叶片尺度光合荧光实验设计

,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,叶片尺度光合,/,荧光实验设计,ECOTEK,不同时间尺度上的实验,1,季节尺度动态变化,2,日尺度动态变化,3,小时,尺度动态变化,4,分钟尺度动态变化,5,秒尺度动态变化,2,1,季节尺度动态变化,1.1,光合参数季节动态,1.2,荧光参数季节动态,3,1.1,光合参数季节动态,(David A. Ramrez,et al.,2012),4,光合参数季节动态解释的问题,不同种植物对降水格局变化的响应,The relationship between water availability, and RGR and,carbon assimilation,in,C. ladanifer, and the lack of any relationship in,E. arborea,suggest that the former has an enhanced capacity to harness unpredictable rainfall pulses compared with the latter.,改变群落中植物种的优势地位,These contrasting responses to water availability indicate that the projected changes in rainfall with global warming could alter the competitive ability of these two species, and contribute to changes in plant dominance in Mediterranean shrublands.,(David A. Ramrez,et al.,2012),5,光合参数季节动态实验方法,测量时间：,Net carbon assimilation (A) and stomatal conductance (gs) were measured early in the morning (,08:00 hours solar time,) to avoid midday photo inhibition.,环境条件的控制：,Chamber conditions set to,match typical meteorological conditions in the early morning during the growing season,: photosynthetically active radiation at 1000mmol m,-2,s,-1, reference CO,2,concentration at 400ppm, leaf temperature at 25, and vapour pressure deficit at 1.5-2.0kPa.,(David A. Ramrez,et al.,2012),6,光合参数季节动态实验小结,实验仪器：,Li-6400/XT,；,6400-40,采样：略,时间：定时,重复数：,n=8,光：控制,温度：控制,二氧化碳浓度：控制,湿度,:,维持在一定范围内,(David A. Ramrez,et al.,2012),7,荧光参数季节动态,(Binghua Liu,et,al.,2012),8,荧光参数季节动态解释的问题,砧木的不同导致对干旱的响应不同,Our present study revealed drought-related reductions in Chl content, Pn and,Fv/Fm,the extent of which were more notable in plants on,M. hupehensis.,Thus, the growth and biomass production of apple trees in response to drought depends upon the choice of rootstock.,(Binghua Liu,et,al.,2012),9,荧光参数季节动态实验方法,仪器,Chlorophyll fluorescence was measured with an integrating fluorescence fluorometer (6400-40 Leaf Chamber Fluorometer; Li-Cor).,Fo,的测量,After the samples were adapted to darkness for 1 h, the minimum fluorescence (Fo) was measured with weak modulated irradiation (7,000 molm,-2,s,-1,) was applied to determine the maximum chlorophyll fluorescence yield (Fm) and maximum photochemical efficiency(Fv/Fm).,(Binghua Liu,et,al.,2012),10,荧光参数季节动态小结,实验仪器：,Li-6400/XT,；,6400-40,样品：文中没有提及，但是应该采用相同的标准；,时间：定时；,重复数：,n=6,；,光：测量光,7,000 mol m,-2,s,-1,；,温度：没有提及,二氧化碳浓度：没有提及,湿度,:,没有提及,(Binghua Liu,et,al.,2012),11,2,日尺度动态变化,2.1,光合参数日动态,2.2,荧光参数日动态,12,光合,/,荧光参数日动态,（孙东宝，王庆锁，,2012,）,13,光合,/,荧光参数日动态解释的问题,灌水改变了苜蓿叶片,Pn,的日变化格局。,灌水较多的处理,(W3,和,W2),苜蓿叶片没有出现光合“ 午休” 现象, Pn,的日变化趋势呈现“ 单峰” 型。,而灌水较少和不灌水的处理,(W1,和,W0),苜蓿叶片出现了明显的光合“午休” 现象,其,Pn,的日变化进程呈现“双峰”型。,（孙东宝,&,王庆锁，,2012,）,14,光合,/,荧光参数日动态解释的问题,灌水提高了苜蓿叶片的,Fv/ Fm, Fv/Fm,随着灌水量的增加而增加。,以初花期为例, W1,、,W 2,和,W 3,处理的苜蓿叶片,Fv/ Fm,的日均值分别比,W0,提高了,1.3%,、,6.5%,和,7.9%,。,灌水对苜蓿叶片,Fv/ Fm,的影响是在早晨和傍晚弱,中午前后强,表现在灌水极显著地减轻了中午苜蓿叶片,Fv/Fm,的降低幅度。以初花期为例,早晨和傍晚不同灌水处理的苜蓿叶片,Fv/ Fm,均在,0.8,以上,而在中午,12:00, W0,、,W 1,、,W 2,和,W 3,处理的苜蓿叶片,Fv/ Fm,分别降低至,0.58,、,0.66,、,0.77,和,0.78,。,（孙东宝，王庆锁，,2012,）,15,光合,/,荧光参数日动态解释的问题,不同植物,PS II,反应中心对干旱胁迫的敏感程度不同。,在严重干旱胁迫条件下,苜蓿叶片的,Fv/ Fm,降低至了,0.104 (W0,处理在苜蓿再生期,14:00), P n,仍能维持在,5 molm,2,s,1,左右。画眉草在,Fv/ Fm,降低至,0.2,左右时,仍具有一定的光合能力。,而豌豆、菠菜和莴苣缬草在,Fv/Fm,降低至,0.4,左右, PSII,反应中心便完全失去活性。,这说明苜蓿对于干旱胁迫具有较强的抵抗能力。,（孙东宝，王庆锁，,2012,）,16,光合,/,荧光参数日动态测定方法,在第二茬苜蓿的再生期和初花期,选择晴朗天气（,2005,年,6,月,22,日和,2005,年,7,月,23,日）,使用,LI-6400,便携式光合系统测定仪（,LI-COR, Lincoln, USA,）（利用自然光源和田间,CO,2,）对苜蓿叶片的,Pn,进行测定。,测量时选用,6400-15,小叶叶室（,Aradopsis chamber,）,测定面积为,0.785cm,2,。每个小区选择,3,片不同植株上的生长健康、长势一致、光照均一的同一叶位叶片,(,植株顶端第一个完全展开三叶的中间小叶,),于,6:0018:00,每,2h,测一次。测定指标包括苜蓿叶片,Pn,、,Tr,、,Gs,、,PAR,等。,（孙东宝，王庆锁，,2012,）,17,光合,/,荧光参数日动态测定方法,测量前先将叶片暗适应,20-30 min,获取初始荧光,Fo,再用饱和的红蓝光,(5000 molm,2,s,1,),照射,获取最大荧光,Fm,。然后按公式,Fv=FmFo,计算可变荧光,F v,和原初光能转换效率,Fv/Fm,。,（孙东宝，王庆锁，,2012,）,18,光合参数日动态实验小结,实验仪器：,Li-6400/XT,；,透明顶叶室,采样：每个小区选择,3,片不同植株上的生长健康、长势一致、光照均一的同一叶位叶片（植株顶端第一个完全展开三叶的中间小叶）。,时间：定时，,every 2h,重复数：,n=3,光：,sun+sky,温度：不控制,二氧化碳浓度：不控制（用缓冲瓶）,湿度,:,不控制,（孙东宝，王庆锁，,2012,）,19,荧光参数日动态实验小结,实验仪器：,Li-6400/XT,；,6400-40,；,采样：每个小区选择,3,片不同植株上的生长健康、长势一致、光照均一的同一叶位叶片（植株顶端第一个完全展开三叶的中间小叶）；,时间：定时,every 2h,；,重复数：,n=3,光：暗适应,30,分钟,温度：不控制,/,控制,二氧化碳浓度：不控制（用缓冲瓶）,湿度,:,不控制,20,3,小时,尺度动态变化,3.1,光响应曲线（,Light curve,）,3.2,温度响应曲线（,Temperature curve,）,3.3,湿度响应曲线（,Humidity curve,）,3.4,二氧化碳响应曲线（,A-ci curve,）,3.5,荧光 光响应曲线（,Flr Light curve,）,21,光响应曲线,（,Paulo,et al.,2012),22,光响应曲线解释的问题,光补偿点,LCP,的变化,In contrast, increases in SLA were reflected in an increased LUE in the shade, particularly because such increases were accompanied by concomitant physiological,（,increases in Chl concentrations on a mass basis and,lower LCP,）,and morphological (LAR) changes (Tables 2 and 3, Fig. 3) for the optimization of light capture (Poorter and Nagel 2000, Sack and Grubb 2002).,（,Paulo,et al.,2012),23,光响应曲线测量方法,温度控制下的光响应曲线,Photosynthetic light-response curves (A/PAR) were produced by increasing PAR in 12 steps from 0 to 2000mol m,2,s,1,at 25,.,控制二氧化碳浓度,Initially, leaf tissues were exposed to a 5Pa CO,2,partial pressure for 5 min to allow stomatal aperture; subsequently, A/PAR curves were obtained at 40Pa CO,2,partial pressure.,水分利用效率、光补偿点、光饱和点的计算,Light use efficiencies (LUE), light compensating points (LCP) and light saturation points (LSP) were determined from these curves. Non-linear regression techniques for estimating these parameters were followed from Ogren and Evans (1993).,（,Paulo,et al.,2012),24,光响应曲线实验小结,实验仪器：,Li-6400,（,XT,）,6400-40,采样：略,时间：根据设置的光强梯度，大致估算出实验耗时。选择叶片标准要一致。,重复数：,n=5,光适应：没做 ；,改用的是低二氧化碳浓度适应,。,温度：控制,二氧化碳浓度：控制,湿度,:,略,25,温度响应曲线,(Masabumi,et al.,2012),26,温度响应曲线解释的问题,温度与二氧化碳的协同作用,Photosynthetic carbon assimilation rates were synergistically enhanced above 30,under elevated CO,2, because of the higher ETR and suppression of photorespiration under elevated CO,2, (Fig. 2A, Farquhar,et al.,1980).,高温下，二氧化碳对光合的促进现象,It is noteworthy that based on the temperature-dependent photosynthesis, higher photosynthetic rates were observed above 30,in elevated-CO2 -grown plants measured under elevated CO2than in ambient-grown ones measured under ambient CO2.,结论,This suggests that elevated CO2 would boost photosynthetic carbon assimilation at higher temperatures even though photosynthetic down regulation occurred.,(Masabumi,et al.,2012),27,温度响应曲线实验方法,The temperature-dependent net photosynthetic rate (Pn), quantum yield of PSII electron transport ( PSII), photochemical quenching (qP),photochemical efficiency of the open PSII (Fv/F m) and quantum yield of non-regulated Y(NO) and regulated non-photochemical energy loss in PSII Y(NPQ) were measured at a photosynthetic steady state, at various temperatures,（,15, 20, 25, 30, 35 and 40,）, ambient CO,2,concentrations of 400 and 800,mol mol, 1,and saturating PFD of 1000,mol m,2,s,1,.,(Masabumi,et al.,2012),28,温度响应曲线实验小结,实验仪器：,Li-6400,（,XT,）,6400-40,采样：,生长,50,天完全展开的叶片,时间：根据设置的温度梯度，大致估算出实验耗时。,重复数：,n=8,光适应：,30,分钟,温度：适应某个温度至少,30,分钟,二氧化碳浓度：控制,湿度,:,控制（详见原文）。,(Masabumi,et al.,2012),29,湿度响应曲线,（顾礼力等，,2012,）,30,湿度响应曲线解释的问题,设施栽培中需要精细化管理，使大棚中保持适宜的温湿度，从而提高杨梅净光合速率。,31,湿度响应曲线测量方法,为了检验其关系，实验控制光强,1500molm,-2,s,-1,，,CO,2,浓度,375 molmol,-1,，温度,32,，采取,50%,、,55%,、,60%,、,65%,、,70%,、,75%,、,80%,共,7,个湿度梯度测定杨梅净光合速率。,32,湿度响应曲线实验小结,实验仪器：,Li-6400,（,XT,）,6400-40/6400-02B/6400-18,采样：略,时间：略,重复数：略,光适应：,30,分钟,温度：略,二氧化碳浓度：控制,湿度,:,设置湿度梯度,33,二氧化碳响应曲线,(Masabumi,et al.,2012),34,二氧化碳响应曲线解释的问题,生在在高二氧化碳浓度下的植物,Rubisco,羧化能力下降,Plants grown under elevated CO,2, exhibited photosynthetic down regulation, indicated by a decrease in the carboxylation capacity of Rubisco.,白桦树幼苗最大羧化速率和,RuBP,再生速率下降,White birch seedlings grown at elevated CO,2, under limited N supply showed photosynthetic down regulation, indicated by decreases in the maximum rates of RuBP carboxylation (Vcmax) and RuBP regeneration(Jmax ),(Masabumi,et al.,2012),35,二氧化碳响应曲线测量方法,The maximum rates of RuBP carboxylation (Vcmax)and RuBP regeneration were derived from photosynthetic responses to various Ci at a leaf temperature of 25,and saturating photon flux density (PFD) of 1000,mol m,2,s,1, which was provided by a red/blue light emitting diode (LED) array,（,6400-40;Licor,）,with blue light comprising 10% of total PFD .,(Masabumi,et al.,2012),36,二氧化碳响应曲线实验小结,实验仪器：,Li-6400,（,XT,）,6400-40,采样：生长,50,天完全展开的叶片,时间：依据二氧化碳浓度梯度个数而定,重复数：,n=8,光：适应,30,分钟,温度：适应,30,分钟,二氧化碳浓度：梯度,湿度,:,不控,37,荧光,-,光响应曲线,(Mitsutoshi et al. 2012),38,荧光,-,光响应曲线解释的问题,The shade leaves of the Japanese oak grown within a crown were suggested to adjust their N investment to maintain higher photosynthetic capacities compared with those required to maximize the net carbon gain, which may facilitate the dissipation of the excessive light energy of sunflecks to circumvent photoinhibition in cooperation with thermal energy dissipation.,(Mitsutoshi et al. 2012),39,荧光,-,光响应曲线测量方法,Measurements of gas exchange and chlorophyll fluorescence were conducted on 17 mature leaves of the Japanese oak with a portable photosynthesis measuring system (Li-6400, Licor) combined with a leaf chamber fluorometer (6400-40, Licor) in mid-July.,The net photosynthetic rate (Pn), quantum yield of PSII elec-tron transport ( PSII), photochemical quenching (qP)and photochemical efficiency of the open PSII (Fv/Fm)were measured at a photosynthetic steady state, under an ambient CO2 concentration of 360,molmol,1,and at various PPFD (0, 50, 100, 200, 300, 600, 1000,1500 and 2000,mol m,2,s,1,) which was provided by a red/blue LED array (640040, Licor) with blue light comprising 10 % of total PPFD.,(Mitsutoshi,et al.,2012),40,荧光,-,光响应曲线实验小结,实验仪器：,Li-6400,（,XT,）,6400-40,采样：成熟叶片,时间：依据光强梯度个数而定,重复数：略,光：适应,30,分钟,温度：控制,二氧化碳浓度：控制,湿度,:,略,41,4,分钟尺度动态变化,4.1,光合参数分钟动态,（,Auto log,）,4.2,荧光诱导曲线（,Fluorescence induction curve,）,4.3,暗驰豫动力学（,Relaxation of the Kautsky curve,）,42,分钟尺度动态变化,(Veronica,et al.,2012),43,分钟尺度动态变化,解释的问题,Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations.,Together, these results show that tonoplast-localized NHX proteins are essential for active K+ uptake at the tonoplast, for turgor regulation, and for stomatal function.,(Veronica,et al.,2012),44,光合参数,分钟尺度,变化测量方法,Leaf gas exchange was determined using the open gas exchange system Li-6400 (LI-COR) equipped with the chamber head (Li-6400-40;LI-COR) that allowed full control of light, CO,2, and humidity.,Stomatal conductance (gs; mmol m,-2,s,-1,) and the net photosynthetic rate (AN; mmol m,-2,s,-1,) were measured in 3-week-old plants of the wild type and mutant line L2 grown hydroponically in LAK standard solution.,(Veronica,et al.,2012),45,光合参数,分钟尺度,变化测量方法,Leaf responses to osmotic shock were recorded in fully expanded leaves, attached to the plant, under ambient CO,2,and a saturating PPFD of 150mmol m,-2,s,-1,Leaves were allowed to equilibrate under those conditions for at least 10 min and then subjected to osmotic shock by 20% PEG6000 in LAK medium.,Measurements were recorded every 30 s over a period of 120 min. A total of 12 measurements for each genotype (six plants per line and two measurements per plant) were recorded.,(Veronica,et al.,2012),46,光合参数分钟尺度动态测量实验小结,实验仪器：,Li-6400,（,XT,）,6400-40,采样：生长,3,周的叶片,时间：,120mins,重复数：略,光：适应,10,分钟,温度：略,二氧化碳浓度：略,湿度,:,略,47,荧光诱导曲线,(S. DEVACHT,et al.,2011),MT = 16,ML = 100 mol m,2,s,1,MT = 2,ML = 100 mol m,2,s,1,MT = 16,ML = 800 mol m,2,s,1,MT = 2,ML = 800 mol m,2,s,1,48,荧光诱导曲线,(S. DEVACHT,et al.,2011),49,荧光诱导曲线解释的问题,The quenching of the fluorescence signal during light induction depends on the generation of NPQ (Lambrev,et al.,2007).,Our results also demonstrate that the activation of NPQ is not only a light-dependent process (since the function of NPQ is to dissipate excess light energy), it is also strongly affected by temperature.,The combination of light and temperature stress affected the PSII efficiency more than each condition on its own.,(S. DEVACHT,et al.,2011),50,荧光诱导曲线实验方法,The MT was maintained throughout the whole procedure. A Chl fluorescence imaging system ( CFImager, Technologica, UK) was used.,The measurement procedure lasted 2.5 h (Fig. 1). In a first step, the plants were dark-adapted for 30 min.,(S. DEVACHT,et al.,2011),51,荧光诱导曲线实验方法,Fo was measured using a measurement light level of 0.520.85 mol m,2,s,1,. Fm was measured with a saturation pulse of 4,947 mol m,2,s,1,. After 20 s, the plants were exposed to actinic light at the corresponding ML for 1 h.,During this light period a saturation pulse of 4,947 mol m,2,s,1,was given for 800 ms every 2 min for the quenching analysis.,52,荧光诱导曲线实验小结,实验仪器：,CF-Imager,样品：,lue,时间：暗适应,30,分钟,重复数：略,光：暗适应；测量,Fo,和,Fm,，之后,20s,打开,ML,温度：控制,二氧化碳浓度：控制,湿度,:,略,53,暗驰豫动力学,P. LOOTENS,et al.,2011,54,暗驰豫动力学,P. LOOTENS,et al.,2011,55,暗驰豫动力学,P. LOOTENS,et al.,2011,56,暗驰豫动力学,P. LOOTENS,et al.,2011,57,暗驰豫动力学解释的问题,In this paper we discuss the effects of growth temperature (GT), measurement temperature (MT), and measuring light intensity (ML) on the relaxation of the Kautsky curve.,The three components of the nonphotochemical quenching process (NPQE, NPQT, and NPQI) were determined.,（,P. LOOTENS,et al.,2011,）,58,暗驰豫动力学解释的问题,NPQE was not affected by GT but was significantly affected by MT and ML. NPQT and NPQI were affected by all factors and their interactions.,An acclimation effect for plants grown at low GT was detected. Acclimation resulted in lower NPQT and NPQI values.,（,P. LOOTENS,et al.,2011,）,59,暗驰豫动力学解释的问题,The halftime of the inhibition depending on NPQ (NPQI) was not affected by any of the factors investigated.,Based on the data generated, we conclude that NPQI is a valuable parameter for screening the cold sensitivity of young industrial chicory plants.,（,P. LOOTENS,et al.,2011,）,60,暗驰豫动力学实验方法,During this dark period a saturation pulse was given twice after 2.5 min and every five minutes thereafter during 1 h to determine the different components of the relaxation process (NPQE, NPQT, and NPQI).,61,暗驰豫动力学实验方法,The first two pulses after 2.5 min are important to determine NPQE, the “fast” component of the relaxation process (Horton and Hague 1988, Mller,et al.,2001, Bruce,et al.,2004).,62,暗驰豫动力学实验方法,Subsequent pulses were applied with longer intervals, i.e., every 5 min, because the procedure can have an influence on the relaxation of NPQ (Walters and Horton 1991). The application of a saturation pulse every 5 min allowed us to maintain the influence of the pulses on the relaxation kinetics as low as possible, without any loss of information about the dark recovery.,63,暗驰豫动力学实验方法,Within each plate 14 plants were selected at random. This corresponds to the maximum number of traces that the used Chl fluorescence imaging system can record simultaneously. Chl fluorescence measurements were done on the adaxial side of the matures cotyledons. The Chl signals for each of the 14 plants result from the average values of all the pixels of that individual plant.,64,暗驰豫动力学实验小结,实验仪器：,CF-Imager,样品：略,时间：暗适应,2.5,分钟后打两次饱和闪光；之后每,5,分钟打,1,次饱,和闪光，持续,1,个小时。,重复数：,n=14,光：饱和闪光,温度：控制,二氧化碳浓度：略,湿度,:,略,65,5,秒尺度动态变化,5.1,荧光参数直观成像,5.2 Fluoresence Transient,66,荧光参数直观成像,(CHARLES P. CHEN,et al.,2011),67,荧光参数直观成像解释的问题,Both exposure regimes lowered leaf photosynthetic CO,2,uptake about 40% and PSII efficiency ( Fq,/Fm,) by 20% compared with controls, but this decrease was far more spatially heterogeneous in the acute treatment.,(CHARLES P. CHEN,et al.,2011),68,荧光参数直观成像解释的问题,Decline in Fq/Fm in the acute treatment resulted equally from decreases in the maximum efficiency of PSII (Fv/Fm) and the proportion of open PSII centres (Fq/Fv), but in the chronic treatment decline in Fq/Fm resulted only from decrease in Fq/Fv.,(CHARLES P. CHEN,et al.,2011),69,荧光参数直观成像解释的问题,Findings suggest that acute and chronicO,3, exposures do not induce identical mechanisms of O,3,damage within the leaf, and using one fumigation method alone is not sufficient for understanding the full range of mechanisms of O,3,damage to photosynthetic production in the field.,(CHARLES P. CHEN,et al.,2011),70,荧光参数直观成像实验方法,Attached third trifoliates were enclosed in a custom-built temperature, humidity and ambient CO,2,-controlled gas exchange cuvette with a clear IR transmitting window,(Tedlar polyvinyl fluoride film, Dupont, Wilmington,DE,USA) measuring 57.5 cm.,71,荧光参数直观成像实验方法,The chamber was attached to an open gas exchange system incorporating infrared CO,2,and water analyzers to allow measurement of photosynthetic CO,2,uptake (A) and stomatal conductance to water,vapour ( gs) (LI-6400, Li-Cor Inc., Lincoln, NE, USA).,72,荧光参数直观成像实验方法,The ambient CO,2,concentration (Ca) was held at 400 ppm and leaf temperature at 25,for all measurements. Steady-state A and gs were recorded while fluorescence images were being captured. The leaf chamber was placed within the imaging area of a pulse-modulated CFI system (CF Imager, Technologica Ltd.) to enable simultaneous measurements of gas exchange and fluorescence.,73,荧光参数直观成像实验方法,The imaging system used 1600 blue LEDs (peak wavelength = 470 nm) to provide actinic illumination, measuring pulses and saturating pulses. The system employed a CCD camera which captured 8-bit images at a spatial resolution of 150150 m per pixel and a maximum count of 704520 pixels per image.,74,荧光参数直观成像实验方法,Plants were dark-adapted for 30 min, after which a single saturating pulse was applied to capture Fo and Fm images. Plants were then exposed to an actinic photon flux of 500mol m,-2,s,-1,. Light-adapted fluorescence images were taken by applying a saturating pulse(3500mol m,-2,s,-1,) every 2.5 min until the mean Fq/Fm reached steady state. Typically, this required 30 min and roughly coincided with attainment of steady-state A.,75,荧光参数直观成像实验小结,实验仪器：,CF-Imager LI-6400,自制叶室,样品：略,时间：暗适应,30,分钟后，每,2.5,分钟打一次饱和闪光，持续,30,分钟,重复数：,n=14,光强：略,温度：控制,二氧化碳浓度：控制,湿度,:,略,76,77,