四通道動態(tài)LED陣列近紅外光譜儀

DUAL-KLAS-NIR

同步測量PSII活性（葉綠素?zé)晒猓?/span>和PSI活性（P700）

PC（質(zhì)體藍(lán)素）Fd（鐵氧還蛋白）的氧化還原變化

相較于經(jīng)典的雙通道葉綠素?zé)晒鈨xDUAL-PAM-100測量系統(tǒng)，新一代四通道動態(tài)LED陣列近紅外光譜儀DUAL-KLAS-NIR，在光合作用電子傳遞鏈組分質(zhì)體藍(lán)素(PC)、光系統(tǒng)I反應(yīng)中心(P700)及鐵氧還蛋白(Fd)的氧化還原測量方面實(shí)現(xiàn)了重大技術(shù)突破。它創(chuàng)新性的采用了四波長對近紅外探測技術(shù)，成功解決了圍繞光系統(tǒng)I供體側(cè)和受體側(cè)電子傳遞精準(zhǔn)解析的難題，為光合作用研究開辟了一條嶄新的道路。

作為PSI的電子供體和電子受體，PC(質(zhì)體藍(lán)素)和Fd(鐵氧還蛋白)對PSI的氧化還原起著至關(guān)重要的調(diào)控作用。但一直缺乏科學(xué)便捷的手段對其運(yùn)轉(zhuǎn)狀態(tài)進(jìn)行檢測。DUAL-KLAS-NIR采用先進(jìn)的去卷積技術(shù)(一種根據(jù)來源不同對信號進(jìn)行分離的技術(shù))，能夠同時測量4組不同波長對(780-820nm，820-870nm，840-965nm，870-965nm)的信號，實(shí)現(xiàn)對P700(PSI反應(yīng)中心)、PC和Fd的氧化還原狀態(tài)的同步分析。另外，它還可以測量由540nm和460nm光化光激發(fā)的葉綠素?zé)晒?。還有， DUAL-KLAS-NIR四通道動態(tài)LED陣列近紅外光譜儀也可以擴(kuò)展P515/535模塊，測量跨類囊體膜的質(zhì)子梯度ΔpH和跨膜電位ΔΨ，分析與電子傳遞耦合的跨類囊體膜質(zhì)子轉(zhuǎn)移，質(zhì)子動力勢pmf形成?？梢詳U(kuò)展NADPH/9-AA模塊，測量NADP+的還原程度。最后，DUAL-KLAS-NIR也可以通過聯(lián)用葉室3010-DUAL與GFS-3000光合儀聯(lián)用，同步測量光反應(yīng)電子傳遞和暗反應(yīng)CO2同化，系統(tǒng)全面的研究光合作用機(jī)理。

從2016年2月Photosynthesis Research雜志發(fā)表Schreiber博士團(tuán)隊(duì)標(biāo)題為“Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer”的研究論文，隆重介紹了最新設(shè)計(jì)的DUAL-KLAS-NIR四通道動態(tài)LED陣列近紅外光譜儀。時至今日，四通道動態(tài)LED陣列近紅外光譜儀DUAL-KLAS-NIR已累計(jì)發(fā)表論文超過80篇。其中不乏Nature Plants，Nature Communications，The Plant Cell，New Phytologist，Plant Physiology，The Plant Journal等植物學(xué)領(lǐng)域的專業(yè)高分雜志文章(詳見附錄)。

突出特點(diǎn)：

1. 可測量活體植物葉片或葉綠體/類囊體/藻類懸浮液，對P700、PC和Fd分別進(jìn)行連續(xù)實(shí)時的分析。

2. 藍(lán)光460nm和綠光540nm雙波長調(diào)制葉綠素?zé)晒鉁y量，可分別測量葉片表層和深層細(xì)胞的光能轉(zhuǎn)換。

3. 通過板載芯片LED技術(shù)設(shè)計(jì)了高度緊湊的固態(tài)照明系統(tǒng)，可提供635nm，460nm的光化光和740nm的遠(yuǎn)紅光，以及635nm單周轉(zhuǎn)和多周轉(zhuǎn)飽和閃光。

4. 光學(xué)部件的幾何結(jié)構(gòu)完美兼容3010-DUAL聯(lián)用葉室，可與GFS-3000光合儀組合，在可控條件(光照，溫度，濕度，CO2濃度)下同步測量氣體交換相關(guān)的CO2同化和電子傳遞相關(guān)的氧化還原。

5. 測量光頻率范圍廣(1- 400 kHz)，允許連續(xù)評估Fo，也可以在高時間分辨率下記錄葉綠素?zé)晒饪焖賱討B(tài)瞬變(如多相熒光上升動力學(xué)或脈沖弛豫動力學(xué))。

6. 專業(yè)數(shù)據(jù)記錄軟件，入門特別簡單。

主要功能：

1. 測量暗適應(yīng)樣品的PC，P700和Fd最大氧化/還原量，根據(jù)光譜特征可計(jì)算PC/P700和Fd/P700的比值，評估PSI及其與供體側(cè)和受體側(cè)的氧化還原平衡，用于PSI復(fù)合體組裝中間體功能的研究。

2. 測量并記錄光適應(yīng)條件下光合電子傳遞過程中質(zhì)體藍(lán)素(PC)，PS I反應(yīng)中心(P700)和鐵氧還蛋白(Fd)的氧化還原比例，評估PSI及其與供體側(cè)和受體側(cè)的相互關(guān)系和協(xié)調(diào)性。

3. 可以通過藍(lán)色和/或綠色PAM熒光測量葉片表層和深層細(xì)胞的光能轉(zhuǎn)換，非常適合與整個葉子的NIR吸收測量進(jìn)行對比分析。

4. 完整的慢速誘導(dǎo)動力學(xué)曲線和快速誘導(dǎo)動力學(xué)曲線測量功能，慢速誘導(dǎo)動力學(xué)可進(jìn)行飽和脈沖分析、淬滅分析，誘導(dǎo)曲線，光合控制，快速光曲線和暗弛豫曲線測量；快速誘導(dǎo)動力學(xué)可進(jìn)行Qa_Decay，Poly300ms等十幾種程序測量。

5. 可使用軟件的自動測量程序?qū)嶒?yàn)，也可以編輯腳本(Script)或者保存手動測量程序(Trigger)，輕松執(zhí)行復(fù)雜的測量協(xié)議?？勺远x測量動作用于特殊誘導(dǎo)過程動力學(xué)曲線數(shù)據(jù)獲取和分析，如狀態(tài)轉(zhuǎn)換和波動光曲線等。

6. 擴(kuò)展P515/535模塊，可測量跨類囊體膜的質(zhì)子梯度ΔpH和跨膜電位ΔΨ，分析與電子傳遞耦合的跨類囊體膜質(zhì)子轉(zhuǎn)移，質(zhì)子動力勢pmf形成。

7. 擴(kuò)展NADPH/9-AA模塊，可測量NADP+的還原程度。

軟件界面：

擴(kuò)展模塊：

1、擴(kuò)展P515/535模塊，可測量跨類囊體膜的質(zhì)子梯度ΔpH和跨膜電位ΔΨ，分析與電子傳遞耦合的跨類囊體膜質(zhì)子轉(zhuǎn)移，質(zhì)子動力勢pmf形成。

2、擴(kuò)展NADPH/9-AA模塊，可測量NADP+的還原程度。

3、擴(kuò)展3010-DUAL聯(lián)用葉室，可與GFS-3000光合儀組合，在可控條件(光照，溫度，濕度，CO2濃度)下同步測量氣體交換相關(guān)的CO2同化和電子傳遞相關(guān)的氧化還原。

應(yīng)用領(lǐng)域：

1. 光合電子傳遞鏈復(fù)合體的氧化還原狀態(tài)深入剖析，類囊體膜蛋白組分功能研究，如光系統(tǒng)I的裝。

2. 光合合成生物學(xué)研究相關(guān)的植物學(xué)，植物生理學(xué)，分子生物學(xué)，農(nóng)學(xué)，林學(xué)、園藝的領(lǐng)域。

3. 人工光合作用和能源相關(guān)領(lǐng)域，如生物光伏等

應(yīng)用案例：

案例1. 德國Christian-Albrechts大學(xué)的科學(xué)家Jens Appel使用四通道動態(tài)LED陣列近紅外光譜儀Dual-KLAS-NIR，測量藍(lán)藻Synechocystis sp.PCC 6803圍繞PSI的質(zhì)體藍(lán)素、P700和FeS簇（包括鐵氧還蛋白）的氧化還原狀態(tài)，首次以絕對值量化了光合生物中通過光系統(tǒng)I的電子流。該研究確定了線性和環(huán)式電子傳遞的比例：環(huán)式電子傳遞占通過PSI的電子流的35%。

Marius L. , et al.2020, BBA – Bioenergetics

https://doi.org/10.1016/j.bbabio.2020.148353

案例2. 德國WALZ的應(yīng)用科學(xué)家Gert Schansker博士使用四通道動態(tài)LED陣列近紅外光譜儀DUAL-KLAS-NIR測量33種植物的光曲線，探測和表征光合控制的光強(qiáng)度依賴性。研究發(fā)現(xiàn)， PC在光強(qiáng)≤400 μmolm-2s-1時完全氧化(陰生植物的葉片的光照強(qiáng)度低于陽生植物葉片)。qP和還原態(tài)P700之間的關(guān)系可以用于衡量光合控制的程度。除了測量光曲線，也可以使用單個中等光強(qiáng)度來表征葉片之間的相對狀態(tài)。進(jìn)一步的發(fā)現(xiàn)，在一些適應(yīng)陰生環(huán)境的葉片中，F(xiàn)d在高光強(qiáng)度下變得更加氧化表明從PQ庫到P700的電子傳遞無法跟上PS I受體側(cè)的電子流出。與光合控制的誘導(dǎo)相比，NPQ誘導(dǎo)需要較低的光強(qiáng)度(類囊體腔酸化程度低)。測量結(jié)果還可以用于比較qP和qL，比較的結(jié)果是qP是與光合控制更相關(guān)的葉綠素?zé)晒鈪?shù)。

Gert Schansker, 2022, Photosynthesis Research

https://doi.org/10.1007/s11120-022-00934-7

案例3. 芬蘭圖爾庫大學(xué)的Tapio Lempi?inen等人通過對正常溫度下培養(yǎng)的擬南芥設(shè)置5種不同的光處理：(1).不處理，(2).60% PSI光抑制，(3).85% PSI光抑制, (4) .60% PSI光抑制后在生長條件下“恢復(fù)”24小時，(5).85% PSI 光抑制后在生長條件下“恢復(fù)”24 小時。然后對不同處理樣品的主要光合復(fù)合物、光合光反應(yīng)的功能和調(diào)節(jié)、ATP 合酶和碳同化進(jìn)行分析。研究功能性PSII和PSI之間的不平衡是否會誘導(dǎo)光合作用適應(yīng)PSI受限的條件。探索植物短期和長期的馴化機(jī)制。研究發(fā)現(xiàn)，抑制后直接測量可探測短期適應(yīng)機(jī)制，包括將激發(fā)能量重定向到PSI 的類囊體蛋白磷酸化，光合作用反饋調(diào)節(jié)的變化，比如放松光合作用控制(Photosynthetic Control)和激發(fā)能淬滅。處理后恢復(fù)24小時測量可以有效探測光合機(jī)構(gòu)的長期適應(yīng)機(jī)制，比如基質(zhì)氧化還原系統(tǒng)的重建以及ATP合酶和細(xì)胞色素b6f豐度的增加。植物在適應(yīng)了PSI限制條件后無需進(jìn)行大量PSI修復(fù)即可恢復(fù)CO2同化能力。對 PSI 抑制的反應(yīng)表明植物有效地適應(yīng)了光合機(jī)構(gòu)中發(fā)生的變化，這可能是植物適應(yīng)不利環(huán)境條件的關(guān)鍵組成部分。

Lempi?inen, T., et al. 2022.

https://doi.org/10.1111/pce.14400

案例4. 德國亥姆霍茲環(huán)境研究中心Lai Bin團(tuán)隊(duì)通過四通道動態(tài)LED陣列近紅外光譜儀Dual-KLAS-NIR系統(tǒng)地研究了在BPV系統(tǒng)中培養(yǎng)的藍(lán)藻集胞藻的光合電子流，揭示了電子傳遞鏈中各組分的氧化還原狀態(tài)，并描繪了相應(yīng)的電子流向各種匯。該研究表明，EET與PSI下游的類Mehler反應(yīng)競爭電子。在高濃度下，亞鐵氰化物對電子傳遞鏈的影響與微量氰化物相似，突出了精心設(shè)計(jì)BPV實(shí)驗(yàn)的必要性。此外，該團(tuán)隊(duì)另外一項(xiàng)研究還通過Dual-KLAS-NIR測量PSI、質(zhì)體藍(lán)素和鐵氧還蛋白的氧化還原變化。闡明了生物光伏藍(lán)藻集胞藻動態(tài)切換電子源，并根據(jù)生理和環(huán)境條件，利用不同的細(xì)胞外轉(zhuǎn)移途徑將電流輸出到外部電子匯的機(jī)理。

Jianqi Yuan., et al. 2024, https://doi.org/10.1016/j.ese.2024.100519.

Schneider, H., et al. 2025, https://doi.org/10.1111/tpj.17225

案例5. 英國謝菲爾德大學(xué)Matthew P Johnson課題組利用基于CRISPR/Cas9的基因編輯技術(shù)，在萊茵衣藻中構(gòu)建了通過PSAF將FNR錨定于PSI的嵌合型突變體。使用DUAL-PAM-100雙通道葉綠素?zé)晒鈨xP515/535模塊檢測電致變色位移(ECS)，使用 DUAL-KLAS-NIR四通道動態(tài)LED陣列近紅外光譜儀以類似方法測量P700氧化。研究發(fā)現(xiàn)，相較于野生型，嵌合突變體因NADPH還原速率降低導(dǎo)致光合生長受限、線性電子傳遞受阻，且PSI受體側(cè)限制增強(qiáng)。但該突變體同時表現(xiàn)出增強(qiáng)的跨膜質(zhì)子梯度(ΔpH)和非光化學(xué)淬滅(NPQ)，表明CET活性顯著提升。因此，將FNR錨定于PSI并未促進(jìn)光合線性電子傳遞，反而通過犧牲線性電子傳遞與CO?固定效率優(yōu)先支持環(huán)式電子傳遞。這一發(fā)現(xiàn)揭示了FNR定位對光合電子流向分配的關(guān)鍵調(diào)控作用。

Emrich-Mills, T. Z., et al. 2025.

https://doi.org/10.1093/plcell/koaf042

產(chǎn)地：德國WALZ

代表文獻(xiàn)：

數(shù)據(jù)來源：光合作用文獻(xiàn)Endnote數(shù)據(jù)庫

原始數(shù)據(jù)來源：Google Scholar

 2025

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