Spectral characterization of NAD(P)H fluorescence in intact isolated chloroplasts and leaves: Effect of chlorophyll concentration on reabsorption of blue-green fluorescence

In the present communication we report a spectral analysis of the blue-green fluorescence related to changes in NAD(P) redox state in chloroplasts and leaves. To assess the contribution of reabsorption and the inner filter effect, we compared transmission and fluorescence at different chloroplast concentrations, and showed that reabsorption by the photosynthetic pigments (chlorophylls and carotenoids) was at the origin of the two peaks in the emission spectrum in vivo. The absence of potential green-emitting fluorophores in chloroplasts was determined by measuring variable and time-resolved fluorescence at different wavelengths. We defined the conditions which optimize the UV-excited blue-green fluorescence signal dependent on NAD(P)H, and we present an example of monitoring of NAD(P)H fluorescence in intact leaves.     

Mercury inhibits the non-photochemical reduction of plastoquinone by exogenous NADPH and NADH: evidence from measurements of the polyphasic chlorophyll a fluorescence rise in spinach chloroplasts

Chlorophyll a fluorescence rise kinetics (from 50 μs to 1 s) were used to investigate the non-photochemical reduction of the plastoquinone (PQ) pool in osmotically broken spinach chloroplasts (Spinacia oleracea L.). Incubation of the chloroplasts in the presence of exogenous NADPH or NADH resulted in significant changes in the shape of the fluorescence transient reflecting an NAD(P)H-dependent accumulation of reduced PQ in the dark, with an extent depending on the concentration of NAD(P)H and the availability of oxygen; the dark reduction of the PQ pool was saturated at lower NAD(P)H concentrations and reached a higher level when the incubation took place under anaerobic conditions than when it occurred under aerobic conditions. Under both conditions NADPH was more effective than NADH in reducing PQ, however only at sub-saturating concentrations. Neither antimycin A nor rotenone were found to alter the effect of NAD(P)H. The addition of mercury chloride to the chloroplast suspension decreased the NAD(P)H-dependent dark reduction of the PQ pool, with the full inhibition requiring higher mercury concentrations under anaerobic than under aerobic conditions. This is the first time that this inhibitory role of mercury is reported for higher plants. The results demonstrate that in the dark the redox state of the PQ pool is regulated by the reduction of PQ via a mercury-sensitive NAD(P)H-PQ oxidoreductase and the reoxidation of reduced PQ by an O₂-dependent pathway, thus providing additional evidence for the existence of a chlororespiratory electron transport chain in higher plant chloroplasts. This revised version was published online in June 2006 with corrections to the Cover Date.     

Intrathylakoidal crystal appearance during the vital cycle of spinach chloroplasts

Summary The entire vital cycle of chloroplasts was followed in spinach leaves, in order to determine the plastidial situations leading to the build-up of intrathylakoidal crystals. This kind of inclusion was noticed in the juvenile ontogenetic stages as well as in the senescent chloroplast stage, while intrathylakoidal crystals were not present in the mature organelles. The appearance of such crystals, which are known to consist of ribulose 1,5-diphosphate carboxylase, is interpreted as a consequence of an imbalance between enzyme level and membrane function.Supported by a C.N.R. grant.     

Characterisation of the effects of Antimycin A upon high energy state quenching of chlorophyll fluorescence (qE) in spinach and pea chloroplasts

High energy state quenching of chlorophyll fluorescence (qE) is inhibited by low concentrations of the inhibitor antimycin A in intact and osmotically shocked chloroplasts isolated from spinach and pea plants. This inhibition is independent of any effect upon pH (as measured by 9-aminoacridine fluorescence quenching). A dual control of qE formation, by pH and the redox state of an unidentified chloroplast component, is implied. Results are discussed in terms of a role for qE in the dissipation of excess excitation energy within photosystem II.Abbreviations 9-AA_max = Maximum yield of 9-aminoacridine fluorescence - DCMU = 3(3,4-dichlorophenyl)-1,1-dimethylurea; F_max ± Maximum yield of chlorophyll fluorescence - hr = hour - PAR = Photosynthetically Active Radiation - Q_A = Primary stable electron acceptor within photosystem II - qE = High energy state quenching of chlorophyll fluorescence - qI = quenching of chlorophyll fluorescence related to photoinhibition - qP = Quenching of chlorophyll fluorescence by oxidised plastoquinone - qQ = photochemical quenching of chlorophyll fluorescence - qR = (F_max—maximum level of chlorophyll fluorescence induced by the addition of saturating DCMU) - qT = Quenching of chlorophyll fluorescence attributable to state transitions     

Purification and characterization of a salinity induced alkaline protease from isolated spinach chloroplasts

In cynobacteria and higher plants, salinity is known to inhibit the activity of several enzymes involved in photosynthesis and hence decreases the overall photosynthetic rate. This gave us an impetus to search for a protease, which may be involved in the turnover of non-functional enzymes produced under salinity stress. Taking the possible changes in pH gradient of the chloroplast under consideration, we have tried to identify a protease, which is induced under salinity and characterized it as an alkaline protease using spinach (Spinacia oleracea) leaves as a model system. The HIC-HPLC purified homogeneous alkaline serine protease from the isolated spinach chloroplasts had two subunits of molecular weight 63 and 32 kDa. The enzyme was maximally active at pH 8.5 and 50°C. The enzyme showed the property to hydrolyze the synthetic substrate like azocaesin and had sufficient proteolytic activity in gelatin bound native PAGE. The enzyme activity was also dependent upon the presence of divalent cations and reduced environment. The active site residues were identified and the homogeneous alkaline serine protease had cysteine, lysine and tryptophan residues at its active site.     

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae,Codium andCaulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO₂-dependent and ferricyanide-dependent O₂ evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O₂ evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO₂-dependent O₂ evolution in both species, but this was partially restored in theCodium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when theCodium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen.Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as inCodium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar,Caulerpa chloroplasts could not regain their capacity for CO₂-dependent O₂ evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids.Caulerpa chloroplasts were less sensitive to detergent than those ofCodium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.     

The role of the gas phase in the greening and growth of illuminated cell suspension cultures of spinach (Spinacia oleracea,L.)

Summary The gas phase developed above spinach suspension cultures critically affected their growth and greening. Ethylene accumulation inhibited greening; this effect of ethylene was antagonised when the culture gas phase was enriched with carbon dioxide. Greening was enhanced by reducing the partial pressure of oxygen below the air level; this effect was observed when oxygen supply did not restrict growth. One of the authors (C.C.D.) was supported by an S.R.C. studentship grant during this work.     

Presence in the stroma of chloroplasts of a large pool of a ribosomal protein not structurally related to any Escherichia coli ribosomal protein

Summary A search was made for the presence of a pool of free ribosomal proteins in the stroma of the spinach chloroplast. The results showed that a relatively large amount of one protein, CS-S5, is present in the stroma. Immunoprecipitation experiments showed that this protein is encoded by the nuclear genome. Clones were isolated from a cDNA library constructed in the expression vector lambda gtll, using specific antibodies raised against the CS-S5 protein. A full-length cDNA was sequenced which contains an open reading frame (ORF) for the precursor of the CS-S5 protein, as shown by immunoprecipitation. This precursor contains a putative transit peptide of 66 amino acids and the mature product has no significant homology with any of the Escherichia coli ribosomal proteins, in contrast to the other ribosomal protein gene products so far identified in spinach chloroplasts.     

Non-photochemical quenching of chlorophyll a fluorescence in isolated chloroplasts under conditions of stressed photosynthesis

Non-photochemical quenching of chlorophyll a fluorescence after short-time light, heat and osmotic stress was investigated with intact chloroplasts from Spinacia oleracea L. The proportions of non-photochemical fluorescence quenching (q _N ) which are related (q _E ) and unrelated (q _I ) to the transthylakoid proton gradient (pH) were determined. Light stress resulted in an increasing contribution of q _Ito total q _N.The linear dependence of q. _Eand pH, as seen in controls, was maintained. The mechanisms underlying this type of quenching are obviously unaffected by photoin-hibition. In constrast, q _Ewas severely affected by heat and osmotic stress. In low light, the response of q _Eto changes in pH was enhanced, whereas it was reduced in high light. The data are discussed with reference to the hypothesis that q _Eis related to thermal dissipation of excitation energy from photosystem II. It is shown that q _Eis not only controlled by pH, but also by external factors.Abbreviations and symbols 9-AA 9-aminoacridine - F _o basic chlorophyll fluorescence - F _o variable chlorophyll fluorescence - L ₂ saturating light pulse - PS photosystem - q _E pH-dependent, non-photochemical quenching of fluorescence - q _I pH-independent, non-photochemical quenching - q _N entire non-photochemical quenching - q _Q photochemical quenching     

Chlorophyll luminescence as an indicator of stress-induced damage to the photosynthetic apparatus. Effects of heat-stress in isolated chloroplasts

A brief review is given of investigations on stres-induced alterations of ms-to s-luminescence yield of chlorophyll in plants. Three different approaches are considered: phytoluminography, luminescence-temperature curves, and luminescence induction curves. The remainder of this article presents new results of the effect of heat stress on luminescence induction curves of isolated chloroplasts. Three parameters with widely different heat resistances were resolved from induction curves. A fast valinomycin sensitive transient, L'_i, with a 50% inhibition temperature of 33 to 34°C was correlated with the magnitude of the light-induced membrane potential after heat pretreatment. A slower nigericin sensitive transient, L'_m, with a 50% inhibition temperature of 39 to 40°C was mainly correlated with the light-induced proton gradient. An uncoupler resistant part of the induction curve, L₀, was enhanced by heat stress (half maximum after pretreatment at 46°C) and was correlated with the degree of inhibition of oxygen evolution. Since L₀ was also raised by other treatments impairing the oxygen evolving enzyme system, and since this rise was inhibited by DCMU and hydroxylamine, this type of luminescence was ascribed to the intrinsic backreaction. We conclude that luminescence induction curves can serve as an useful indicator of the intactness of the membrane potential, the proton gradient, and the oxygen evolving enzyme system in isolated chloroplasts after heat stress.Abbreviations 9-AA 9-aminoacridine - CCCP carbonylcyanide m-chlorophenylhydrazone - A₅₁₈ light-induced absorbance change at 518 nm - A_on, A_off rapid A₅₁₈ upon switching actinic light on or off, respectively - L_i, L_m, L₀ in this order, initial spike, main maximum, uncoupler insensitive transient of luminescence induction curve - L'_i = L_i – L₀ - L'_m = L_m – L₀ - P, P680 primary donor of PS II - PFD photon flux density (400–700 nm) - Q_A primary acceptor of PS II     

The effects of low temperature acclimation and photoinhibitory treatments on Photosystem 2 studied by thermoluminescence and fluorescence decay kinetics

The effects of low temperature acclimation and photoinhibitory treatment on Photosystem 2 (PS 2) have been studied by thermoluminescence and chlorophyll fluorescence decay kinetics after a single turnover saturating flash. A comparison of unhardened and hardened leaves showed that, in the hardened case, a decrease in overall and B-band thermoluminescence emissions occurred, indicating the presence of fewer active PS 2 reaction centers. A modification in the form of the B-band emission was also observed and is attributed to a decrease in the apparent activation energy of recombination in the hardened leaves. The acclimated leaves also produced slower Q_A ^– reoxidation kinetics as judged from the chlorophyll fluorescence decay kinetics. This change was mainly seen in an increased lifetime of the slow reoxidation component with only a small increase in its amplitude. Similar changes in both thermoluminescence and fluorescence decay kinetics were observed when unhardened leaves were given a high light photoinhibitory treatment at 4°C, whereas the hardened leaves were affected to a much lesser extent by a similar treatment. These results suggest that the acclimated plants undergo photoinhibition at 4°C even at low light intensities and that a subsequent high light treatment produces only a small additive photoinhibitory effect. Furthermore, it can be seen that photoinhibition eventually gives rise to PS 2 reaction centers which are no longer functional and which do not produce thermoluminescence or variable chlorophyll fluorescence.Abbreviations D1 The 32 kDa protein of Photosystem 2 reaction center - F_m maximum chlorophyll fluorescence yield - F₀ minimal chlorophyll fluorescence yield obtained when all PS 2 centers are open - F_i intermediate fluorescence level corresponding to PS 2 centers which are loosely or not connected to plastoquinone (non-B centers) - F_v maximum variable chlorophyll fluorescence yield (F_v=F_m–F₀) - PS 2 Photosystem 2 - Q_A and Q_B respectively, primary and secondary quinonic acceptors of PS 2 - S1, S2 and S3 respectively, the one, two and three positively charged states of the oxygen evolving system - Z secondary donor of PS 2     

Improved rates of CO2-fixation by intact chloroplasts isolated in media with KCl as the osmoticum

Intact chloroplasts were isolated from spinach leaves using media with either 330 mM sorbitol or 200 mM KCl as the osmoticum. Chloroplasts isolated in KCl exhibited higher rates of CO₂-dependent oxygen evolution in nine out of ten experiments, the average increase being 43%. Chloroplasts isolated in KCl routinely achieved rates of CO₂-dependent oxygen evolution of 200–300 mol·mg chlorophyll^-1·hour^-1 at 20°C. Intact chloroplasts were also isolated in media with 200 mM NaCl or choline chloride but the rates of CO₂ fixation were not superior to those isolated in sorbitol media. The K⁺ content of chloroplasts isolated in KCl media was higher than for chloroplasts isolated in sorbitol. It is suggested that the use of KCl as an osmoticum prevents the loss of chloroplast K⁺ which can occur during isolation in sorbitol media. Chloroplasts isolated in KCl lost, on average, 36% of the initial CO₂ fixation activity after storage for four hours on ice, compared to 24% loss of activity for chloroplasts isolated in sorbitol. This increased loss of activity was not observed if KCl was used in the grinding medium and sorbitol or glycinebetaine in the resuspension media. For measurement of the maximum photosynthetic capacity in vitro, the use of KCl in the grinding medium may be better than sorbitol.Abbreviations BSA bovine serum albumin - Chl chlorophyll - Pi inorganic orthophosphate - EDTA ethlenediamine tetraacetic acid     

The quantum efficiency of photosystem II and its relation to non-photochemical quenching of chlorophyll fluorescence; the effect of measuring-and growth temperature

The relation between the quantum yield of oxygen evolution of open photosystem II reactions centers (_p), calculated according to Weis and Berry (1987), and non-photochemical quenching of chlorophyll fluorescence of plants grown at 19°C and 7°C was measured at 19°C and 7°C. The relation was linear when measured at 19°C, but when measured at 7°C a deviation from linearity was observed at high values of non-photochemical quenching. In plants grown at 7°C this deviation occurred at higher values of non-photochemical quenching than in plants grown at 19°C. The deviations at high light intensity and low temperature are ascribed to an increase in an inhibition-related, non-photochemical quenching component (q_I).The relation between the quantum yield of excitation capture of open photosystem II reaction centers (_exe), calculated according to Genty et al. (1989), and non-photochemical quenching of chlorophyll fluorescence was found to be non-linear and was neither influenced by growth temperature nor by measuring temperature.At high PFD the efficiency of overall steady state electron transport measured by oxygen-evolution, correlated well with the product of q _N and the efficiency of excitation capture (_exe) but it deviated at low PFD. The deviations at low light intensity are attributed to the different populations of chloroplasts measured by gas exchange and chlorophyll fluorescence and to the light gradient within the leaf.Abbreviations F₀ basic fluorescence - F₀ basic fluorescence, thylakoid in energized state - F_m maximal fluorescence - F_m maximum fluorescence in energized state - F_s steady state fluorescence - F_v maximal variable fluorescence - PFD photon flux density - PS II_rc Photosystem II reaction center - q_F0 quenching of basic fluorescence - q_E energy related quenching - q_N non-photochemical quenching:-q_f-total quenching - q_I inhibition-related quenching - q_p photochemical quenching - q_r quenching due to state transition - R_d dark respiration - _p PS II efficiency of excitation capture of open PS II_rc - _pe extrapolated minimal value of _p - _p0 extrapolated maximal value of _p - _si quantum efficiency of linear electron transport, calculated from gas exchange measurements based on incident light - _sf quantum efficiency of linear electron transport, calculated from fluorescence measurements, based on incident measuring light     

The stereospecificity,purification, and characterization of an NADH-ferricyanide reductase from spinach leaf plasma membrane

Summary The stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane, peroxisomal membrane, plasma membrane and tonoplast are all specific for the -hydrogen of NADH whereas the reductases in the ER, the Golgi and the outer mitochondrial membrane are -specific. This shows unequivocally that the NADH-ferricyanide activity in the plasma membrane is not caused by ER contamination. In all the membranes one or several polypeptides with an apparent size of 45–50 kDa cross-react with antibodies raised against a microsomal NADH-ferricyanide reductase. An NADH-ferricyanide reductase was purified from spinach leaf plasma membranes. The enzyme was released from the membrane by CHAPS solubilization and purified 360-fold by ion-exchange chromatography followed by affinity chromatography and size exclusion chromatography on FPLC. A major band of 45 kDa was detected by SDS-PAGE and it cross-reacted with the anti-NADH-ferricyanide reductase antibodies. The native size of the enzyme is 160 kDa as determined by size-exclusion chromatography indicating that it is a tetramer. Isoelectric focusing revealed three isoenzymes between pH 5.3 and 5.6. The enzyme shows typical FAD fluorescence spectra with excitation peaks at 371 and 468 nm and an emission peak at 525 nm. It is specific for the -hydrogen of NADH and prefers NADH over NADPH as electron donor. It is highly specific for ferricyanide as electron acceptor and it is therefore unlikely to be the enzyme responsible for iron reduction on the outer surface of the plasma membrane.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammoniol]-1-propanesulfonate - DQ duroquinone - FPLC fast protein liquid chromatography; Ferricyanide hexacyanoferrate(III) - NEM N-ethylmaleimide - PCMB p-chloromercurobenzoate - SHAM salicylhydroxamic acid - SMP submitochondrial particles     

Thylakoid and grana formation during the development of pea chloroplasts,illuminated by white,red, and blue low intensity light

Summary We analyzed the formation of thylakoids and grana during the development of pea chloroplasts, illuminated by white, red and blue low intensity light. The total length of granal and intergranal thylakoids, and the length of granal thylakoids per unit area of plastid section were measured. Initially the greatest increase in length of granal thylakoids and the highest incidence of grana with large thylakoid content occurred in red light. On the other hand, with illumination times of over 12 hours blue light appeared to be more efficient in stimulating grana formation and thylakoid growth.     

Betaine aldehyde dehydrogenase polymorphism in spinach: Genetic and biochemical characterization

Spinach (Spinacia oleracea L.) has a major chloroplastic isozyme of betaine aldehyde dehydrogenase (BADH) and a minor cytosolic one. Among a diverse collection of spinach accessions, three electrophoretic banding patterns of chloroplastic BADH were found: two were single banded and one was triple banded. Genetic analysis of these patterns indicated that chloroplastic BADH is encoded by a single, nuclear gene with two alleles, designated slow (S) and fast (F), and that products of these alleles can hybridize to form either homodimers or a heterodimer. The S allele was by far the most common among the accessions examined. Native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the SS and FF homodimers differ in charge but not molecular weight.This research was supported by the U.S. DOE under Contract DE-AC02-76ERO-1338, by USDA-CRGO Grant 87-CRCR-1-2460, and by grants from the State of Michigan REED-Biotechnology Program, from CIBA-GEIGY Corporation, and from the Beet Sugar Development Foundation.     

蓝桉凋落叶分解对菠菜生长及光合特性的影响

通过盆栽试验,模拟蓝桉(Eucalyptus globulus)凋落叶在土壤中分解对受体植物菠菜(Spinacia oleracea)生长及光合特性的影响,设置L30(30 g/盆)、L60(60 g/盆)、L90(90 g/盆)和L120(120 g/盆)4个凋落叶施用水平,对照(CK)不加凋落叶。结果表明:(1)在分解30 d时,低量的凋落叶(L30)未抑制菠菜生物量的积累,而中量和高量处理(L60、L90和L120)下其生物量显著降低(P0.05);在凋落叶分解50 d后,蓝桉凋落叶的化感抑制效应已经减弱,仅L120仍然抑制菠菜生物量的积累;(2)一定量的凋落叶(L30和L60)对菠菜叶绿素的合成有促进作用,而凋落叶超过一定量时(L120)转为抑制作用,在凋落叶分解28 d时相对不明显,40 d后逐渐显现;(3)经凋落叶处理的菠菜叶片胞间CO2浓度(Ci)、气孔导度(Gs)及蒸腾速率(Tr)均显著高于对照(CK)(P0.05),而净光合速率(Pn)在L30处理下最高,L60和L90处理与CK差异不显著,仅L120处理显著低于CK(P0.05);(4)光响应与CO2响应曲线的特征参数表观量子效率(AQY)、最大净光合速率(Pmax)、光饱和点(Lsp)、光补偿点(Lcp)、暗呼吸速率(Rd)、Ru BP表观羧化效率(CE)和光呼吸速率(Rp)在L30处理下高于CK,其余凋落叶处理均低于CK,而CO2饱和点(Csp)、CO2补偿点(Ccp)随凋落叶量的增加而升高。综合各指标可见,少量的蓝桉凋落叶分解(L30)可通过增加叶绿素含量,提高对光和CO2的利用能力促进菠菜的光合作用,进而促进其生长,而凋落叶超过一定量时(L90—L120)则起到相反的作用。     

菠菜性别相关的ISSR标记

菠菜为雌雄异株植物,用CTAB法提取其雌、雄株成株幼嫩叶片DNA,分别构建雌、雄株DNA池,以之为模板,用已优化的ISSR体系扩增,在74条ISSR引物中,I62扩增出一条约1 200 bp雌性连锁标记,回收纯化该特异扩增片段,将其连接于pUCm-T载体,转化进大肠杆菌JM109菌株,并检测及测序。回收克隆和测序后发现该片段全长1 176 bp,富含AT,AT占57.0%。根据测序结果设计1对25 bp的特异引物将这个雌性连锁的ISSR标记转化为稳定性和特异性更好的SCAR标记。该特异引物对随机选取的雌雄菠菜单株进行PCR扩增,在雌株中均有1 176 bp的特异条带,而雄株中均无。此特异条带的获得为菠菜性别相关基因的克隆奠定基础。     

Diurnal changes in leaf gas exchange and chlorophyll fluorescence in tropical tree species with contrasting light requirements

Interspecific ecophysiological differences in response to different light environments are important to consider in regeneration behavior and forest dynamics. The diurnal changes in leaf gas exchange and chlorophyll fluorescence of two dipterocarps, Shorea leprosula (a high light-requiring) and Neobalanocarpus heimii (a low light-requiring), and a pioneer tree species (Macaranga gigantea) growing in open and gap sites were examined. In the open site, the maximum net photosynthetic rate (P_n), photosystem II (PSII) quantum yield (; F/Fm), and relative electron transport rate (r-ETR) through PSII at a given photosynthetic photon flux density (PPFD) was higher in S. leprosula and M. gigantea than in N. heimii, while non-photochemical quenching (NPQ) at a given PPFD was higher in N. heimii. The maximum values of net photosynthetic rate (P_n) in M. gigantea and S. leprosula was higher in the open site (8–11 mol m^–2 s^–1) than in the gap site (5 mol m^–2 s^–1), whereas that in N. heimii was lower in the open site (2 mol m^–2 s^–1) than in the gap site (4 mol m^–2 s^–1), indicating that N. heimii was less favorable to the open site. These data provide evidence to support the hypothesis that ecophysiological characteristics link with plants regeneration behavior and successional status. Although P_n and stomatal conductance decreased at midday in M. gigantea and S. leprosula in the open site, both r-ETR and leaf temperature remained unchanged. This indicates that stomatal closure rather than reduced photochemical capacity limited P_n in the daytime. Conversely, there was reduced r-ETR under high PPFD conditions in N. heimii in the open site, indicating reduced photochemical capacity. In the gap site, P_n increased in all leaves in the morning before exposure to direct sunlight, suggesting a relatively high use of diffuse light in the morning.