叶片尺度光合荧光实验设计.ppt

叶片尺度光合/荧光实验设计,ecotek,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,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,ecotek,1.1 光合参数季节动态,(david a. ramrez et al. 2012),ecotek,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),ecotek,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-2s-1, reference co2 concentration at 400ppm, leaf temperature at 25, and vapour pressure deficit at 1.5-2.0kpa. (david a. ramrez et al. 2012),ecotek,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),ecotek,荧光参数季节动态,(binghua liu et al. 2012),ecotek,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),ecotek,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-2s-1) was applied to determine the maximum chlorophyll fluorescence yield (fm) and maximum photochemical efficiency(fv/fm). (binghua liu et al. 2012),ecotek,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-2s-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),ecotek,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,ecotek,光合/荧光参数日动态,（孙东宝，王庆锁，2012）,ecotek,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）,ecotek,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）,ecotek,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 molm2s1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）,ecotek,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co2）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0.785cm2。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）,ecotek,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 molm2s1)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）,ecotek,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）,ecotek,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,ecotek,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）,ecotek,光响应曲线,（paulo et al. 2012),ecotek,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),ecotek,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 m2s1at 25. 控制二氧化碳浓度 initially, leaf tissues were exposed to a 5pa co2 partial pressure for 5 min to allow stomatal aperture; subsequently, a/par curves were obtained at 40pa co2 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),ecotek,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,ecotek,温度响应曲线,(masabumi et al. 2012),ecotek,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 co2 because of the higher etr and suppression of photorespiration under elevated co2 (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),ecotek,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 co2 concentrations of 400 and 800mol mol 1 and saturating pfd of 1000 mol m2s1. (masabumi et al. 2012),ecotek,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),ecotek,湿度响应曲线,（顾礼力等，2012）,ecotek,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。,ecotek,湿度响应曲线测量方法,为了检验其关系，实验控制光强 1500molm-2s-1，co2 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。,ecotek,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,ecotek,二氧化碳响应曲线,(masabumi et al. 2012),ecotek,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 co2 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 co2 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),ecotek,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 1000mol m2s1, 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),ecotek,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,ecotek,荧光-光响应曲线,(mitsutoshi et al. 2012),ecotek,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),ecotek,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 360molmol1and at various ppfd (0, 50, 100, 200, 300, 600, 1000,1500 and 2000 mol m2s1) which was provided by a red/blue led array (640040, licor) with blue light comprising 10 % of total ppfd. (mitsutoshi et al. 2012),ecotek,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,ecotek,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）,ecotek,分钟尺度动态变化,(veronica et al. 2012),ecotek,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),ecotek,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, co2, and humidity. stomatal conductance (gs; mmol m-2s-1) and the net photosynthetic rate (an; mmol m-2s-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),ecotek,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 co2 and a saturating ppfd of 150mmol m-2s-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),ecotek,光合参数分钟尺度动态测量实验小结,ecotek,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,荧光诱导曲线,(s. devacht et al. 2011),ecotek,mt = 16 ml = 100 mol m2 s1,mt = 2 ml = 100 mol m2 s1,mt = 16 ml = 800 mol m2 s1,mt = 2 ml = 800 mol m2 s1,荧光诱导曲线,(s. devacht et al. 2011),ecotek,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),ecotek,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),ecotek,荧光诱导曲线实验方法,fo was measured using a measurement light level of 0.520.85 mol m2s1. fm was measured with a saturation pulse of 4,947 mol m2s1. 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 m2s1was given for 800 ms every 2 min for the quenching analysis.,ecotek,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,ecotek,暗驰豫动力学,p. lootens et al.,2011,ecotek,暗驰豫动力学,p. lootens et al.,2011,ecotek,暗驰豫动力学,p. lootens et al.,2011,ecotek,暗驰豫动力学,ecotek,p. lootens et al.,2011,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）,ecotek,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）,ecotek,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）,ecotek,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).,ecotek,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).,ecotek,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.,ecotek,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.,ecotek,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,ecotek,5 秒尺度动态变化,5.1 荧光参数直观成像 5.2 fluoresence transient,ecotek,荧光参数直观成像,(charles p. chen et al. 2011),ecotek,荧光参数直观成像解释的问题,both exposure regimes lowered leaf photosynthetic co2 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),ecotek,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),ecotek,荧光参数直观成像解释的问题,findings suggest that acute and chronico3 exposures do not induce identical mechanisms of o3 damage within the leaf, and using one fumigation method alone is not sufficient for understanding the full range of mechanisms of o3 damage to photosynthetic production in the field. (charles p. chen et al. 2011),ecotek,荧光参数直观成像实验方法,attached third trifoliates were enclosed