生長(zhǎng)素調(diào)控水稻穎花開(kāi)放的效應(yīng)研究

何永明 張 芳

生長(zhǎng)素調(diào)控水稻穎花開(kāi)放的效應(yīng)研究

何永明 張 芳*

江西農(nóng)業(yè)大學(xué) / 作物生理生態(tài)與遺傳育種教育部重點(diǎn)實(shí)驗(yàn)室 / 江西省作物生理生態(tài)與遺傳育種重點(diǎn)實(shí)驗(yàn)室, 江西南昌 330045

穎花開(kāi)放由漿片膨大所啟動(dòng), 對(duì)水稻授粉受精具有直接影響。生長(zhǎng)素是調(diào)節(jié)花藥開(kāi)裂、花粉育性和種子起始等生殖發(fā)育過(guò)程的重要激素。為闡明生長(zhǎng)素在水稻穎花開(kāi)放中的調(diào)控作用, 本研究以粳稻品種中花11為試驗(yàn)材料, 調(diào)查了外源生長(zhǎng)素及其抑制劑對(duì)穎花開(kāi)放的影響, 以及內(nèi)源生長(zhǎng)素水平和生長(zhǎng)素信號(hào)通路基因表達(dá)的動(dòng)態(tài)變化。結(jié)果表明, IAA (10～20 mmol L–1)、NAA (0.05～0.50 mmol L–1)浸穗處理將推遲水稻穎花開(kāi)放, 其中高濃度(0.5 mmol L–1) NAA能使穎花開(kāi)放推遲3 d, 并表現(xiàn)出穎花張開(kāi)時(shí)間延長(zhǎng)和結(jié)實(shí)率下降的現(xiàn)象。IAA極性運(yùn)輸抑制劑TIBA及其作用抑制劑PCIB也抑制穎花開(kāi)放。NAA預(yù)處理后, 增施茉莉酸甲酯(MeJA)能有效恢復(fù)穎花開(kāi)放。水稻穎花中IAA含量在自然開(kāi)放前2 h迅速下降, 比開(kāi)穎前1 d、2 d分別降低了65.85%、74.27%。與IAA水平變化相對(duì)應(yīng), 穎花開(kāi)放時(shí)漿片IAA生物合成基因(、)表達(dá)下調(diào), 而催化IAA結(jié)合失活的酶基因(、)、IAA輸出載體基因(、)以及IAA極性運(yùn)輸正向調(diào)節(jié)因子基因均顯著上調(diào)表達(dá)。此外, 我們也鑒定到13個(gè)差異表達(dá)的IAA早期響應(yīng)基因(、、), 其中10個(gè)上調(diào)表達(dá), 3個(gè)下調(diào)表達(dá)。綜上表明, 水稻穎花開(kāi)放受內(nèi)源生長(zhǎng)素調(diào)控, 但提高漿片生長(zhǎng)素水平則抑制其開(kāi)放。

水稻; 穎花開(kāi)放; 生長(zhǎng)素; 抑制效應(yīng)

水稻是世界重要的糧食作物之一, 其花器官的發(fā)育為人類提供了主要的糧食來(lái)源。近年來(lái), 我國(guó)秈粳亞種間雜種優(yōu)勢(shì)利用取得重大突破, 以甬優(yōu)、春優(yōu)、浙優(yōu)等系列為代表的粳型不育系/秈型恢復(fù)系(“粳不秈恢”)雜交組合, 雜種優(yōu)勢(shì)更強(qiáng), 發(fā)展勢(shì)頭良好[1-2]; 但其親本在日間開(kāi)花時(shí)間的差異, 導(dǎo)致制種產(chǎn)量低下, 嚴(yán)重制約了秈粳雜交水稻的推廣[3-4]。因此, 明確水稻穎花開(kāi)放調(diào)控機(jī)制, 解決秈粳品種的“花時(shí)不遇”成為提高雜交制種產(chǎn)量的關(guān)鍵?；〞r(shí)是指水稻穎花在1 d中的開(kāi)放時(shí)間, 通常以開(kāi)花高峰表示。成熟穎花的開(kāi)放通常發(fā)生在09:00—12:00, 但與品種遺傳特性(秈稻花時(shí)一般比粳稻早)和環(huán)境因素(溫度、濕度、光照、風(fēng)力和CO2等)密切相關(guān), 例如增溫和CO2處理均可促進(jìn)水稻開(kāi)穎, 搓揉稻穗等機(jī)械刺激也能引起水稻開(kāi)花, 田間風(fēng)速增加會(huì)延遲水稻穎花開(kāi)放[5-7]。

植物激素是植物細(xì)胞接受特定環(huán)境信號(hào)誘導(dǎo)的信號(hào)分子, 調(diào)控著植物生長(zhǎng)發(fā)育及環(huán)境適應(yīng)的各個(gè)過(guò)程。曾曉春等研究發(fā)現(xiàn)茉莉酸(JA)及其甲酯(MeJA)對(duì)水稻開(kāi)穎表現(xiàn)出強(qiáng)烈誘導(dǎo)效應(yīng), 該效應(yīng)在雄性不育系表現(xiàn)更明顯[8-10]。1 mmol L–1水楊酸(SA)能抑制MeJA誘導(dǎo)的水稻開(kāi)穎, 而MeJA的再次處理能解除SA的抑制作用[10]。水稻漿片JA生物合成在穎花開(kāi)放前大幅上升, 閉穎后急劇下降[9,11-12], 水稻JA缺失突變體[13-14]、[15-16]以及JA不足的不育系ZS97A[17]表現(xiàn)出花時(shí)分散的現(xiàn)象, 而MeJA在人工改善水稻花時(shí)方面具有良好效果[18-20]。脫落酸(ABA)處理可明顯加快已開(kāi)穎花的閉穎過(guò)程, 縮短開(kāi)閉歷時(shí)[21]。

生長(zhǎng)素主要以吲哚乙酸(indole-3-acetic acid, IAA)形式存在于植物體內(nèi), 是最早被發(fā)現(xiàn)且作用最廣泛的植物激素, 對(duì)水稻花器官發(fā)育的調(diào)控發(fā)揮著重要作用。水稻控制依賴于色氨酸的生長(zhǎng)素合成, 該基因缺失后,突變體胚胎致死, 花器官異常發(fā)育[22]。編碼的雙加氧酶催化IAA不可逆氧化生成OxIAA, 該基因缺失后花藥和子房中游離IAA增加, 突變體表現(xiàn)出雄性不育[23]。參與水稻生長(zhǎng)素極性運(yùn)輸?shù)恼{(diào)控, 過(guò)量表達(dá)后, 轉(zhuǎn)基因水稻雄蕊減少, 雌蕊柱頭增加, 漿片發(fā)育異常[24]。眾多研究證明, JA、MeJA能調(diào)控水稻花時(shí), 但生長(zhǎng)素除了調(diào)控水稻花器官的發(fā)育外, 是否也參與調(diào)控水稻穎花開(kāi)放？為此, 本研究以中花11為試驗(yàn)材料, 采用生長(zhǎng)素及其抑制劑處理開(kāi)花前的稻穗, 對(duì)其進(jìn)行開(kāi)花動(dòng)態(tài)調(diào)查、內(nèi)源生長(zhǎng)素水平測(cè)定和基因表達(dá)分析等, 從而摸清生長(zhǎng)素對(duì)水稻穎花開(kāi)放的調(diào)控效應(yīng), 以期進(jìn)一步揭示水稻穎花開(kāi)放的內(nèi)在控制機(jī)制, 為水稻花時(shí)的人工調(diào)節(jié)提供理論基礎(chǔ)和技術(shù)路徑。

1 材料與方法

1.1 試驗(yàn)材料與試劑

水稻品種為中花11, 分批種植于江西農(nóng)業(yè)大學(xué)科技園, 常規(guī)管理, 抽穗期為7月上旬至9月上旬。吲哚乙酸(indole-3-acetic acid, IAA)、萘乙酸(naphthaleneacetic acid, NAA)、三碘苯甲酸(2,3,5-triiodobenzoic acid, TIBA)和對(duì)氯苯氧異丁酸(p-chlorophenoxyisobutyric acid, PCIB) (國(guó)藥集團(tuán)化學(xué)試劑有限公司), 以0.1 mol L–1KOH溶解; 茉莉酸甲酯(methyl jamonate, MeJA)(Sigma公司)以95%乙醇溶解。試劑配成高濃度母液, 避光存放于4℃冰箱, 使用時(shí)取母液稀釋成相應(yīng)的施用濃度, 并用0.1 mmol L–1HCl調(diào)節(jié)生長(zhǎng)素類溶液pH至6.5。

1.2 藥劑處理和穎花開(kāi)花動(dòng)態(tài)調(diào)查

穎花開(kāi)花調(diào)查試驗(yàn)前1 d下午18:00—19:00, 選擇頂部已有少量穎花開(kāi)放的主穗, 以一定濃度的生長(zhǎng)素類(IAA、NAA、TIBA、PCIB)溶液(含0.2‰吐溫-20)浸蘸1 min, MeJA溶液在開(kāi)花調(diào)查當(dāng)天早上08:00處理, 以H2O (含0.2‰吐溫-20)作對(duì)照。08:30—15:00時(shí)間內(nèi)統(tǒng)計(jì)處理稻穗的穎花開(kāi)放數(shù), 每30 min統(tǒng)計(jì)1次, 以記號(hào)筆標(biāo)記已開(kāi)的穎花, 每處理6穗。單朵穎花的開(kāi)閉歷時(shí)調(diào)查為在10:00— 13:00之間記錄單朵穎花的開(kāi)放和閉合時(shí)間, 每處理記錄8朵(2朵穗–1)。

1.3 單穗穎花結(jié)實(shí)率考查

試驗(yàn)前1 d下午, 選取已抽出1/2左右的主穗, 每株選1穗, 用記號(hào)筆標(biāo)記已開(kāi)放的穎花(不計(jì)入結(jié)實(shí)率), 然后用一定濃度的IAA、NAA溶液處理稻穗(方法同1.2), 隨后套袋, 25 d后考查單穗穎花結(jié)實(shí)率,每處理8穗。

1.4 穎花取樣與IAA測(cè)定

根據(jù)穎花著生位置和雄蕊長(zhǎng)度判斷穎花的成熟度。剪取稻穗上開(kāi)花前不同時(shí)間點(diǎn)的穎花, 液氮速凍后保存于–80℃冰箱。穎花樣品在液氮中研磨至粉末, 準(zhǔn)確稱取0.1 g, 放置于1.5 mL離心管, 加入500 μL預(yù)冷的激素提取液(甲醇∶水∶乙酸=90∶9∶1), 于避光的4℃平板搖床中劇烈振蕩提取24 h, 低溫高速離心(4℃, 12,000轉(zhuǎn) min–1, 15 min), 收集上清液備用; 繼續(xù)加400 μL提取液重復(fù)提取2次(提取時(shí)間分別為12 h、6 h), 然后合并3次提取所獲上清液于室溫下以氮?dú)獯蹈? 加200 μL甲醇溶解, 再經(jīng)0.22 μm尼龍濾膜過(guò)濾, 保存于–20℃冰箱中待測(cè)。按Liu等[25]操作, 利用UFLC-ESI-MS系統(tǒng)測(cè)定IAA濃度, 每個(gè)樣品3次生物學(xué)重復(fù)。

1.5 基因表達(dá)分析

在液氮中剝?nèi)∽匀婚_(kāi)放前1 d、0 h漿片, 以Trizol法提取漿片RNA。RNA樣品的純度和完整性經(jīng)Nanodrop 2000、Aglient 2100檢驗(yàn)合格后, 送北京百邁客生物科技有限公司進(jìn)行RNA測(cè)序。測(cè)序平臺(tái)為Illumina公司的HiSeq X-ten高通量測(cè)序系統(tǒng), 測(cè)序讀長(zhǎng)為PE150。測(cè)序數(shù)據(jù)(Reads)與粳稻參考基因組(RGAP7)進(jìn)行比對(duì), 應(yīng)用StringTie軟件進(jìn)行組裝和定量?；虮磉_(dá)水平采用FPKM (每千個(gè)堿基的轉(zhuǎn)錄每百萬(wàn)映射讀取的碎片)指標(biāo)定量。應(yīng)用GO、KEGG數(shù)據(jù)庫(kù)對(duì)IAA途徑差異表達(dá)基因進(jìn)行富集和注釋。

用于qRT-PCR分析的植物RNA樣品經(jīng)DNase I (Invitrogen)去除基因組DNA的污染后, 利用M-MLV 逆轉(zhuǎn)錄酶(Invitrogen)反轉(zhuǎn)錄合成cDNA。qRT-PCR采用SYBR Premix ExII試劑盒(Takara), 反應(yīng)體系20 μL: SYBR Premix Ex10 μL, 正反向引物各0.4 μL, cDNA模板2 μL, ROX Reference Dye (50x) 0.4 μL, ddH2O 6.8 μL。反應(yīng)條件為: 95℃ 30 s; 95℃ 5 s, 60℃ 38s, 循環(huán)數(shù)為40, 以為內(nèi)參基因[26]。目標(biāo)基因、的引物序列參考李金濤等[27],、和分別參見(jiàn)Woo等[28]、Du等[29]和Liu等[30], 結(jié)果根據(jù)2–ΔCt方法計(jì)算分析, 每份樣品3次重復(fù)。

2 結(jié)果與分析

2.1 外源生長(zhǎng)素對(duì)水稻穎花開(kāi)放動(dòng)態(tài)和結(jié)實(shí)的影響

從圖1-A可以看出, 施用外源生長(zhǎng)素(IAA和NAA)后第1天, 水稻穎花開(kāi)放數(shù)均顯著降低。相比清水對(duì)照, 10 mmol L–1、20 mmol L–1IAA和0.05 mmol L–1、0.2 mmol L–1NAA處理下, 穎花開(kāi)放數(shù)分別下降了33.33%、57.14%和71.43%、89.88%, 而0.5 mmol L–1NAA噴施后未觀察到穎花開(kāi)放。隨著時(shí)間的推移, 生長(zhǎng)素的抑制效應(yīng)減弱, 水稻穎花逐漸恢復(fù)開(kāi)放, 低濃度生長(zhǎng)素(10 mmol L–1IAA、0.05 mmol L–1NAA)施用后, 第2天穎花開(kāi)放數(shù)最多(與對(duì)照一致), 而0.2 mmol L–1、0.5 mmol L–1NAA處理的水稻分別在第3天和第4天出現(xiàn)開(kāi)穎高峰。由此可見(jiàn), 外施生長(zhǎng)素將抑制水稻穎花開(kāi)放, 抑制效應(yīng)隨施用濃度的增加而顯著提升, 且NAA的效果比IAA更加強(qiáng)烈。

通過(guò)觀察穎花開(kāi)放最多當(dāng)天的日開(kāi)花動(dòng)態(tài)可知, 低濃度生長(zhǎng)素(10 mmol L–1IAA、0.05 mmol L–1NAA)處理下水稻花時(shí)比對(duì)照(花時(shí)為11:30)推遲了1 h, 于12:30出現(xiàn)開(kāi)穎高峰; 而高濃度生長(zhǎng)素(0.2 mmol L–1NAA)的施用使穎花開(kāi)放集中度下降, 花時(shí)分散, 在11:30—13:30間的穎花開(kāi)放數(shù)無(wú)明顯差異(圖1-B)。這表明外施生長(zhǎng)素將推遲水稻花時(shí), 且推遲效應(yīng)與生長(zhǎng)素濃度正相關(guān)。跟蹤調(diào)查穎花的開(kāi)閉歷時(shí)(圖1-C), 從中可以看出, 生長(zhǎng)素處理顯著延長(zhǎng)了單朵花開(kāi)穎的歷時(shí)(開(kāi)穎到閉穎的時(shí)間), 外施10 mmol L–1IAA和0.1 mmol L–1、0.2 mmol L–1NAA后, 單朵穎花開(kāi)閉歷時(shí)分別比對(duì)照處理增加了28.91%和24.48%、45.13% (圖1-C), 這與Huang等[21]結(jié)果一致。

生長(zhǎng)素處理可顯著推遲穎花開(kāi)放, 是否影響水稻結(jié)實(shí)？為此, 本研究調(diào)查了各處理下的水稻自交結(jié)實(shí)率(圖2), 10 mmol L–1IAA和0.1 mmol L–1、0.2 mmol L–1NAA施用后, 水稻單穗自交結(jié)實(shí)率分別比對(duì)照處理下降35.84%和48.61%、74.22%。這暗示著生長(zhǎng)素處理推遲了穎花開(kāi)放, 可能造成已發(fā)育成熟的雄蕊因未能及時(shí)開(kāi)穎而育性下降, 導(dǎo)致水稻自交結(jié)實(shí)率顯著降低。

2.2 生長(zhǎng)素抑制劑對(duì)水稻穎花開(kāi)放動(dòng)態(tài)的影響

為深入闡明生長(zhǎng)素對(duì)穎花開(kāi)放的調(diào)控作用, 本研究進(jìn)一步調(diào)查了稻穗在施用IAA抑制劑TIBA和PCIB后的開(kāi)花動(dòng)態(tài)。TIBA、PCIB分別是IAA的極性運(yùn)輸抑制劑和作用抑制劑[31]。結(jié)果顯示TIBA處理后, 穎花開(kāi)放的集中度和全天開(kāi)放總數(shù)明顯下降, 下降效應(yīng)隨TIBA施用濃度的增加而增強(qiáng)(圖3-A)。低濃度PCIB (1 mmol L–1)處理的水稻穎花開(kāi)放時(shí)間及數(shù)量與對(duì)照基本無(wú)異, 高濃度(5 mmol L–1、10 mmol L–1) PCIB的施用雖不推遲花時(shí)但抑制穎花開(kāi)放, 花時(shí)開(kāi)穎數(shù)和全天開(kāi)放總量分別減少27.01%、59.12%和18.86%、43.06% (圖3-B)。上述結(jié)果說(shuō)明, 阻礙穎花中IAA的向基運(yùn)輸以及拮抗IAA的生理作用均抑制穎花開(kāi)放, 進(jìn)一步表明內(nèi)源IAA信號(hào)介導(dǎo)穎花開(kāi)放的調(diào)控。

圖1 不同生長(zhǎng)素處理下的水稻穎花開(kāi)放動(dòng)態(tài)

A: 不同生長(zhǎng)素處理后4 d內(nèi)水稻穎花開(kāi)放情況。B: 不同生長(zhǎng)素處理后水稻穎花開(kāi)放最多的當(dāng)天逐時(shí)開(kāi)花情況。C: 不同生長(zhǎng)素處理后單朵穎花開(kāi)閉歷時(shí)。不同小寫字母代表在0.05概率水平差異顯著(Duncan’s檢驗(yàn))。

A: the number of opening florets per panicle after different auxin conditions during 4 days. B: the number of opening florets per panicle in one day that has most opening florets under different auxin conditions. C: the opening duration of single florets under different auxin conditions. Different lowercase letters mean significant difference at the 0.05 probability level (Duncan’s test).

圖2 不同生長(zhǎng)素處理下的單穗自交結(jié)實(shí)率

不同小寫字母代表在0.05概率水平差異顯著(Duncan’s檢驗(yàn))。

Different lowercase letters mean significant difference at the 0.05 probability level (Duncan’s test).

2.3 MeJA對(duì)生長(zhǎng)素抑制穎花開(kāi)放的解除作用

從圖4可以看出, 單一MeJA處理的水稻花時(shí)由11:30 (對(duì)照)提前至08:30, 全天穎花開(kāi)放總數(shù)比對(duì)照高出35.35%, 此結(jié)果與前人研究一致, 表明MeJA能誘導(dǎo)水稻開(kāi)穎。0.1 mmol L–1、0.2 mmol L–1NAA處理后, 穎花開(kāi)放幾乎被完全抑制(與圖1結(jié)果一致); 而在此處理上增施MeJA, 水稻穎花恢復(fù)開(kāi)放, 且花時(shí)分別提早至上午09:00、09:30, 當(dāng)天穎花開(kāi)放總數(shù)也與對(duì)照無(wú)明顯差異。這表明生長(zhǎng)素對(duì)水稻穎花開(kāi)放的抑制效應(yīng)可被MeJA所逆轉(zhuǎn), 這也提示外源生長(zhǎng)素處理并未損傷穎花內(nèi)漿片等組織。

2.4 水稻穎花中IAA含量的動(dòng)態(tài)變化

圖5顯示, 水稻穎花中IAA水平波動(dòng)較大: 開(kāi)穎前2 d (BF2d)高水平積累, 含量達(dá)到最大值(近90 mg g–1); 而隨著開(kāi)花的臨近, IAA含量逐步下降, 且在穎花開(kāi)放前數(shù)小時(shí)內(nèi)快速下降。與BF2d相比, 穎花中IAA在開(kāi)穎前1 d (BF1d)降低了24.67%, 而在開(kāi)穎花前2 h (BF2h)降幅高達(dá)74.27%, 且含量持續(xù)下降, 于穎花開(kāi)放時(shí)(BF0h)達(dá)到最低值, 僅為BF2d的19.96% (圖5)。這表明水稻花器官發(fā)育可能需要高濃度IAA, 于開(kāi)花前2 d大量積累IAA, 而穎花開(kāi)放需要低水平IAA, 前期積累于穎花的IAA在穎花開(kāi)放前2 h內(nèi)快速下降。

圖3 TIBA (A)和PCIB (B)處理下水稻穎花的開(kāi)放動(dòng)態(tài)

圖4 MeJA對(duì)生長(zhǎng)素抑制水稻穎花開(kāi)放的影響

圖5 水稻穎花開(kāi)放前不同時(shí)間的IAA含量

BF2d、BF1d、BF2h和BF0h分別表示開(kāi)花前2 d、1 d、2 h和穎花開(kāi)放時(shí)。不同小寫字母代表在0.05概率水平差異顯著(Duncan’s檢驗(yàn))。

BF2d, BF1d, BF2h, and BF0h means the time of 2 d, 1 d, 2 h before flowering and floret opening, respectively. Different lowercase letters mean significant difference at the 0.05 probability level (Duncan’s test).

2.5 水稻穎花開(kāi)放前漿片IAA信號(hào)通路變化

穎花基部的一對(duì)微小漿片在推動(dòng)外稃張開(kāi)過(guò)程中發(fā)揮關(guān)鍵作用[17], 為進(jìn)一步揭示水稻穎花開(kāi)放過(guò)程中漿片IAA水平的動(dòng)態(tài)變化, 本研究對(duì)漿片進(jìn)行轉(zhuǎn)錄組測(cè)序, 并對(duì)IAA信號(hào)通路差異表達(dá)基因進(jìn)行富集分析(圖6-A)。植物體內(nèi)最基本的生長(zhǎng)素合成途徑是以色氨酸為前提的吲哚丙酮酸途徑, 需要色氨酸氨基轉(zhuǎn)移酶(TAR/TAA)和黃素單加氧酶(YUCCA)的催化[31]。與IAA合成和代謝途徑相關(guān)的基因, 我們富集到了7個(gè)差異基因。與開(kāi)花前1 d (BF1d)相比, 水稻穎花開(kāi)放時(shí)(BF0h)漿片IAA合成關(guān)鍵基因、均顯著下降表達(dá), 但表達(dá)上調(diào)。IAA的結(jié)合態(tài)是非活性形式, 催化IAA與氨基酸不可逆結(jié)合的GH3蛋白編碼基因和[32]表達(dá)分別增加了約12和2倍。我們也富集到3個(gè)與IAA極性運(yùn)輸有關(guān)的差異基因, 介導(dǎo)IAA輸出的PIN1蛋白基因和, 以及IAA極性運(yùn)輸正調(diào)節(jié)因子基因[30]均大幅增強(qiáng)表達(dá)。以上基因表達(dá)變化表明, 穎花開(kāi)放前漿片游離態(tài)IAA水平會(huì)下降, 這與圖5穎花IAA水平大幅下降相一致。13個(gè)差異表達(dá)的生長(zhǎng)素信號(hào)途徑早期響應(yīng)基因(、、)也被鑒定, 其中10個(gè)上調(diào)表達(dá), 3個(gè)下調(diào)表達(dá), 這提示IAA信號(hào)介導(dǎo)漿片膨大過(guò)程。為進(jìn)一步驗(yàn)證轉(zhuǎn)錄組測(cè)序結(jié)果, 對(duì)其中10個(gè)基因的表達(dá)水平進(jìn)行qRT-PCR鑒定(圖6-B), 從中可以看出, 這10個(gè)基因的差異表達(dá)變化與轉(zhuǎn)錄組測(cè)序結(jié)果基本吻合。

圖6 漿片中IAA信號(hào)通路相關(guān)基因的表達(dá)分析

A: 水稻穎花開(kāi)放前1 d和穎花開(kāi)放時(shí)漿片轉(zhuǎn)錄組分析。B:,,和等10個(gè)選擇基因的表達(dá)變化值qRT-PCR檢測(cè)與RNA-Seq的相關(guān)性分析。橫坐標(biāo)為qRT-PCR的相對(duì)表達(dá)水平, 縱坐標(biāo)為RNA-Seq的差異表達(dá)水平, 二者均取log2的對(duì)數(shù)。BF1d和BF0h分別表示開(kāi)花前1 d和穎花開(kāi)放時(shí)。

A: transcriptional analysis of rice lodicules at the time of 1 d before flowering and floret opening. B: the correlation analysis on the expression changes of 10 selected genes including,,, andwith the methods of qRT-PCR and RNA-seq. The values of log2for relative expression level of qRT-PCR and differential expression levels of RNA-Seq were as the horizontal and vertical coordinates, respectively. BF1d and BF0h means the time of 1 d before flowering and floret opening, respectively.

3 討論

生長(zhǎng)素作為最重要的植物激素, 參與調(diào)控植物生長(zhǎng)發(fā)育的各個(gè)過(guò)程[32-33]。然而, 生長(zhǎng)素在植物花開(kāi)放中的調(diào)控作用卻研究較少。前人發(fā)現(xiàn), 生長(zhǎng)素(IAA、NAA)促進(jìn)鳶尾()和睡蓮()的花朵開(kāi)放, 而其極性運(yùn)輸抑制劑TIBA卻抑制開(kāi)放[34-35]。但是, 外施生長(zhǎng)素及極性運(yùn)輸抑制劑(TIBA、NPA)對(duì)水稻胚根、擬南芥初生根的生長(zhǎng)只表現(xiàn)出抑制作用, 沒(méi)有促進(jìn)效應(yīng)[31,36]。本研究顯示, 外源生長(zhǎng)素(IAA、NAA)對(duì)水稻穎花開(kāi)放具有明顯抑制作用, 抑制效應(yīng)與生長(zhǎng)素濃度正相關(guān)(圖1-A, B)。這與水稻突變體植株因活性IAA大幅增加而表現(xiàn)出穎花不開(kāi)和花藥不裂的現(xiàn)象[23]相符合。本研究進(jìn)一步發(fā)現(xiàn), IAA運(yùn)輸、作用抑制劑TIBA、PCIB也顯著抑制穎花開(kāi)放(圖3), 而且穎花自然開(kāi)放前2 h內(nèi)源IAA含量顯著下降(圖5)。這些結(jié)果表明, 內(nèi)源IAA介導(dǎo)水稻穎花開(kāi)放的調(diào)控, 但穎花開(kāi)放時(shí)漿片IAA含量已下降至低水平, 提高IAA水平則推遲穎花開(kāi)放。

已知JA誘導(dǎo)穎花開(kāi)放, 并且穎花開(kāi)放前漿片JA水平會(huì)顯著上升[11,17]。在擬南芥花器官晚期發(fā)育以及側(cè)根發(fā)育中, IAA信號(hào)途徑對(duì)JA生物合成均存在負(fù)調(diào)控作用[37-38]。有趣的是, MeJA能有效恢復(fù)生長(zhǎng)素抑制的穎花開(kāi)放(圖4), 這提示高水平IAA對(duì)漿片JA生物合成可能有抑制作用, 兩者相互作用的分子機(jī)制有待進(jìn)一步研究。

作為高效的發(fā)育信號(hào), 激素必須壽命短并且不能長(zhǎng)時(shí)間積累, 細(xì)胞內(nèi)IAA穩(wěn)態(tài)(homeostasis)受到合成、代謝、結(jié)合和運(yùn)輸?shù)鹊木C合調(diào)控[23,32]。YUCCA是催化IPA (吲哚-3-丙酮酸)合成IAA的限速酶, 對(duì)生長(zhǎng)素的生物合成至關(guān)重要, 并在眾多植物研究中獲得證實(shí)。研究表明, 擬南芥YUCCA基因家族11個(gè)成員均參與IAA的生物合成[39]; HvYUCCA4特異表達(dá)于大麥花粉, 負(fù)責(zé)活性生長(zhǎng)素的合成[40]; 水稻中多數(shù)超表達(dá)后都導(dǎo)致轉(zhuǎn)基因植株IAA水平顯著增加, 并表現(xiàn)出相似的異常表型[41]。本研究中的表達(dá)下降, 尤其是, 該基因可能直接調(diào)控漿片IAA的生物合成。Lee等[42]研究發(fā)現(xiàn)擬南芥中超量表達(dá)還能提高植株的抗旱性。本研究結(jié)果顯示, 穎花開(kāi)放時(shí)漿片表達(dá)量顯著增加(圖6), 但I(xiàn)AA含量(圖5)并未積累, 推測(cè)穎花開(kāi)放時(shí)柱頭和花藥暴露在外, 需忍受一定程度的干旱脅迫, 從而誘導(dǎo)了該基因的增強(qiáng)表達(dá)。

生長(zhǎng)素的分解代謝保證了活性激素的濃度超過(guò)最適水平或激素反應(yīng)完成時(shí)活性激素的降解。是生長(zhǎng)素早期基因(、和)之一, 編碼生長(zhǎng)素酰胺合成酶, 催化游離的IAA不可逆降解生成OxIAA, 在IAA的酶促降解以及植株體內(nèi)IAA穩(wěn)態(tài)調(diào)控中起著非常重要的作用。和過(guò)量表達(dá)導(dǎo)致擬南芥?zhèn)雀∩偾野? 植株體內(nèi)高水平積累IAA; 而基因敲除后, 突變體對(duì)IAA非常敏感[43-44]。Ding等[45]研究發(fā)現(xiàn)水稻中GH3.8能催化IAA-氨基酸的合成從而抑制生長(zhǎng)素的作用。本研究顯示水稻穎花自然開(kāi)放時(shí), 漿片中和顯著增強(qiáng)表達(dá)(圖6), 表明IAA的不可逆降解增強(qiáng), 這與穎花中IAA水平低下(圖5)相符。

生長(zhǎng)素的極性運(yùn)輸依賴于3種載體蛋白: 輸入載體AUX1/LAX蛋白、輸出載體PIN蛋白和兼有輸入與輸出功能的ABCB/MDR/PGP蛋白, 植物通過(guò)調(diào)控這些載體蛋白來(lái)調(diào)節(jié)生長(zhǎng)素的極性運(yùn)輸和分布, 進(jìn)而調(diào)控植物的生長(zhǎng)發(fā)育[30]。本研究發(fā)現(xiàn), 穎花開(kāi)放時(shí)漿片和表達(dá)水平上升, 且生長(zhǎng)素極性運(yùn)輸正調(diào)控因子也上調(diào)表達(dá)(圖6), 表明生長(zhǎng)素輸出加快, 這與極性運(yùn)輸抑制TIBA推遲穎花開(kāi)放(圖3-A)符合。最新研究表明, ABCB可獨(dú)立于PIN蛋白作用于生長(zhǎng)素外流; 但PIN介導(dǎo)的生長(zhǎng)素外流主要是通過(guò)ABCB共定位的共依賴外流[46]。研究表明, 在擬南芥中AtABCB1和AtABCB19介導(dǎo)生長(zhǎng)素輸出[47-48], 而AtABCB4和AtABCB21兼具生長(zhǎng)素輸出和輸入雙重運(yùn)輸功能, 運(yùn)輸方向取決于細(xì)胞內(nèi)生長(zhǎng)素水平[49-50]。水稻OsABCB14已被證實(shí)是生長(zhǎng)素輸入載體, 并介導(dǎo)鐵(Fe)穩(wěn)態(tài)[51]。本研究鑒定到穎花開(kāi)放時(shí)漿片中表達(dá)水平顯著下調(diào)(圖6), 其生理功能有待進(jìn)一步研究。

4 結(jié)論

水稻穎花開(kāi)放需要漿片IAA水平的下降, 外源提高IAA水平則抑制穎花開(kāi)放, 但這種抑制效應(yīng)可被MeJA解除。水稻穎花自然開(kāi)放過(guò)程中漿片低水平IAA的形成受到IAA生物合成、結(jié)合、極性運(yùn)輸?shù)认嚓P(guān)途徑的調(diào)節(jié)。

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Study of regulating effect of auxin on floret opening in rice

HE Yong-Ming and ZHANG Fang*

Jiangxi Agricultural University / Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Ministry of Education / Jiangxi Key Laboratory of Crop Physiology, Ecology and Genetic Breeding, Nanchang 330045, Jiangxi, China

Floret opening which is driven by swelling of lodicules has a direct effect on rice pollination and fertilization. Auxin is an essential phytohormone in regulating reproductive development processes such as anther dehiscence, pollen fertility, and seed initiation. To elucidate the role of auxin in floret opening, the effects of exogenous auxin and its inhibitors on floret opening, and dynamic changes of endogenous auxin levels, and gene relative expression levels of auxin pathway in florets and lodicules were investigated incultivar Zhonghua 11. The results showed that the panicles soaked with IAA (10–20 mmol L–1) or NAA (0.05–0.50 mmol L–1) delayed significantly floret opening. Under high concentration of NAA (0.5 mmol L–1), compared with the water-treated panicles, the floret opening was postponed by three days. The prolonged opening duration of single floret and decreased seed-setting rate were also observed after IAA and NAA treatments. Treatments with IAA polar transport inhibitor TIBA and function inhibitor PCIB delayed florets opening as well. Furthermore, the application of methyl jamonate (MeJA) could restore effectively floret opening which was retared by NAA pretreatments. A sharp decline of IAA levels was detected two hours before floret opening in natural condition. Compared to that at 1 d and 2 d before opening, IAA level in florets at 2 hours before opening was decreased by 65.85% and 74.27%, respectively. Corresponding to the changes of IAA levels in florets, the relative expression levels of IAA biosynthetic genes (,) in the lodicules were down-regulated during floret opening, while the expressions of catabolic genes (/) in formation of inactive IAA conjugates, IAA efflux transport genes (,), and its positive regulatorgene were significantly up-regulated. 13 differentially expressed early auxin response genes (,,) were also identified, among which, 10 were up-regulated and 3 down-regulated. In conclusion, rice floret opening was regulated by endogenous auxin, but was inhibited by elevating auxin level in lodicules.

rice; floret opening; auxin; inhibitory effect

10.3724/SP.J.1006.2023.22027

本研究由國(guó)家自然科學(xué)基金項(xiàng)目(31360295, 31801272)和江西省自然科學(xué)基金項(xiàng)目(20212BAB205006, 20202BABL215001)資助。

This study was supported by the National Natural Science Foundation of China (31360295, 31801272) and the Jiangxi Provincial Natural Science Foundation (20212BAB205006, 20202BABL215001).

張芳, E-mail: zhangf0124@126.com

E-mail: hymcom@126.com

2022-05-07;

2022-10-10;

2022-10-26.

URL: https://kns.cnki.net/kcms/detail/11.1809.S.20221025.1811.008.html

This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).