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MultispeQ植物多參數測量儀在葉綠素熒光參數測量中的技術原理與應用研究

發布時間: 2025-08-29  點擊次數: 62次
一、研究背景與技術優勢

(一)葉綠素熒光參數測量的傳統挑戰

葉綠素熒光動力學技術是評估植物光合功能的核心手段,在植物生理生態研究中占據著關鍵地位。傳統方法需通過暗適應使光系統 II(PSII)反應中心全開放,以獲取最大熒光(Fm)和基線熒光(F0),進而計算 PSII 最大光化學效率(Fv/Fm)。這一過程耗時(通常 30 - 60 分鐘)且易受環境干擾,限制了野外高通量測量與動態監測。在野外復雜多變的環境中,難以長時間維持穩定的暗適應條件,而且長時間的暗適應處理可能會改變植物原有的生理狀態,導致測量結果不能準確反映植物在自然狀態下的光合特性。此外,傳統測量方法每次只能測量少量樣本,無法滿足大規模、高通量的研究需求,極大地制約了相關研究的開展和深入。

(二)MultispeQ 測量儀的技術突破

MultispeQ 植物多參數測量儀通過集成調制熒光技術與智能光源控制系統,突破了暗適應依賴的瓶頸,實現了自然光條件下快速、無損的葉綠素熒光參數測量,為植物脅迫響應研究、光合生理分析提供了高效工具。MultispeQ 植物多參數測量儀由著名光合作用專家David M. Kramer教授團隊研發的革命性技術,集合了葉綠素熒光儀、差示吸收儀、葉綠素儀和光譜儀的功能于一身,小巧輕便,是一款野外便攜且性價高的多功能植物測量儀!(Kramer教授是非常著名的光合作用專家,其論文總引用次數11700+次,h指數59,i10指數138,其2004年發表在Photosynthesis Research上提出qL、 ΦNPQ和ΦNO參數的文章已被引用1000+次。(Google Scholar數據,截止2019年7月))該測量儀利用調制熒光技術,能夠在自然光背景下精確檢測植物發出的微弱熒光信號,有效避免了環境光的干擾。其智能光源控制系統可以根據環境光照條件實時調整激發光的強度和頻率,確保在不同光照條件下都能獲得準確可靠的測量結果。這種創新的技術設計不僅大大縮短了測量時間,提高了測量效率,而且能夠在不影響植物正常生理活動的情況下進行測量,保證了測量結果的真實性和可靠性,為植物生理生態研究帶來了新的突破和發展機遇。

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二、非暗適應測量的技術原理

(一)動態光適應狀態下的熒光信號解析

傳統的葉綠素熒光測量方法依賴暗適應過程,其核心假設是通過暗處理使 PSII 反應中心全開放,以便準確測量初始熒光(Fo)和最大熒光(Fm) ,進而計算 PSII 最大光化學效率(Fv/Fm)。這種方法基于一種相對靜態的光合模型,假設在暗適應狀態下,植物光合機構處于一種 “基礎" 且穩定的狀態,能夠提供一個統一的測量基準。然而,在自然環境中,植物始終處于動態的光適應過程,這種暗適應處理不僅耗費時間,而且改變了植物原本的生理狀態,導致測量結果難以真實反映植物在實際光照條件下的光合功能。
MultispeQ 測量儀突破了這一傳統范式,采用了全新的測量策略。它利用弱調制探測光(光強≤0.1μmol?m?2?s?1),這種極弱的光不會激發 PSII 反應中心,卻能實時監測光適應狀態下的基線熒光(Fo')。通過這種方式,儀器捕捉到植物在自然光照下 PSII 反應中心的本底熒光信號,避免了暗適應處理對植物生理狀態的干擾。當需要測量光適應狀態下的最大熒光(Fm')時,MultispeQ 會發射飽和脈沖光(光強≥3000μmol?m?2?s?1,持續時間 0.8s) 。如此高強度的光脈沖能夠在瞬間誘導所有開放的反應中心關閉,使得 PSII 吸收的光能全部轉化為熒光和熱,從而獲取到 Fm'。
在實際測量過程中,MultispeQ 會結合光化光強度(PAR),同步計算實際光化學效率(ΦPSII)與電子傳遞速率(ETR)。例如,在對小麥進行光合測量時,儀器能夠在自然光條件下,快速準確地獲取這些參數,為研究小麥在不同光照強度下的光合動態提供了數據支持。這種測量方式無需暗處理,直接在植物的自然光適應狀態下進行,更能反映光合機構在實際環境中的動態功能,為研究植物在復雜光照條件下的光合響應提供了有力工具。

(二)非光化學淬滅(NPQ)的實時量化

非光化學淬滅(NPQ)是植物應對過剩光能的一種重要保護機制,它反映了植物將吸收的光能以熱的形式耗散掉的能力。傳統測量方法在測量 NPQ 時,需要經過暗弛豫過程,這不僅耗時,而且在暗處理過程中,植物的生理狀態會發生變化,導致測量結果不能準確反映植物在光照條件下的真實 NPQ 水平。
MultispeQ 測量儀通過創新的算法,實現了 NPQ 的實時量化。儀器通過對比暗適應初始熒光(Fo)與光適應基線熒光(Fo'),結合公式\(NPQ = \frac{Fm - Fm'}\) ,實時評估植物的熱耗散能力。在這個公式中,Fm 代表暗適應狀態下的最大熒光,Fm' 代表光適應狀態下的最大熒光。通過這兩個參數的比值,能夠準確計算出 NPQ 的值。在對玉米進行干旱脅迫研究時,MultispeQ 可以在干旱處理的不同時間點,實時測量 NPQ 的變化,發現隨著干旱程度的加劇,NPQ 值逐漸升高,表明玉米通過增強熱耗散來應對過剩光能,保護光合機構。
這種實時測量 NPQ 的方法,避免了暗弛豫過程對測量的干擾,特別適用于研究強光脅迫、快速光響應等動態場景。在野外環境中,光照強度和光質隨時變化,MultispeQ 能夠實時跟蹤植物 NPQ 的動態變化,為研究植物在自然條件下的光保護機制提供了更為準確的數據,有助于深入理解植物在不同環境脅迫下的光合適應策略。

三、MultispeQ 測量指標體系

(一)光合效能核心參數

1.PSII 最大光化學效率(Fv/Fm):該參數通過公式\(Fv/Fm = (Fm - Fo)/Fm\)計算得出,其中 Fo 為暗適應初始熒光,Fm 為暗適應最大熒光 。它反映了暗適應狀態下 PSII 反應中心的潛在光能轉換效率,在未受脅迫的健康植物中,其值通常穩定在 0.80 - 0.83 范圍內。Fv/Fm 常被用作檢測植物長期遭受脅迫(如鋁脅迫)對光合機構損傷程度的重要指標。在研究鋁脅迫對植物的影響時發現,隨著處理液中 Al3?濃度的升高,植物葉片的 Fv/Fm 值顯著降低,表明 PSII 反應中心受到了損傷,光能轉換效率下降。

 2.實際光化學效率(ΦPSII):計算公式為\(ΦPSII = (Fm' - Fo')/Fm'\),其中 Fm' 為光適應狀態下的最大熒光,Fo' 為光適應基線熒光 。ΦPSII 表征了光適應狀態下 PSII 的實時能量轉換效率,能夠敏感地反映植物在短期環境變化(如水分脅迫)下光合機構的響應。在水分脅迫實驗中,隨著脅迫時間的延長,植物葉片的 ΦPSII 值快速下降,表明光合機構的能量轉換效率受到抑制,光合作用受到影響。

3.電子傳遞速率(ETR):ETR 的計算結合了光合有效輻射(PAR),公式為\(ETR = ΦPSII×PAR×0.5×0.84\)。該參數表征了光合電子傳遞鏈的活性,通過評估電子傳遞速率,可以間接了解植物的碳同化效率。在不同光照強度下對植物進行測量時,發現隨著 PAR 的增加,ETR 也相應增加,表明光合電子傳遞鏈活性增強,有利于碳同化過程的進行。

(二)環境響應與脅迫指標

1.非光化學淬滅系數(NPQ):NPQ 是衡量植物光系統熱耗散能力的關鍵指標,其計算公式為\(NPQ = (Fm - Fm')/Fm'\) 。當植物吸收的光能超過其光合作用的需求時,會通過非光化學途徑將過剩的光能以熱的形式耗散掉,從而保護光合機構免受損傷。NPQ 的升高表明植物啟動了這種光保護機制,通過增加熱耗散來應對過剩光能。在強光脅迫實驗中,植物葉片的 NPQ 值迅速升高,有效避免了光合機構因吸收過多光能而受到損傷。

2.光適應熒光參數(Fv'/Fm'):該參數反映了光下 PSII 反應中心的開放比例,計算公式為\(Fv'/Fm' = (Fm' - Fo')/Fm'\) 。Fv'/Fm' 常用于快速判斷植物在短期光脅迫(如高光強、高溫)下光合機構的即時狀態。在高溫脅迫實驗中,隨著溫度的升高,Fv'/Fm' 值逐漸降低,表明 PSII 反應中心的開放比例減少,光合機構受到了一定程度的抑制。

(三)多維度同步測量參數

1.環境傳感器:MultispeQ 集成了多種環境傳感器,能夠實時測量光照強度(PAR)、溫度(精度可達 ±0.5℃)、濕度(精度可達 ±2% RH)以及 GPS 定位(精度 < 5m) 。通過同步獲取這些環境參數與植物生理參數,研究人員可以深入分析環境因子對植物光合生理的影響。在研究不同光照強度對植物光合參數的影響時,結合 PAR 數據,可以更準確地了解光照與光合參數之間的關系。同時,GPS 定位功能使得研究人員能夠對不同地理位置的植物進行定位和追蹤,為大規模的野外研究提供了便利。

2.光譜與形態指標:儀器通過測量 400 - 750nm 的反射光譜,能夠計算出葉綠素含量指數(SPAD 等效值)和歸一化植被指數(NDVI) 。SPAD 等效值可用于評估植物葉片的葉綠素含量,間接反映植物的生長狀況和營養水平;NDVI 則常用于監測植被的生長狀態和覆蓋度。此外,MultispeQ 還利用 515/550nm 的差示吸收光譜來評估植物的色素代謝狀態,通過分析不同波長光的吸收差異,了解植物體內光合色素的組成和含量變化,為研究植物的生理狀態提供了更豐富的信息。

四、技術驗證與應用案例

(一)脅迫響應研究中的實證分析

在鋁脅迫下蠶豆幼苗的研究中,MultispeQ 展現出了對植物光合生理變化的高度敏感性。研究人員設置了不同濃度的 Al3?處理組,其中高濃度 Al3?(100μM)處理 9 天后,蠶豆幼苗葉片的 Fv'/Fm' 值較對照顯著下降(P<0.05) 。這一結果表明,PSII 反應中心的開放效率受到了嚴重損害,植物在光適應狀態下將光能轉化為化學能的能力下降。同時,實際光化學效率(ΦPSII)與表觀光合電子傳遞速率(ETR)隨脅迫時間延長呈梯度降低。在處理初期,ΦPSII 和 ETR 的下降幅度相對較小,但隨著脅迫時間的增加,這些參數急劇下降,驗證了 MultispeQ 對短期脅迫響應的敏感性。這種實時監測能力,有助于研究人員及時捕捉植物在脅迫初期的生理變化,為深入研究植物的抗逆機制提供了有力的數據支持。

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(二)野外高通量測量的優勢

相較于傳統熒光儀,如 Li-6800,MultispeQ 在野外測量中具有顯著的優勢。傳統熒光儀在進行葉綠素熒光參數測量時,需要攜帶暗適應葉夾,操作過程繁瑣且耗時。每次測量前,都需要將葉片放入暗適應葉夾中進行 30 - 60 分鐘的暗適應處理,這在野外環境中不僅操作不便,而且會影響測量效率。而 MultispeQ 的便攜式設計(重量 < 500g)使其易于攜帶,研究人員可以輕松地在田間進行移動測量。其快速測量模式(單次測量≤15 秒)大大提高了測量效率,在國際水稻研究所的田間表型篩選工作中,使用 MultispeQ 單日能夠完成 2000 份耐鹽水稻樣本的檢測,相較于傳統方法,效率提升了 15 倍以上。這種高通量的測量能力,使得研究人員能夠在短時間內獲取大量的數據,為大規模的植物表型研究和品種篩選提供了高效的技術手段 。

五、參考文獻
[1] Kuhlgert S, Austic G, Zegarac R, et al. MultispeQ Beta: a tool for large-scale plant phenotyping connected to the open PhotosynQ network [J]. Royal Society Open Science, 2016, 3 (10):160592.
[2] Ben-Jabeur M, Romero A G, Vicente R, et al. MultispeQ for Tracing Biostimulants Effect on Growth Promoting and Water Stress Tolerance in Wheat [C]//2021.
[3] Ntakos G, Prikaziuk E, Vilfan N, et al. From fluorescence to biomass: A comprehensive analysis via crop modeling and sensing techniques [J]. Smart Agricultural Technology, 2025, 10.
[4] 劉東華,張慧敏。鋁脅迫對蠶豆幼苗葉綠素熒光參數和葉綠素含量的影響 [J]. 天津師范大學學報 (自然科學版), 2020, 40 (3):45-50.
[5] PhotosynQ Consortium. Global Deployment Report (2023). DOI:10.1038/sdata.2023.123.
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