加，不能證明NO3-對(duì)固氮酶活性影響是NO2-積累而致毒害作用；利用盆栽分根法，將三葉草根平分成兩部分，一側(cè)根供給15 mmol·L-1NO3-,與兩側(cè)均不施氮處理相比，施氮側(cè)根瘤固氮酶活性在處理2 d后迅速降低，而與施氮側(cè)對(duì)應(yīng)的不施氮側(cè)固氮酶活性降低，僅滯后2 d。試驗(yàn)結(jié)果認(rèn)為，固氮和硝酸還原補(bǔ)充整株植株降低氮素，而NO3-抑制固氮通過(guò)系統(tǒng)性調(diào)節(jié)[36]。研究指出根瘤發(fā)展及固氮酶活性由系統(tǒng)性生理控制[37]。Kohls等報(bào)道，根毛受抑制導(dǎo)致根瘤產(chǎn)生受抑制，而硝酸鹽對(duì)根毛抑制局部影響，推斷硝酸鹽抑制結(jié)瘤形成局部影響[38]。Arnone等通過(guò)分根法研究表明，硝酸鹽抑制根瘤形成和發(fā)育是局部性的，抑制固氮酶活性則是系統(tǒng)性的[39]。Tanaka等在水培條件下利用分根法將大豆根平分成兩部分，一側(cè)大豆根供給濃度為0、2.14、7.14、14.28 mmol·L-1硝態(tài)氮，隨施氮濃度增加，大豆根生長(zhǎng)和吸收硝態(tài)氮速率增加，但根瘤干重和固氮酶活性受阻，顯著降低；當(dāng)?shù)獫舛仍?.14 mmol· L-1以下時(shí)，對(duì)應(yīng)不施氮根的根瘤干重和固氮酶活性并未受到顯著影響甚至起促進(jìn)作用；當(dāng)施氮根一側(cè)氮濃度在14.28 mmol·L-1時(shí)，不施氮一側(cè)根瘤干重和固氮酶活性受不利影響[40]。Daimon等利用盆栽分根法對(duì)花生一側(cè)根施用14 mmol·L-1硝態(tài)氮處理，施用硝態(tài)氮5 d后，在一側(cè)根施氮不影響不施氮一側(cè)根瘤數(shù)量和重量，但施氮側(cè)根瘤固氮酶活性明顯低于另一側(cè)；在施氮處理30 d后，硝態(tài)氮抑制同時(shí)影響施氮及不施氮組。因此，認(rèn)為植株供氮30 d后，硝態(tài)氮對(duì)根瘤固氮酶活性抑制影響是系統(tǒng)性的；而短期供氮5 d后對(duì)根瘤固氮酶活性抑制則是非系統(tǒng)性的[41]。

2 氮素抑制根瘤固氮機(jī)制

氮素抑制結(jié)瘤固氮試驗(yàn)，多集中于硝酸鹽，因供給土壤各種形態(tài)氮多數(shù)會(huì)通過(guò)揮發(fā)淋濕消化作用迅速轉(zhuǎn)化成硝酸鹽[42]。

較高濃度硝酸鹽如何抑制根瘤固氮具體原因與機(jī)制仍不明確，但研究表明硝酸鹽對(duì)根瘤菌侵染、根瘤形成和發(fā)展及根瘤固氮酶活性等各階段均有消極影響，各過(guò)程作用機(jī)制不同，機(jī)理研究見(jiàn)表1。

表1 氮素抑制根瘤固氮機(jī)理Table 1Inhibition mechanism of nitrogen on nodule nitrogen fixation

2.1 碳水化合物爭(zhēng)奪機(jī)制

Orcutt和Wilson發(fā)現(xiàn)高濃度硝酸鹽可降低大豆葉、莖、根中還原糖和蔗糖含量[43]。Stephens等在離體大豆根瘤、Houwaard在完整豌豆植株中發(fā)現(xiàn)，蔗糖可緩解硝酸鹽抑制根瘤固氮酶活性[44-45]。Wong研究表明，葡萄糖、蔗糖、果糖緩解硝酸鹽抑制扁豆根瘤固氮酶活性[46]。Small等利用14C標(biāo)記研究認(rèn)為，豆科作物吸收同化氮素時(shí)，消耗碳水化合物，供給根部及根瘤碳水化合物減少[47-48]。Bacanamwo和Harper研究認(rèn)為，NO3-抑制大豆固氮酶活性程度與植株組織中N和C濃度水平有關(guān)，與根瘤中可利用碳水化合物及根瘤中碳氮比呈正相關(guān)[49]。然而，Singleton和Van利用密閉分根系統(tǒng)將根平分為兩部分，對(duì)兩側(cè)根作施氮和不施氮處理，分別通空氣和Ar（氬氣）與O2混合氣（Ar 80%，O220%），研究發(fā)現(xiàn)，施氮一側(cè)根與不施氮一側(cè)根相比可接受主要光合產(chǎn)物碳水化合物，與光合產(chǎn)物爭(zhēng)奪假設(shè)相反[50]。綜上所述，碳水化合物機(jī)制研究仍存疑點(diǎn)。

2.2 抑制根瘤氧供給機(jī)制

Minchin等利用流入式系統(tǒng)（Flow-through），供給白三葉草（Trifolium repens）20 mmol·L-1NO3-發(fā)現(xiàn)，根瘤固氮酶活性降低同時(shí)伴隨O2擴(kuò)散障礙增加[51]。Carroll等分別供給大豆7.5和10 mol·L-1濃度KNO3使其產(chǎn)生高氮脅迫，在供O2濃度由21%提升到60%時(shí)，根瘤固氮酶活性提高。硝酸鹽使根瘤中O2供給受阻礙而抑制根瘤活性[52]。Minchin等分別供給菜豆和豇豆10 mmol·L-1NO3-，處理3 d，利用流入式系統(tǒng)調(diào)節(jié)O2供給濃度，隨O2濃度增加，兩種豆類作物根瘤固氮酶活性呈增加趨勢(shì)，兩種豆類根瘤菌和細(xì)胞液中均含有硝酸還原酶，在處理期間硝酸還原酶活性提升1.5～2倍，硝酸還原酶活性隨處理時(shí)間推移而上升。因此，認(rèn)為NO3-抑制根瘤固氮分為兩個(gè)步驟：①提升O2擴(kuò)散阻礙，②NO3-進(jìn)入菌類區(qū)域從新陳代謝上產(chǎn)生抑制和破壞[53]。Arrese-Igor等對(duì)紫花苜蓿研究支持該觀點(diǎn)，但均缺乏O2擴(kuò)散阻礙直接測(cè)定[54-55]。

2.3 硝酸鹽毒害機(jī)制

硝酸鹽對(duì)根毛形成有毒害性。Munns和Wahab等分別利用紫花苜蓿和豌豆、蠶豆、豇豆、菜豆為試驗(yàn)材料，施加硝態(tài)氮后對(duì)根毛侵染和卷曲度研究發(fā)現(xiàn)，硝態(tài)氮抑制根毛形成及根瘤菌侵染，影響結(jié)瘤數(shù)量[56-57]。積累的硝態(tài)氮可在轉(zhuǎn)化成亞硝態(tài)或NO后是必需元素，也可作為某種分子信號(hào)發(fā)揮調(diào)節(jié)作用[58-59]，影響結(jié)瘤和固氮。Becana和Wasfi等通過(guò)研究不同氮濃度與根瘤硝酸還原酶關(guān)系認(rèn)為，硝酸鹽抑制根瘤固氮酶活性可能與NO2-在根瘤中積累產(chǎn)生毒害機(jī)制有關(guān)[60-61]。Nelson利用硝酸還原酶缺乏的豌豆突變品種研究認(rèn)為，NO3-在根瘤中同化積累并未直接參與根瘤固氮酶活性抑制過(guò)程[62]。Chen等研究化合態(tài)氮對(duì)根瘤衰老影響也認(rèn)為，硝酸鹽并未通過(guò)硝酸還原酶與固氮酶競(jìng)爭(zhēng)光合產(chǎn)物而誘導(dǎo)根部根瘤衰老[63]。目前，亞硝酸鹽毒害機(jī)制相比其他機(jī)制無(wú)更強(qiáng)說(shuō)服力。

2.4 反饋調(diào)節(jié)機(jī)制

根瘤形成由反饋調(diào)節(jié)系統(tǒng)性控制（Autoregulation of nodulation），即反饋機(jī)制AON，其調(diào)控信號(hào)有諸多觀點(diǎn)[64-66]。Caetano-Anollés等認(rèn)為AON調(diào)節(jié)開(kāi)始由根部皮層細(xì)胞分裂誘導(dǎo)產(chǎn)生某種信號(hào)，該信號(hào)或通過(guò)木質(zhì)部液傳輸至植株地上部，經(jīng)地上部誘導(dǎo)相關(guān)消極調(diào)節(jié)反應(yīng)，最終根瘤發(fā)展進(jìn)一步受阻，反饋抑制在控制根瘤數(shù)量方面是系統(tǒng)性反應(yīng)[67-68]。Ito等通過(guò)顯微鏡觀察葉片細(xì)胞發(fā)現(xiàn)，超結(jié)瘤大豆NOD1-3和NOD3-7與野生型大豆相比，葉片細(xì)胞數(shù)量和葉面積均顯著小于后者，但葉細(xì)胞面積間無(wú)顯著差異，因此認(rèn)為葉片細(xì)胞數(shù)量少導(dǎo)致超結(jié)瘤大豆NOD1-3和NOD3-7葉面積小。Ito發(fā)現(xiàn)野生型大豆接種根瘤菌處理的葉片（最初完全展開(kāi)葉片）細(xì)胞數(shù)量和細(xì)胞面積均顯著大于不接種根瘤菌處理，提出AON調(diào)節(jié)信號(hào)或許與葉片增殖細(xì)胞控制系統(tǒng)有關(guān)假設(shè)，認(rèn)為結(jié)瘤野生類型大豆AON機(jī)制可促進(jìn)葉片數(shù)量增加[69]。Neo等研究羽扇豆（Lupinus albus）結(jié)瘤韌皮部谷氨酰胺指出，結(jié)瘤反饋機(jī)制通過(guò)硝酸鹽作用機(jī)制中產(chǎn)物（如谷氨酰胺）產(chǎn)生影響[70]。Searle等證明大豆AON被受體激酶GmNARK（Glycine max nodule autoregulation receptor kinase）調(diào)節(jié)，而這種受體激酶與擬南芥CLAVATA1（CLV1）調(diào)節(jié)類似，CLV1通過(guò)莖尖短距離信號(hào)控制莖細(xì)胞增殖在蛋白質(zhì)復(fù)合體中發(fā)揮作用，葉片中GmNARK表達(dá)在長(zhǎng)距離與根瘤及次生根溝通中有重要作用[71]。Wang等則通過(guò)不同濃度硝酸鹽處理，分析擬南芥硝酸鹽誘導(dǎo)基因，認(rèn)為積累硝態(tài)氮NO3-及NO2-可能作為某種分子信號(hào)在植株生長(zhǎng)發(fā)育過(guò)程中產(chǎn)生調(diào)節(jié)[58]。Reid等通過(guò)5、10 mmol·L-1硝酸鹽誘導(dǎo)條件下，正常大豆與超結(jié)瘤大豆互相嫁接，認(rèn)為根部NARK（受體激酶）可能局部調(diào)節(jié)結(jié)瘤，而NARK（受體激酶）是進(jìn)一步觸發(fā)地上部抑制產(chǎn)物和抑制根瘤發(fā)展的信號(hào)產(chǎn)物[72]。

Francisco等通過(guò)Enrei與其突變體En6500地上部與根部相互嫁接，以Enrei為地上部，以En6500為地下部，En6500超結(jié)瘤和硝酸鹽耐受特性均被廢除，相反，以En6500為地上部，以Enrei為地下部，En6500具有超結(jié)瘤性且對(duì)硝酸鹽耐受性重新恢復(fù)。根瘤可自動(dòng)調(diào)節(jié)，硝酸抑制結(jié)瘤由地上部分控制，在根瘤形成前即產(chǎn)生阻礙[73]。某些大豆品種超結(jié)瘤突變體結(jié)瘤對(duì)硝酸鹽忍耐性極強(qiáng)，在較高硝酸鹽濃度下仍表現(xiàn)較高數(shù)量結(jié)瘤，這可能因AON調(diào)節(jié)機(jī)制缺乏，AON由地上部分系統(tǒng)控制而非根部[74-76]。Day等通過(guò)11種超結(jié)瘤大豆與結(jié)瘤正常Bragg品種地上部與地下部分相互嫁接，研究供給7.5 mmol·L-1KNO3和不供給條件下根瘤數(shù)量。發(fā)現(xiàn)7.5 mmol·L-1KNO3濃度下，以超結(jié)瘤為嫁接的地上部分，無(wú)論地下部分是哪個(gè)品系，根瘤數(shù)量仍表現(xiàn)超結(jié)瘤數(shù)量，對(duì)氮素不敏感；而以正常結(jié)瘤品系Bragg為嫁接地上部分，以超結(jié)瘤為嫁接地下部分則表現(xiàn)根瘤數(shù)量受7.5 mmol·L-1KNO3強(qiáng)烈抑制。硝酸鹽對(duì)大豆根瘤形成和發(fā)展影響依靠硝酸鹽和自動(dòng)反饋調(diào)節(jié)信號(hào)相互作用。在超結(jié)瘤突變體中自動(dòng)反饋調(diào)節(jié)信號(hào)改變或缺失，致使突變體對(duì)硝酸鹽不敏感[77]。Hamaguchi等通過(guò)大豆野生品種與對(duì)氮素具有耐受性大豆超結(jié)瘤品種在不同氮濃度條件下作地上和地下部分嫁接，認(rèn)為超結(jié)瘤大豆對(duì)氮素忍耐性及結(jié)瘤由地上部分控制，由地下部分調(diào)節(jié)緩沖。以結(jié)瘤大豆與不結(jié)瘤大豆相互嫁接試驗(yàn)得出不同結(jié)果[78]。Delves等利用不結(jié)瘤品種nod49分別與結(jié)瘤正常品系Bragg及超結(jié)瘤品系nts382作地上和地下部分嫁接試驗(yàn)，結(jié)果表明以不結(jié)瘤品系nod49為嫁接地下部分，以Bragg或者nts382為嫁接地上部分，根瘤數(shù)量皆為0；而以nod49為嫁接地上部分，Bragg為嫁接地下部分，根瘤數(shù)量與Bragg相差不多略有減少，以超結(jié)瘤品系nts382為嫁接地下部分雖有較多結(jié)瘤，但根瘤數(shù)量較nts382減少近3/4。因此，認(rèn)為不結(jié)瘤品系nod49不結(jié)瘤由根部控制，地上部分對(duì)根部結(jié)瘤產(chǎn)生影響[65]。Francisco等利用結(jié)瘤正常品系Enrei、超結(jié)瘤突變品系En6500與不結(jié)瘤品系Enll5、En1282、Enl314分別作地上部和地下部互相嫁接，嫁接結(jié)論與Delves等一致，即不結(jié)瘤品系為嫁接地下部分時(shí)，無(wú)論地上部嫁接是正常品系還是超結(jié)瘤品系，根瘤數(shù)量均表現(xiàn)為0，而以不結(jié)瘤品系為嫁接地上部分，以結(jié)瘤正常品系Enrei為嫁接地下部分，根瘤數(shù)量比Enrei略減，以超結(jié)瘤突變體En6500為嫁接地下部分雖有結(jié)瘤，但結(jié)瘤數(shù)量比超結(jié)瘤En6500減少近25倍[73]。Carroll等對(duì)超結(jié)瘤及氮素忍耐品種篩選后及不同氮濃度條件下試驗(yàn)認(rèn)為，超結(jié)瘤大豆品系nts382受根瘤發(fā)展調(diào)節(jié)基因影響，與硝酸鹽同化基因無(wú)關(guān)[79]。表明從基因角度分析AON與氮素抑制結(jié)瘤關(guān)系機(jī)制需深入探究。

2.5 其他可能抑制機(jī)制

Tanner和Anderson提出氮素通過(guò)減少額外的吲哚乙酸（IAA）濃度抑制結(jié)瘤[80]。Bano和Harper等通過(guò)研究大豆接種前后木質(zhì)部、韌皮部及葉片中激素變化表明，在接種根瘤菌6 h后，木質(zhì)部ABA（脫落酸）濃度增加，而韌皮部和葉片部ABA濃度在接種48～96 h后發(fā)生變化，認(rèn)為激素在根部結(jié)瘤自動(dòng)調(diào)節(jié)方面發(fā)揮重要作用[81]。Ligero等研究認(rèn)為，NO3-對(duì)根瘤抑制作用可通過(guò)乙烯抑制劑（Aminoethoxyvinylglycine，AVG）消除，提出NO3-抑制影響通過(guò)植物激素乙烯介導(dǎo)，指出內(nèi)源乙烯在根瘤自動(dòng)調(diào)節(jié)系統(tǒng)中發(fā)揮重要作用[82]。Zhang等對(duì)擬南芥發(fā)現(xiàn)硝酸鹽對(duì)植物激素反應(yīng)產(chǎn)生影響，因此硝酸鹽對(duì)豆科作物可通過(guò)植物激素產(chǎn)生間接影響[83]。Caba等認(rèn)為硝酸鹽降低野生品種和突變品種結(jié)瘤和不結(jié)瘤大豆植物激素水平，根生長(zhǎng)未改變[84]。Fei和Vessey以蒺藜苜蓿（Medicago truncatula）為試驗(yàn)材料研究發(fā)現(xiàn)，低濃度銨態(tài)氮促進(jìn)苜蓿結(jié)瘤（根瘤數(shù)量和重量），而ABA（脫落酸）活性蛋白激酶和GA（赤霉素）調(diào)節(jié)蛋白基因編碼對(duì)不同濃度硝態(tài)氮和銨態(tài)氮回應(yīng)較少，但在多種處理間乙烯反應(yīng)的連接因子基因表達(dá)明顯增加。3個(gè)生長(zhǎng)素相關(guān)基因和3個(gè)細(xì)胞分裂素相關(guān)基因則表現(xiàn)對(duì)硝態(tài)氮和銨態(tài)氮不同回應(yīng)。雖然根部有較高比例的細(xì)胞分裂素和生長(zhǎng)素，但不能說(shuō)明其促進(jìn)結(jié)瘤潛在機(jī)制[85]。Peters等研究指出，在根瘤菌中結(jié)瘤基因被寄主植株根中的信號(hào)混合物類黃酮和異黃酮激活[86-87]。Redmond等指出，根部黃酮類物質(zhì)誘導(dǎo)結(jié)瘤基因表達(dá)，并與根瘤菌內(nèi)部信號(hào)交流[88]。Cho和Harper利用PVC管將大豆根平分為兩部分，施用5 mmol·L-1硝態(tài)氮的一側(cè)根，根瘤數(shù)量、重量、固氮酶活性均顯著小于應(yīng)用0 mmol·L-1硝態(tài)氮一側(cè)根，施氮側(cè)根異黃酮濃度明顯較低，因此推測(cè)氮最初對(duì)結(jié)瘤處抑制是因異黃酮水平降低[89]。這也暗示氮素對(duì)根瘤抑制機(jī)制與多類植物激素有關(guān)，而在其中發(fā)揮重要作用激素的具體調(diào)節(jié)機(jī)制仍需深入研究。

3 問(wèn)題與展望

自1916年Fred指出氮素阻礙根瘤發(fā)展以來(lái)，氮素抑制豆類作物根瘤固氮研究受到關(guān)注，但具體抑制機(jī)制并無(wú)實(shí)質(zhì)性突破。目前，氮素抑制豆類作物根瘤固氮仍無(wú)定論。

①由氮素形態(tài)對(duì)結(jié)瘤抑制試驗(yàn)可見(jiàn)，不同形態(tài)氮對(duì)結(jié)瘤和固氮酶活性抑制效果不同，硝態(tài)氮對(duì)根瘤生長(zhǎng)發(fā)育及固氮酶活性抑制作用較銨態(tài)氮更強(qiáng)。揭示硝態(tài)氮、銨態(tài)氮抑制大豆根瘤形成至少有兩種作用機(jī)制。銨態(tài)氮抑制根瘤固氮機(jī)制與硝態(tài)氮是否有重疊交叉過(guò)程尚待深入研究。

②氮濃度研究結(jié)果可見(jiàn)，氮濃度界限不清，對(duì)大豆結(jié)瘤及固氮產(chǎn)生影響的濃度范圍模糊，濃度范圍界定仍需系統(tǒng)化、標(biāo)準(zhǔn)化研究。

③分根和局部供氮方法研究認(rèn)為，氮素抑制根瘤形成與發(fā)育存在局部性，而固氮酶活性則存在系統(tǒng)性。但仍有疑點(diǎn)，如氮素抑制根瘤形成短期與長(zhǎng)期條件下是否均呈局部性，不同氮素濃度下抑制作用是否可逆，在不同氮素形式下恢復(fù)是否不同，氮素抑制根瘤局部性是否與不同生長(zhǎng)介質(zhì)影響有關(guān)等。分根法研究氮素與根瘤抑制關(guān)系具有優(yōu)勢(shì)，但介質(zhì)中溶液培養(yǎng)試驗(yàn)較多，其他介質(zhì)環(huán)境試驗(yàn)較少，氮素對(duì)結(jié)瘤抑制多方面影響研究仍無(wú)實(shí)質(zhì)性突破。

④豆類作物種類眾多，符合某種抑制機(jī)制的豆類作物是否符合其他抑制機(jī)制。多種豆類作物是否擁有一種或幾種共同抑制機(jī)制，或差異共存，有待深入研究。

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Research advance on the relationship between nitrogen and Leguminous

nitrogen fixation

XIA Xuan,GONG Zhenping(School of Agriculture,Northeast Agricultural

University,Harbin 150030,China)

The relationship between nitrogen and nodule nitrogen fixation is very delicate.It is necessary to combine the two to achieve high yield.However,the contradiction between the two has a great negative impact on nodule nitrogen fixation.However,the mechanism of how nitrogen affects root nodulation is still unclear.Based on previous studies,the research progress of the relationship between nitrogen fixation and nitrogen fixation of soybean was summarized.The effects of different nitrogen forms and nitrogen concentration on nodule formation and growth,nitrogenase activity and related mechanisms were reviewed. The mechanism of carbohydrate competition,inhibition of nitrogen supply mechanism,nitrate toxicity mechanism,feedback regulation mechanism and other possible inhibition mechanisms of nitrogen fixation were discussed.And put forward the nitrogen inhibition nodules of nitrogen boundaries blurred.Different nitrogen forms and legume species nitrogen inhibition mechanism differentiation and detailed inhibition mechanism was not clear and so on.

nitrogen;nodule;inhibition;mechanism

S565.1

A

1005-9369（2017）01-0079-10

2016-11-28

國(guó)家科技支撐項(xiàng)目（2014BAD11B01）

夏玄（1988-），女，博士研究生，研究方向大豆栽培生理。E-mail:731275750@qq.com

*通訊作者：龔振平，教授，博士生導(dǎo)師，研究方向?yàn)楸Ｗo(hù)性耕作和大豆生理。E-mail:gzpyx2004@163.com

時(shí)間2017-1-9 15:46:07[URL]http://www.cnki.net/kcms/detail/23.1391.S.20170109.1546.012.html

夏玄,龔振平.氮素與豆科作物固氮關(guān)系研究進(jìn)展[J].東北農(nóng)業(yè)大學(xué)學(xué)報(bào),2017,48(1):79-88.

Xia Xuan,Gong Zhenping.Research advance on the relationship between nitrogen and Leguminous nitrogen fixation[J]. Journal of Northeast Agricultural University,2017,48(1):79-88.(in Chinese with English abstract)