四通道动态LED阵列近红外光谱仪
DUAL-KLAS-NIR
同步测量PSII活性（叶绿素荧光）和PSI活性（P700）
PC（质体蓝素）Fd（铁氧还蛋白）的氧化还原变化
2016年2月Photosynthesis Research杂志发表了Schreiber博士团队的研究文章Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer，隆重介绍了DUAL-KLAS-NIR四通道动态LED阵列近红外光谱仪。之后2016年4月，2017年3月Schreiber博士团队再次发表文章，进一步阐述DUAL-KLAS-NIR的实际应用。
作为PSI的电子供体和电子受体，PC（质体蓝素）和Fd（铁氧还蛋白）对PSI的氧化还原起着至关重要的调控作用。但一直缺乏科学便捷的手段对其运转状态进行检测。集成以DUALl-PAM-100为标志的第二代PAM的基本功能，采用先进的去卷积技术（一种根据来源不同对信号进行分离的技术），WALZ公司推出了可以测量PC和Fd氧化还原状态的新一代PAM荧光仪—DUAL-KLAS-NIR四通道动态LED阵列近红外光谱仪。
DUAL-KLAS-NIR不但集成了Dual-PAM-100的基本功能，可以同时测量PSP和PSI，而且能够测量4组不同波段（780-820nm，820-870nm，840-965nm，870-965nm）的信号，实现对P700（PSI反应中心）、PC和Fd的氧化还原状态分别测量。另外，它还可以测量由540nm和460nm光化光激发的叶绿素荧光。利用DUAL-KLAS-NIR四通道动态LED阵列近红外光谱仪，可以准同步地测量各种不同的信号，不仅在驰豫动力下，还可持续地在自然稳态下同时获取各组分的信息。
突出特点
Ø 可测量活体叶片或悬浮液，对P700、PC和Fd分别进行连续的实时的去卷积分析。
Ø 同时测量分别由540nm（整个叶片）和460nm（表层细胞层）波段激发的两种叶绿素荧光。
Ø 通过集成发光二极管技术，独创高度紧凑的固态照明系统，提供635nm，460nm的光化光和740nm波段远红光，以及635nm单周转和多周转饱和闪光。
Ø 拥有和DUAL-PAM-100相似的光学部件几何结构，可与3010-DUAL兼容，结合GFS-3000光合仪，在可控条件（光照，温度，湿度，CO2浓度）下，同步测量气体交换和电子传递相关的氧化还原。
Ø 测量光频率范围广（1 - 400 kHz），允许连续评估Fo，可以在高时间分辨率下记录快速动态瞬变（如多相荧光上升动力学或脉冲弛豫动力学）。
主要功能
Ø 测定质体蓝素（PC），PS I反应中心（P700）和铁氧还蛋白（Fd）的氧化还原变化。
Ø 通过应用创新的分析方法获得PC，P700和Fd光谱特征。在线监测P700，PC和Fd的氧化还原变化，并确定PC / P700和Fd / P700的比值。
Ø 可以通过绿色或蓝色PAM测量光来激发荧光。绿光比蓝光更深入到叶子中。因此，绿色激发的荧光包括来自更深叶层的信息，因此非常适合与整个叶子的NIR吸收测量进行对比分析。
Ø 专业数据记录软件，入门特别简单。可使用DUAL-KLAS-NIR软件的自动测量程序实验，也可以编辑脚本（Script）或者保存手动测量程序（Trigger），轻松执行复杂的测量协议。可自定义测量动作用于特殊诱导过程动力学曲线数据获取和分析。
Ø 兼具慢速动力学曲线（饱和脉冲分析、诱导曲线和光响应曲线）和快速动力学曲线（饱和脉冲动力学曲线、高达30µs分辨率的驰豫动力学曲线）。
DUAL-KLAS-NIR软件近红外测量光设置 |
同步测量Fluo, P700, PC, Fd慢速诱导动力学曲线 |
应用领域
光合作用电子传递过程各复合体的氧化还原状态深入剖析，类囊体膜蛋白组分功能研究。
可广泛应用于光合合成生物学研究相关的植物学，植物生理学，分子生物学，农学，林学的领域。
应用案例
DUAL-KLAS-NIR为光合作用开辟了一个全新的研究领域，实时显示P700，PC和Fd在活体材料中的氧化还原状态，在线解卷积氧化还原信号。完美实现PS I及其供体侧和受体侧氧化还原动力学的同步测量，从而了解它们围绕光系统I的复杂相互作用，另外还可以探究PS I周围的循环电子传递的信息。
在DUAL-KLAS-NIR出现之前，测量光系统I的有效量子产量，P700信号总是会掺杂Fd的贡献和PC的变量。上图中图C显示了不同光强梯度下甘蓝型油菜叶片PSI的有效PSI量子产量Y(I)，PSII的有效量子产量Y(II)和经PSI荧光修正后的PSII的有效量子产率Y(II)corr。经过修正后，Y(II)corr和Y(I)在低光强下相似（小于500μmol m-2 s-1）。然而，当光强大于500μmol m-2 s-1时，Y(I)明显高于Y(II)，Y(I)/Y(II)最高可达1.45.
光系统I的有效天线尺寸测量。植物样品从在黑暗条件转移到光下时，在PSI附近，首先PC被氧化，开始积累，之后才是P700被氧化。单纯的PC信号变化的初始斜率可以用作PS I的有效天线尺寸的度量。
| 右图是放大后的PC（红色）和P700（蓝色）初始吸光度变化，显示了他们初始斜率的巨大差异。对于黑暗适应的叶子，转到光下的短时间内，光系统I受体侧未活化，Fd还原的初始斜率也也说明了这一点。 |  |
DUAL-KLAS-NIR软件设有一个窗口显示P700和PC氧化还原状态的相对变化。该功能可以用来计算PC和P700之间的表观平衡常数。这对研究P700与其供体侧的相互关系是非常重要的。 |  |
对暗适应的叶子施加饱和脉冲，测量Fd氧化还原动力学。我们不难发现，饱和脉冲产生的电子将Fd还原，饱和脉冲之后的黑暗中，Fd被缓慢再氧化。之后，PSI的受体侧的电子流被激活，再氧化动力学变得更快。在激活PSI的受体侧之后，可以通过监测脉冲后Fd再氧化的速率来研究Fd的暗灭活。这些动力学变化可以通过指数拟合程序拟合。图A给出了Fd再氧化动力学曲线指数拟合程序拟合的实例，图B显示了常春藤叶片不同暗适应时间后的PSI受体侧的暗灭活动力学差异。
PC，P700和Fd的最大NIR透射率变化与这些复合物的在样品中的含量成比例，并且PC，P700和Fd的消光系数的比率是恒定的。这可以用于探究不同物种或不同生长条件下（例如阳生/阴生，胁迫/非胁迫）样品的PC / P700和Fd / P700比率，以及PC和Fd库的相对大小。现已观察到高PC / P700比率与高电子传递速率（ETR）值相关。上图显示，在常春藤阳生和阴生叶片中，相对于P700，它们PC和Fd含量有着显著的不同。
主要测量参数：
Ø 叶绿素荧光测量：Fo, Fm, Fm’, F, Fo’, Fv/Fm, Y(II), qP, qL, qN, NPQ, Y(NO), Y(NPQ) , ETR(II)等参数，以及各种荧光动力学曲线。
Ø P700测量：必须能够测量Pm, Pm’, Y(I), ETR(I)， Y(ND)和Y(NA)等参数，以及各种P700动力学曲线。
Ø PC测量：PCm, PCm’, PCox, Rel PCox
Ø Fd测量：Fdm, Fdm’, Fdred, Rel Fdred, Fd/PC
Ø 实时显示数据采集，可以连续显示数据采集过程即完整的动力学曲线过程
Ø 软件程序：慢速动力学曲线，快速动动力学曲线，曲线拟合
产地：德国WALZ
代表文献
数据来源：光合作用文献Endnote数据库
原始数据来源：Google Scholar
2022
Santana-Sánchez, A., et al. (2022). "Flv3A facilitates O2 photoreduction and affects H2 photoproduction independently of Flv1A in diazotrophic Anabaena filaments." New Phytol n/a(n/a).
https://doi.org/10.1111/nph.18506
Lazár, D., et al. (2022). "Insights on the regulation of photosynthesis in pea leaves exposed to oscillating light." Journal of Experimental Botany 73(18): 6380–6393.
https://doi.org/10.1093/jxb/erac283
Lucius, S., et al. (2022). "CP12 fine-tunes the Calvin-Benson cycle and carbohydrate metabolism in cyanobacteria." 13.
https://doi.org/10.3389/fpls.2022.1028794
Khruschev, S. S., et al. (2022). "Machine learning methods for assessing photosynthetic activity: environmental monitoring applications." Biophysical Reviews.
https://doi.org/10.1007/s12551-022-00982-2
Penzler, J.-F., et al. (2022). "Commonalities and specialties in photosynthetic functions of PROTON GRADIENT REGULATION5 variants in Arabidopsis." Plant Physiology.
https://doi.org/10.1093/plphys/kiac362
Appel, J., et al. (2022). "Evidence for Electron Transfer from the Bidirectional Hydrogenase to the Photosynthetic Complex I (NDH-1) in the Cyanobacterium Synechocystis sp. PCC 6803." Microorganisms 10(8): 1617.
https://doi.org/10.3390/microorganisms10081617
Lempiäinen, T., et al. (2022). "Plants acclimate to Photosystem I photoinhibition by readjusting the photosynthetic machinery." Plant Cell Environ.
https://doi.org/10.1111/pce.14400
Schansker, G. (2022). "Determining photosynthetic control, a probe for the balance between electron transport and Calvin–Benson cycle activity, with the DUAL-KLAS-NIR." Photosynthesis Research.
https://doi.org/10.1007/s11120-022-00934-7
Burgstaller, H., et al. (2022). "Synechocystis sp. PCC 6803 Requires the Bidirectional Hydrogenase to Metabolize Glucose and Arginine Under Oxic Conditions." Front Microbiol 13: 896190.
https://doi.org/10.3389/fmicb.2022.896190
Rodriguez-Heredia, M., et al. (2022). "Protection of photosystem I during sudden light stress depends on ferredoxin:NADP(H) reductase abundance and interactions." Plant Physiology.
https://doi.org/10.1093/plphys/kiab550
Wang, Y., et al. (2022). "Pyruvate:ferredoxin oxidoreductase and low abundant ferredoxins support aerobic photomixotrophic growth in cyanobacteria." eLife 11.
https://doi.org/10.7554/eLife.71339
Niu, Y., et al. (2022). "A plant’s capacity to cope with fluctuating light depends on the frequency characteristics of non-photochemical quenching and cyclic electron transport." bioRxiv: 2022.2002.2009.479783.
https://doi.org/10.1101/2022.02.09.479783
Schmidtpott, S. M., et al. (2022). "Scrutinizing the Impact of Alternating Electromagnetic Fields on Molecular Features of the Model Plant Arabidopsis thaliana." International Journal of Environmental Research and Public Health 19(9): 5144.
https://doi.org/10.3390/ijerph19095144
2021
Furutani, R., et al. (2021). "The difficulty of estimating the electron transport rate at photosystem I." Journal of Plant Research.
https://doi.org/10.1007/s10265-021-01357-6
Rodriguez-Heredia, M., et al. (2021). "Protection of photosystem I during sudden light stress depends on ferredoxin:NADP(H) reductase abundance and interactions." Plant Physiology.
https://doi.org/10.1093/plphys/kiab550
Santana-Sánchez, A. (2021). "DYNAMIC REGULATION OF OXYGENIC PHOTOSYNTHESIS IN CYANOBACTERIA BY FLAVODIIRON PROTEINS."
https://www.utupub.fi/handle/10024/152715
Balti, H., et al. (2021). "Differences in Ionic, Enzymatic, and Photosynthetic Features Characterize Distinct Salt Tolerance in Eucalyptus Species." Plants 10(7): 1401.
https://www.mdpi.com/2223-7747/10/7/1401
Castell, C., et al. (2021). "New Insights into the Evolution of the Electron Transfer from Cytochrome f to Photosystem I in the Green and Red Branches of Photosynthetic Eukaryotes." Plant and Cell Physiology.
https://doi.org/10.1093/pcp/pcab044
Hepworth, C., et al. (2021). "Dynamic thylakoid stacking and state transitions work synergistically to avoid acceptor-side limitation of photosystem I." Nature Plants.
https://doi.org/10.1038/s41477-020-00828-3
Mattila, H., et al. (2021). "Singlet oxygen, flavonols and photoinhibition in green and senescing silver birch leaves." Trees.
https://doi.org/10.1007/s00468-021-02114-x
Miyake, C. (2021). "Photosynthetic Linear Electron Flow Drives CO2 Assimilation in Maize Leaves." International journal of molecular sciences 22.
https://doi.org/10.3390/ijms22094894
Ohnishi, M., et al. (2021). "Photosynthetic Parameters Show Specific Responses to Essential Mineral Deficiencies." Antioxidants 10(7): 996.
https://www.mdpi.com/2076-3921/10/7/996
Rühle, T., et al. (2021). "PGRL2 triggers degradation of PGR5 in the absence of PGRL1." Nature communications 12(1): 3941.
https://doi.org/10.1038/s41467-021-24107-7
2020
Nikkanen L, Santana Sánchez A, Ermakova M, Rögner M, Cournac L, Allahverdiyeva Y: Functional redundancy and crosstalk between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803.BioRxiv
https://doi.org/10.1101/2019.12.23.886929
Shimakawa, G., et al. (2020). "Near-infrared in vivo measurements of photosystem I and its lumenal electron donors with a recently developed spectrophotometer." Photosynthesis Research 144(1): 63-72.
https://doi.org/10.1007/s11120-020-00733-y
Flannery, S. E., et al. (2021). "Developmental acclimation of the thylakoid proteome to light intensity in Arabidopsis." The Plant Journal 105(1): 223-244.
https://onlinelibrary.wiley.com/doi/abs/10.1111/tpj.15053
Furutani, R., et al. (2020). "Intrinsic Fluctuations in Transpiration Induce Photorespiration to Oxidize P700 in Photosystem I." Plants 9(12): 1761.
https://doi.org/10.3390/plants9121761
Kato, H., et al. (2020). "Characterization of a giant photosystem I supercomplex in the symbiotic dinoflagellate Symbiodiniaceae." Plant Physiology: pp.00726.02020.
https://doi.org/10.1104/pp.20.00726
Nikkanen, L., et al. (2020). "Functional redundancy between flavodiiron proteins and NDH-1 in Synechocystis sp. PCC 6803." The Plant Journal n/a(n/a).
https://doi.org/10.1111/tpj.14812
Sétif, P., et al. (2020). "Identification of the electron donor to flavodiiron proteins in Synechocystis sp. PCC 6803 by in vivo spectroscopy." Biochimica et Biophysica Acta (BBA) - Bioenergetics 1861(10): 148256.
https://doi.org/10.1016/j.bbabio.2020.148256
Theune, M. L., et al. (2020). "In-vivo quantification of electron flow through photosystem I – cyclic electron transport makes up about 35 % in a cyanobacterium." Biochimica et Biophysica Acta (BBA) - Bioenergetics: 148353.
https://doi.org/10.1016/j.bbabio.2020.148353
2019
Kumar V, Vogelsang L, Seidel T, Schmidt R, Weber M, Reichelt M, Meyer A, Clemens S, Sharma SS, Dietz K-J: Interference between arsenic-induced toxicity and hypoxia. Plant Cell and Environment 42: 574-590.
https://doi.org/10.1111/pce.13441
Kadota K, Furutani R, Makino A, Suzuki Y, Wada S, Miyake C: Oxidation of P700 induces alternative electron flow in photosystem I in wheat leaves. Plants 8: 152.
https://doi.org/10.3390/plants8060152
Lima-Melo Y, Gollan PJ, Tikkanen M, Silveira JAG, Aro E-M: Consequences of photosystem-I damage and repair on photosynthesis and carbon use in Arabidopsis thaliana. Plant Journal 97: 1061-1072.
https://doi.org/10.1111/tpj.14177
Nikkanen L, Guinea Diaz M, Toivola J, Tiwari A, Rintamäki E: Multilevel regulation of non-photochemical quenching and state transitions by chloroplast NADPH-dependent thioredoxin reductase. Physiologia Plantarum 166: 211-225.
https://doi.org/10.1111/ppl.12914
Sétif P, Boussac A, Krieger-Liszkay A: Near-infrared in vitro measurements of photosystem I cofactors and electron-transfer partners with a recently developed spectrophotometer. Photosynthesis Research 142: 307-319.
https://doi.org/10.1007/s11120-019-00665-2
Telman W, Liebthal M, Dietz K-J: Redox regulation by peroxiredoxins is linked to their thioredoxin-dependent oxidase function.Photosynthesis Research, in press.
https://doi.org/10.1007/s11120-019-00691-0
2018
Nikkanen L, Toivola J, Trotta A, Guinea Diaz M, Tikkanen M, Aro E-M, Rintamäki E: Regulation of cyclic electron flow by chloroplast NADPH-dependent thioredoxin system. Plant Direct 2: e00093.
https://doi.org/10.1002/pld3.93
Shimakawa G, Miyake C: Changing frequency of fluctuating light reveals the molecular mechanism for P700 oxidation in plant leaves. Plant Direct 2: e00073.
https://doi.org/10.1002/pld3.73
Takagi D, Miyake C: PROTON GRADIENT REGULATION 5 supports linear electron flow to oxidize photosystem I. Physiologia Plantarum 164: 337–348.
https://doi.org/10.1111/ppl.12723
Vaseghi M-J, Chibani K, Telman W, Liebthal MF, Gerken M, Schnitzer H, Müller SM, Dietz K-J: The chloroplast 2-cysteine peroxiredoxin functions as thioredoxin oxidase in redox regulation of chloroplast metabolism. eLife 7: e38194.
https://doi.org/10.7554/eLife.38194
2017
Schreiber U: Redox changes of ferredoxin, P700, and plastocyanin measured simultaneously in intact leaves. Photosynthesis Research 134: 343–360.
https://doi.org/10.1007/s11120-017-0394-7
2016
Klughammer C, Schreiber U: Deconvolution of ferredoxin, plastocyanin, and P700 transmittance changes in intact leaves with a new type of kinetic LED array spectrophotometer.
Photosynthesis Research 128: 195–214.
https://doi.org/10.1007/s11120-016-0219-0
Schreiber U, Klughammer C: Analysis of photosystem I donor and acceptor sides with a new type of online-deconvoluting kinetic LED-array spectrophotometer. Plant and Cell Physiology 57: 1454–1467
https://doi.org/10.1093/pcp/pcw044
