歡迎關(guān)注「漢莎科技集團(tuán)」微信公眾號！
1.快速葉綠素?zé)晒庹T導(dǎo)動力學(xué)分析(JIP-test)
近二十年來，基于“生物膜能量通量理論”的活體快速葉綠素 a 熒光誘導(dǎo)動力學(xué)OJIP曲線和JIP-test分析，由于其無損、精確、快速等特性，已被廣泛而成功地用做研究植物生理狀態(tài)的有力工具(Strasser et al.,1995, 2004)。植物快速葉綠素?zé)晒庹T導(dǎo)曲線（OJIP曲線）中包含著大量關(guān)于PSⅡ反應(yīng)中心原初光化學(xué)反應(yīng)的信息，植物在不同脅迫處理后OJIP曲線會發(fā)生特異性變化(Strasser et al., 2004)。OJIP曲線對不同的環(huán)境變化極為敏感，例如光脅迫、化學(xué)物質(zhì)影響、熱脅迫、低溫或凍害、干旱脅迫、重金屬或鹽脅迫、營養(yǎng)不良、大氣CO2或臭氧升高和病害。通過對曲線熒光參數(shù)的分析，可以知道在環(huán)境因子影響下植物光合機(jī)構(gòu)的變化。
從動力學(xué)曲線上可以得到大量的原始數(shù)據(jù)，為了能更好地反映動力學(xué)曲線和被測樣品的關(guān)系，Strasser RJ(1995)以生物膜能量流動為基礎(chǔ)，通過計(jì)算能量流和能量比率來衡量在給定物理狀態(tài)下樣品材料內(nèi)部變化，建立了高度簡化的能量流動模型圖。
圖1. 高度簡化的能量在光合器官中的流動模型圖(Strasser BJ, Strasser RJ, 1995)依照能量流動模型，天線色素(Chl)吸收的能量(Absorption, ABS)的一部分以熱能和熒光(F)的形式耗散掉，另一部分則被反應(yīng)中心(Reaction Centre, RC，在JIP-test中RC指有活性的反應(yīng)中心)所捕獲(Trapping, TR)，在反應(yīng)中心激發(fā)能被轉(zhuǎn)化為還原能，將QA還原為QA-，后者又可以被重新氧化，從而產(chǎn)生電子傳遞(electron transport,ET)，把傳遞的電子用于固定CO2或其它途徑。
在此基礎(chǔ)上發(fā)展起來的數(shù)據(jù)處理稱為“JIP-test”(Strasser etal. 1995; Krüger et al. 1997; Strasser et al. 2000, 2004)。JIP-test為我們提供了被測樣品的大量信息，如光合器官在不同環(huán)境條件下的結(jié)構(gòu)和功能的變化(Strivastava & Strasser1996; Jiang et al. 2003; Hermans et al. 2003; van Heerden et al. 2003, 2004)。圖2. 葉綠素?zé)晒庀嚓P(guān)聯(lián)合作者網(wǎng)絡(luò)（注意R.Strasser和R.J.Strasser是同一個人）。從黃色到紅色，協(xié)作性更強(qiáng)，中心性更高(K. HU et al, 2020)學(xué)術(shù)界對JIP-test方法的研究和應(yīng)用熱度在不斷增加，而對脈沖調(diào)制式(PAM)方法的興趣在逐漸減弱。這是什么意思？乍一看，一個可能的解釋是源于對OJIP動力學(xué)實(shí)驗(yàn)測量可用性的增加，主要是因?yàn)椋?mpchecktext id="1588896360267_0.021774839277353797" contenteditable="false">1）研究者有新的熒光檢測方法可用，2）JIP-test已明顯證明是基于半經(jīng)驗(yàn)合理假設(shè)的穩(wěn)健分析工具(robust analysis tool based on semi-empiricalreasonable assumptions)。圖3：Strasser教授和Hansatech初代PEA植物效率分析儀（Rodriguez, 2000年)
由Reto J.Strasser教授發(fā)明授權(quán)英國Hansatech公司生產(chǎn)的PEA植物效率分析儀系列產(chǎn)品(Handy PEA、M-PEA...)是目前世界上可以完美真實(shí)測定OJIP曲線的成熟商品化設(shè)備。近20年來，JIP-test方法的不斷發(fā)展及其在野外應(yīng)用和實(shí)驗(yàn)室研究中的應(yīng)用呈現(xiàn)出顯著的增長趨勢。近期發(fā)表文章《能量流理論慶祝40年：走向系統(tǒng)生物學(xué)概念？》(The energy flux theory celebrates 40 years: toward a systems biology concept?" Photosynthetica, April 2019, 57(2):521-522.)詳細(xì)闡述了這一研究熱點(diǎn)趨勢。2019年末國際光合作用研究雜志(Photosynthetica)推出榮耀特刊，刊發(fā)30余篇榮耀文章以表彰紀(jì)念Strasser教授在JIP-test理論方向做出的卓越貢獻(xiàn)。榮耀特刊文獻(xiàn)預(yù)覽及下載請點(diǎn)擊以下鏈接文章：
2.主成分分析(PCA)簡介
主成分分析(Principal Components Analysis)也稱主分量分析，旨在利用“降維”的思想，把多指標(biāo)轉(zhuǎn)化為少數(shù)幾個綜合指標(biāo)。在許多研究領(lǐng)域中，通常需要對含有多個變量的數(shù)據(jù)進(jìn)行觀測，收集大量數(shù)據(jù)后進(jìn)行分析尋找規(guī)律。多變量大數(shù)據(jù)集為研究提供了豐富的信息，而在多數(shù)情況下，許多變量之間可能存在相關(guān)性，從而增加了問題分析的復(fù)雜性。如果分別對每個指標(biāo)進(jìn)行分析，分析往往是孤立的，不能完全利用數(shù)據(jù)中的信息，因此盲目減少指標(biāo)會損失很多有用的信息，從而產(chǎn)生錯誤的結(jié)論。鑒于各變量之間存在一定的相關(guān)關(guān)系，因此可以考慮將關(guān)系緊密的變量變成盡可能少的新變量，使這些新變量是兩兩不相關(guān)的，那么就可以用較少的綜合指標(biāo)分別代表存在于各個變量中的各類信息。主成分分析PCA就屬于這類降維算法，將高維度的數(shù)據(jù)保留下最重要的一些特征，去除噪聲和不重要的特征，從而實(shí)現(xiàn)提升數(shù)據(jù)處理速度的目的。圖4a. 數(shù)據(jù)點(diǎn)降維的信息損失與矯正：X軸投影
如何降維？我們以最簡單的二維轉(zhuǎn)一維為例，如圖4中就是把二維平面上不同位置上的點(diǎn)投影到同一條直線上（X軸或Y軸）。但是仔細(xì)觀察前兩個圖，我們就會發(fā)現(xiàn)，有些點(diǎn)在投影過后，位置是重合的，也就是說，存在不同的點(diǎn)在壓縮過后表示的信息是完全一樣的，投影到x軸，有兩個點(diǎn)重合，投影到y(tǒng)軸，有三個點(diǎn)重合。
圖4b. 數(shù)據(jù)點(diǎn)降維的信息損失與矯正：Y軸投影這就是當(dāng)所有點(diǎn)集中至一條軸上時，另一維度或另一軸上的信息就會丟失，這是不可逆的過程，這一信息的損失也是必然的。這不是我們想要的結(jié)果，最終我們還是希望點(diǎn)與點(diǎn)之間間隔盡可能的遠(yuǎn)，保留的信息盡可能的多，讓所有的點(diǎn)能夠盡可能的進(jìn)行區(qū)分。
圖4c. 數(shù)據(jù)點(diǎn)降維的信息損失與矯正：X/Y軸矯正最好的結(jié)果應(yīng)該是我們依然選擇了某個直線，并把點(diǎn)投影到這條直線上，但是點(diǎn)之間沒有重合，點(diǎn)與點(diǎn)的間隔也比較遠(yuǎn)。看到這里，我們就知道PCA到底要做什么了，沒錯，就是找到這條直線，并求出投影到這條直線的點(diǎn)的坐標(biāo)（當(dāng)然二維降一維是直線，三維降二維就是平面了，更多維度也是類似的）。
3.主成分分析在JIP-test中的應(yīng)用
主成分分析（PCA）是深度分析JIP-test眾多熒光參數(shù)的有效方法。通過PCA對JIP-test熒光參數(shù)進(jìn)行二次處理，對其數(shù)量、精度和復(fù)雜性進(jìn)行分析，可以識別熒光參數(shù)大數(shù)據(jù)中內(nèi)的隱藏信息，而傳統(tǒng)方法則是無法有效進(jìn)行的(Samborska et al.2014)。
使用PEA系列植物效率分析儀，每個樣品僅需2秒鐘，即可獲得完整OJIP曲線和50多個熒光參數(shù)，包括(i)OJIP曲線特征位點(diǎn)FJ、FI、Area等，(ii)比活性參數(shù)ABS/RC、TRM/RC等，(iii)性能指數(shù)PIABS、PItotal等和(iiiii)推動力DFABS等。JIP-test每個熒光參數(shù)并不是完全獨(dú)立的，因?yàn)镴IP-test熒光參數(shù)是根據(jù)熒光瞬態(tài)曲線點(diǎn)計(jì)算的，其中一些參數(shù)由于其數(shù)學(xué)表達(dá)式（如φDo和φPo）而具有很高的相關(guān)性。通過主成分分析PCA評估植物在不同環(huán)境下的生理或脅迫效應(yīng)，以確定對植物光合生理反應(yīng)最敏感的參數(shù)，這種方法允許將一組測量參數(shù)轉(zhuǎn)換成較少的變量，以確定植物生理狀態(tài)的變化(Jolliffe，2002; Legendre and Legendre 2012; Goltsev etal. 2012)。 圖5：羽狀短柄草(Brachypodium pinnatum)不同林分密度對54個JIP-test熒光參數(shù)的PCA分析(Baba，未發(fā)表)
如圖5中JIP-test熒光數(shù)據(jù)來自于不同生長年齡短柄草(隨著生長年齡的增大，其林分密度隨之增大)。首先第一PCA軸(Dim1)向上，兩個極值分別為：VI和單位PSⅡ活性反應(yīng)中心比通量參數(shù)(TRo/RC、ETo/RC、REo/RC)。
同時第二PCA軸(Dim2)向上，可以看到參數(shù)Fv/Fo和PSⅡ原初最大量子產(chǎn)率(ΦPo)的增大。
通過這種方法，我們發(fā)現(xiàn)了四個最重要的參數(shù)(而不是最初的54個)來描述光合機(jī)構(gòu)的狀態(tài)，它們與短柄草的林分密度的增加顯著相關(guān)。
圖6. 缺肥條件下玉米葉片JIP-test參數(shù)變異性的主成分分析(Kalaji,2014)
圖6中對不同施肥處理的玉米JIP-test熒光數(shù)據(jù)進(jìn)行PCA分析，使其分為了5個分離簇。第一類為對照組和缺磷植株。此簇位于Comp1和Comp2均為正值的第一象限，結(jié)果表明與對照組相比，缺磷處理對玉米光合機(jī)構(gòu)的影響不顯著。第二類是均勻分布在坐標(biāo)系原點(diǎn)附近的缺氮、缺鎂和缺硫樣品。缺氮、缺硫植株的參數(shù)點(diǎn)略有向正方向移動，缺鎂植株的參數(shù)點(diǎn)向負(fù)方向移動。這意味著盡管JIP-test熒光參數(shù)變化具有相似性，但仍有足夠的特征可用作區(qū)分組內(nèi)樣本的熒光表型標(biāo)記。第三類主要由植物缺鉀樣品組成，位于Comp1和Comp2的負(fù)區(qū)。這意味著玉米中鉀的缺乏可以通過JIP-test來很容易地確定。第四和第五個簇是由缺鐵和缺鈣植株形成的，即當(dāng)玉米缺鐵或缺鈣時，具有相似的JIP-test參數(shù)，并且它們與其他缺肥處理有很好的分離。圖7. 不同環(huán)境條件下5個玉米雜交種葉片JIP試驗(yàn)參數(shù)變異性的主成分分析：對照（C）、弱光（LL）、田間（F）、冷（Co）、熱（H）和高溫（SH）(Frani M et al. 2020)
圖7為不同環(huán)境條件下5個玉米雜交種葉片JIP試驗(yàn)參數(shù)變異性的主成分分析：前三主成分占總方差的95.9%，選擇的14個參數(shù)對環(huán)境效應(yīng)的敏感性不同，因而對主成分形成的貢獻(xiàn)也不同(數(shù)據(jù)見原文)。
所有五種處理都是獨(dú)立的簇，并位于坐標(biāo)系的不同區(qū)域。SH處理對玉米植株的熱脅迫最為分散，通過JIP-test熒光參數(shù)的變化可以看出熱脅迫對玉米植株的嚴(yán)重性。
PC1與DIo/RC(0.98)和RC/ABS(–0.96)的相關(guān)性最強(qiáng)，因此可以認(rèn)為PC1是一個功能反應(yīng)中心的量度，其兩端極值處理組為C和SH。與PC2兩極相關(guān)性最強(qiáng)的參數(shù)為(VJ，-0.90)和ΨEo(0.87)。
在第二主成分兩端的是F、Co和LL處理組，其中LL和Co的主要特征參數(shù)是VJ和VI，F(xiàn)處理組的特征是解釋電子傳遞通量的ΨEo和ETo/RC。在最近對幾種植物的環(huán)境影響分類的研究中，也顯示了相似的JIP參數(shù)分組(Bussotti et al. 2020)。
此例中PIABS似乎只提供了一個軸向的分類，而其他JIP-test熒光參數(shù)可用于檢測各個環(huán)境條件下對玉米的特定影響。例如，第一主成分的相對側(cè)顯示了玉米植株受到的兩個環(huán)境極值：冷脅迫處理組(Co)-主要由VJ和VI參數(shù)表征，而高溫脅迫處理組(SH)-主要由K、Mo、REo/RC和DIo/RC表征。
Stirbet(Stirbet et al. 2018)等人也證實(shí)了這一點(diǎn)，同時建議設(shè)計(jì)新參數(shù)以表征已知特定條件反應(yīng)的JIP-test參數(shù)。同時Galic等人(Galic et al. 2019)表明，PIABS可以有效地用于熱脅迫環(huán)境下的糧食產(chǎn)量選擇。
總的來說通過PCA我們可以分類植物對各種環(huán)境因素的不同反應(yīng)：(i)找到特定處理下植物樣品OJIP曲線發(fā)生的特異性變化(ii)篩選出發(fā)生顯著變化的JIP-test熒光參數(shù)及其變化特征，可更好對植物樣品光合機(jī)構(gòu)發(fā)生的變化（傷害）進(jìn)行定位分析，如PSⅡ供體側(cè)/受體測或PSⅡ活性中心等。(iii)我們還可以將JIP-test熒光數(shù)據(jù)與其他環(huán)境數(shù)據(jù)或生理參數(shù)進(jìn)行聚類結(jié)合(Goltsev et al. 2012)。(iv)此外Tyystjärvi等人應(yīng)用PCA等人工智能方法分析不同類型光照(低光強(qiáng)、飽和脈沖、遠(yuǎn)紅色等)激發(fā)的JIP-test熒光數(shù)據(jù)，可識別植物物種(Tyystjärvi et al. 1999; Keränen et al. 2003; Codrea et al. 2003;Kirova et al. 2009)。(v)Kalaji等人利用JIP-test、主成分分析(PCA)和一種新的機(jī)器學(xué)習(xí)方法建立了一種無創(chuàng)檢測和監(jiān)測大田條件下油菜籽微量和大量營養(yǎng)素缺乏的方法(Kalaji et al. 2017)。鑒于篇幅限制，我們將在下期文章中篩選數(shù)篇應(yīng)用PCA方法分析JIP-test熒光數(shù)據(jù)具有代表性的文章進(jìn)行詳細(xì)介紹，期待您的關(guān)注，謝謝！
4.引用文獻(xiàn)
[1] Appenroth, K.J., Stöckel, J., Srivastava, A.,Strasser, R.J., 2001. Multiple effects of chromate on the photosyntheticapparatus of Spirodela polyrhiza as probed by OJIP chlorophyll a fluorescencemeasurements. Environ. Pollut. 115, 49–64.[2] Bussotti F, Gerosa G, Digrado A, Pollastrini M, 2020.Selection of chlorophyll fluorescence parameters as indicators of photosyntheticefficiency in large scale plant ecological studies. Ecol Indic 108: 105686.[3] Bussotti, F., Strasser, R.J., Schaub, M., 2007.Photosynthetic behavior of woody species under high ozone exposure probed withthe JIP-test: a review. Environ. Pollut. 147, 430–437.[4] Ceppi, M.G., Oukarroum, A., Cicek, N., Strasser,R.J., Schansker, G., 2012. The IP amplitude of the fluorescence rise OJIP issensitive to changes in the photosystem I content of leaves: a study on plantsexposed to magnesium and sulfate deficiencies, drought stress and salt stress. Physiol.Plant 144, 277–288.[5] Chen, S.G., Xu, X.M., Dai, X.B., Yang, C.L., Qiang,S., 2007. Identification of tenuazonic acid as a novel type of naturalphotosystem II inhibitor binding in QB-site of Chlamydomonasreinhardtii. Biochim. Biophys. Acta 1767, 306–318.[6] Chen, S.G., Zhou, F.Y., Yin, C.Y., Strasser, R.J.,Qiang, S., Yang, C.L., 2011. Application of fast chlorophyll a fluorescencekinetics to probe action target of 3-acetyl-5-isopropyltetramic acid. Environ.Exp. Bot. 71, 269–279.[7] Christen, D., Schönmann, S., Jermini, M., Strasser,R.J., Défago, G., 2007. Characterization and early detection of grapevine (Vitisvinifera) stress responses to esca disease by in situ chlorophyllfluorescence and comparison with drought stress. Environ. Exp. Bot. 60,504–514.[8] Clark, A.J., Landolt, W., Bucher, J.B., Strasser,R.J., 2000. Beech (Fagus sylvatica) response to ozone exposure assessedwith a chlorophyll a fluorescence performance index. Environ. Pollut.109, 501–507.[9] Codrea C, Aittokallio T, Keränen M et al(2003) Feature learning with a genetic algorithm for fluorescencefingerprinting of plant species. Pattern Recognit Lett 24:2663–2673.[10] Demetriou, G., Neonaki, C., Navakoudis, E.,Kotzabasis, K., 2007. Salt stress impact on the molecular structure andfunction of the photosynthetic apparatus—the protective role of polyamines. Biochim.Biophys. Acta 1767, 272–280.[11] Frani M, Jambrovi A, Zduni Z, et al. Photosyntheticproperties of maize hybrids under different environmental conditions probed bythe chlorophyll a fluorescence[J]. Maydica, 2020, 64(3):M25.[12] Galić V, Mazur M, Šimić D, Zdunić Z, Franić M, 2019.Plant biomass in salt-stressed young maize plants can be modelled with photosyntheticperformance. Photosynthetica 57: 9-19.[13] Goltsev V, Zaharieva I, Chernev P et al (2012)Drought-induced modifications of photosynthetic electron transport in intactleaves: analysis and use of neural networks as a tool for a rapid non-invasiveestimation. Biochim Biophys Acta-Bioenerg 1817:1490–1498.[14] Gururani, M.A., Venkatesh, J., Ganesan, M.,Strasser, R.J., Han, Y., Kim, J.I., Lee, H.Y., Song, P.S., 2015. In vivoassessment of cold tolerance through chlorophyll-a fluorescence in transgeniczoysiagrass expressing mutant phytochrome A. PLoS One 10, e0127200.[15] Hermans C, Smeyers M, Rodriguez RM, Eyletters M,Strasser RJ, Delhaye JP (2003). Quality assessment of urban trees: Acomparative study of physiological characterization, airborne imaging and onsite of fluorescence monitoring by the OJIP-test. J Plant Physiol, 160:81–90.[16] Hermans, C., Johnson, G.N., Strasser, R.J.,Verbruggen, N., 2004. Physiological characterisation of magnesium deficiency insugar beet: acclimation to low magnesium differentially affects photosystems Iand II. Planta 220, 344–355.[17] Hu, K., Govindjee, G., Tan, J., Xia, Q., Dai, Z. andGuo, Y. Co-author and co-cited reference network analysis for chlorophyllfluorescence research from 1991 to 2018. Photosynthetica, 2020, vol. 58,iss. 1, p. 110-124.[18] Jiang CD, Gao HY, Zou Q (2003). Changes of donorand accepter side in photosystem II complex induced by iron deficiency inattached soybean and maize leaves. Photosynthetica, 41: 267–271.[19] Jolliffe, I.T., 2002. Graphical representation ofdata using principal components. In: Jolliffe, I.T. (Ed.), Principal ComponentAnalysis, Springer Series in Statistics. Springer, New York, pp. 78-110.[20] Kalaji H M, BaBa W , Gediga K , et al. Chlorophyllfluorescence as a tool for nutrient status identification in rapeseedplants[J]. Photosynthesis Research, 2017.[21] Kalaji H M, Oukarroum A, Alexandrov V, et al.Identification of nutrient deficiency in maize and tomato plants by in vivochlorophyll a fluorescence measurements[J]. Plant Physiology &Biochemistry, 2014, 81:16-25.[22] Kalaji, H.M., Carpentier, R., Allakhverdiev, S.L.,Bosa, K., 2012. Fluorescence parameters as early indicators of light stress inbarley. J. Photochem. Photobiol. B: Biol. 112, 1–6.[23] Keränen M, Aro EM, Tyystjärvi E, Nevalainen O(2003) Automatic plant identification with chlorophyll fluorescencefingerprinting. Precis Agric 4:53–67.[24] Kirova M, Ceppi G, Chernev P et al (2009)Using artificial neural networks for plant taxonomic determination based onchlorophyll fluorescence induction curves. Biotechnol Biotechnol Equip23:941–945.[25] Krüger, G.H.J., Tsimilli-Michael, M., Strasser,R.J.,1997. Light stress provokes plastic and elastic modifications instructureand function of photosystem II in camellia leaves. Physiol. Plant. 101,265–277.[26] Lazár, D., 2003. Chlorophyll a fluorescence riseinduced by high light illumination of dark-adapted plant tissue studied bymeans of a model of photosystem II and considering photosystem IIheterogeneity. J. Theor. Biol. 220, 469–503.[27] Legendre P, Legendre L (2012) Numerical ecology,3rd edn. Elsevier, Amsterdam.[28] Li, X., Zhang, L., 2015. Endophytic infectionalleviates Pb2+ stress effects on photosystem II functioning of Oryzasativa leaves. J. Hazard. Mater. 295, 79–85.[29] Lu, C.M., Zhang, J.H.,1999. Heat-induced multipleeffects on PSII in wheat Plants. J. Plant Physiol. 156, 259–265.
[30] Mathur, S., Allakhverdiew, S.I., Jajoo,A.,2011.Analysis of high temperature stress on the dynamic of antenna size andreducing side heterogeneity of photosystem II in wheat leaves (Triticumaestivum). Biochim. Biophys. Acta 1807, 22–29.[31] Meinander, O., Somersalo, S., Holopainen, T.,Strasser, R.J., 1996. Scots pines after exposure to elevated ozone and carbondioxide probed by reflectance spectra and chlorophyll a fluorescencetransients. J. Plant Physiol. 148, 229–236.[32] Misra, A.N., Srivastava, A., Strasser, R.J., 2001.Utilization of fast chlorophyll a fluorescence technique in assessing thesalt/ion sensitivity of mung bean and Brassica seedlings. J. Plant Physiol.158, 1173–1181.[33] Nussbaum, S., Geissmann, M., Eggenberg, P.,Strasser, R.J., Fuhrer, J., 2001. Ozone sensitivity in herbaceous species asassessed by direct and modulated chlorophyll fluorescence techniques. J.Plant Physiol. 158, 757–766.[34] Oukarroum, A., Madidi, S. E., Schansker, G.,Strasser, R.J., 2007. Probing the responses of barley cultivars (Hordeumvulgare L.) by chlorophyll a fluorescence OLKJIP under drought stress andre-watering. Environ. Exp. Bot 60, 438–446.[35] Oukarroum, A., Schansker, G., Strasser, R.J., 2009.Drought stress effects on photosystem I content and photosystem IIthermotolerance analyzed using Chl a fluorescence kinetics in barley varietiesdiffering in their drought tolerance. Physiol. Plant 137, 188–199.[36] Ouzounidou, G., Moustakas, M., Strasser, R.J.,1997. Sites of action of copper in the photosynthetic apparatus of maizeleaves: kinetics analysis of chlorophyll fluorescence, oxygen evolution,absorption changes and thermal dissipation as monitored by photoacousticsignals. Aust. J. Plant Physiol. 24, 81–90.[37] Pollastrini, M., Desotgiu, R., Camin, F., Ziller,L., Gerosa, G., Marzuoli, R., Bussotti, F., 2014. Severe drought eventsincrease the sensitivity to ozone on poplar clones. Environ. Exp. Bot.100, 94–104.[38] Pontes. D, Ontes, M., Rodriguez, R. and Santiago,E.F. Letter to The Editor. The energy flux theory celebrates 40 years: toward asystems biology concept? Photosynthetica, 2019, vol. 57, iss. 2, p.521-522.[39] Rivera-Becerril, F., Calantzis, C., Turnau, K.,Caussanel, J., Belimov, A. A., Gianinazzi,S., Strasser, R.J., Gianinazzi-Pearson,V., 2002. Cadmium accumulation and buffering of cadmium-induced stress byarbuscular mycorrhiza in three Pisum sativum L. genotypes. J. Exp.Bot. 53, 1177–1185.[40] Roccotiello, E., Manfredi, A., Drava, G., Minganti,V., Mariotti, M.G., Berta, G., Cornara, L., 2010. Zinc tolerance andaccumulation in the ferns Polypodium cambricum L. and Pteris vittataL. Ecotoxicol. Environ. Saf. 73, 1264–1271.[41] Samborska IA, Alexandrov V, Sieczko L et al (2014)Artificial neural networks and their application in biological and agriculturalresearch. Sigpost Open Access J Nano Photo Bio Sciences 2:14–30.[42] Schansker, G., Tóth, S.Z., Strasser, R.J., 2005.Methylviolegen and dibromothymoquinone treatments of pea leaves reveal the roleof photosystem I in the Chl a fluorescence rise OJIP. Biochim. Biophys. Acta1706, 250–261.[43] Sekhar, K.M., Rachapudi, V.S., Mudalkar, S., Reddy,A.R., 2014. Persistent stimulation of photosynthesis in short rotation coppicemulberry under elevated CO2 atmosphere. J. Photochem. Photobiol.B: Biol. 137, 21–30.[44] Srivastava, A., Guissé, B., Greppin, H., Strasser,R.J., 1997. Regulation of antenna structure and transport in photosystem II of Pisumsativum under elevated temperature probed by fast polyphasic chlorophyll afluorescence transient: OKJIP. Biochim. Biophys. Acta 1320, 95–106.[45] Srivastava, A., Jüttner, F., Strasser, R.J., 1998.Action of the allelochemical, fischerellin A, on photosystem II. Biochim.Biophys. Acta 1364, 326–336.[46] Srivastava, A., Strasser, R.J., Govindjee, 1995.Differential effects of dimethylbenzoquinone and dichlorobenzoquinone onchlorophyll fluorescence transient in spinach thylakoids. J. Photochem.Photobiol. B: Biol. 31, 163–169.[47] Stirbet A, Lazár D, Kromdijk J, Govindjee, 2018. Chlorophylla fluorescence induction: Can just a one-second measurement be used to quantifyabiotic stress responses? Photosynthetica 56: 86-104.[48] Strasser BJ, Strasser RJ (1995). Measuring fastfluorescence transients to address environmental questions: The JIP test. In:Mathis P (eds). Photosynthesis: from Light to Biosphere. Dordrecht: KAPPress, Vol 5: 977-980.[49] Strasser RJ, Srivastava A, Tsimilli-Michael M(2000). The fluorescence transient as a tool to characterize and screenphotosynthetic samples. In: Yunus M, Pathre U, Mohanty P (eds). ProbingPhotosynthesis: Mechanism, Regulationand Adaptation. London: Taylor andFrancis Press, 445–483.[50] Strasser RJ, Tsimill-Michael M, Srivastava A(2004). Analysis of the chlorophyll a fluorescence transient. In: PapageorgiouG, Govindjee(eds). Advances in Photosynthesis and Respiration.Netherlands: KAP Press, 1–42.[51] Strasser, B.J., 1997. Donor side capacity ofPhotosystem II probed by chlorophyll a fluorescence transients. Photosynth.Res. 52, 147–155.[52] Strasser, R.J., Tsimilli-Michael, M., Qiang, S.,Goltsev, V., 2010. Simultaneous in vivo recording of prompt and delayedfluorescence and 820-nm reflection changes during drying and after rehydrationof the resurrection plant Haberlea rhodopensis. Biochim. Biophys. Acta1313–1326.[53] Strauss, A.J., Krüger, G.H.J., Strasser, R.J., vanHeerden, P.D.R., 2006. Ranking of dark chilling tolerance in soybean genotypesprobed by the chlorophyll a fluorescence transient O-J-I-P. Environ. Exp.Bot. 56, 147–157.[54] Strauss, A.J., Krüger, G.H.J., Strasser, R.J., vanHeerden, P.D.R., 2007. The role of low soil temperature in the inhibition ofgrowth and PSII function during dark chilling in soybean genotypes ofcontrasting tolerance. Physiol. Plant 131, 89–105.[55] Strivastava A, Strasser RJ (1996). Stress andstress management of land plants during a regular day. J Plant Physiol,148: 445–455.[56] Susplugas, S., Srivastava, A., Strasser, R.J.,2000. Changes in the photosynthetic activities during several stages ofvegetative growth of Spirodela polyrhiza: effect of chromate. J. PlantPhysiol. 157, 503–512.[57] Tóth, S.Z., Schansker, G., Garab, G., Strasser,R.J., 2007. Photosynthetic electron transport activity in heat-treated barleyleaves: the role of internal alternative electron donors to photosystem II. Biochim.Biophys. Acta 1767, 295–305.[58] Tóth, S.Z., Schansker, G., Kissimon, J., Kovacs,L., Garab, G., Strasser, R.J., 2005b. Biophysical studies of photosystemII-related recovery processes after a heat pulse in barley seedlings (Hordeumvulgare L.). J. Plant Physiol. 162, 181–194.[59] Tóth, S.Z., Schansker, G., Strasser, R.J., 2005a.In intact leaves, the maximum fluorescence level (FM) isindependent of the redox state of the plastoquinone pool: a DCMU-inhibitionstudy. Biochim. Biophys. Acta 1708, 275–282.[60] Tsimilli-Michael, M., Eggenberg, P., Biro, B.,Köves-Pechy, K., Vörös, I., Strasser, R.J., 2000. Synergistic and antagonisticeffects of arbuscular mycorrhizal fungi and Azospirillum and Rhizobiumnitrogen-fixers on the photosynthetic activity of alfalfa, probed by thepolyphasic chlorophyll a fluorescence transient O-J-I-P. Appl. Soil Ecol.15, 169–182.[61] Tyystjärvi E, Koski A, Keränen M, Nevalainen O(1999) The Kautsky curve is a built-in barcode. Biophys J 77:1159–1167.[62] van Heerden PDR, Strasser RJ, Krüger GHJ (2004).Reduction of dark chilling stress in N 2 -fixing soybean by nitrate asindicated by chlorophyll a fluorescence kinetics. Physiol Plant, 121:239–249.[63] van Heerden PDR, Tsimilli-Michael M, Krüger GHJ,Strasser RJ (2003). Dark chilling effects on soybean genotypes duringvegetative development: parallel studies of CO2 assimilation,chlorophyll a fluorescence kinetics O-J-I-P and nitrogen fixation. PhysiolPlant, 117: 476–491.[64] Xia, J.R., Li, Y.J., Zou, D.H., 2004. Effects ofsalinity stress on PSII in Ulva lactuca as probed by chlorophyll fluorescencemeasurements. Aquat. Bot. 80, 129–137.[65] Xiang, M.M., Chen, S.G., Wang, L.S., Dong, Z.Y.,Huang, J.H., Zhang, Y.X., Strasser, R.J., 2013. Effect of vulculic acidproduced by Nimbya alternantherae on the photosynthetic apparatus of Alternanthera.philoxeroides. Plant Physiol. Biochem 65, 81–88.[66] Yadavalli, V., Neelam, S., Rao, A.S.V.C., Reddy,A.R., Subramanyam, R., 2012. Differential degradation of photosystem I subunitsunder iron deficiency in rice. J. Plant Physiol. 169, 753–759.
