Frequently asked questions about chlorophyll fluorescence,the sequel

Using chlorophyll (Chl) a fluorescence many aspects of the photosynthetic apparatus can be studied, both in vitro and, noninvasively, in vivo. Complementary techniques can help to interpret changes in the Chl a fluorescence kinetics. Kalaji et al. (Photosynth Res 122:121–158, 2014a) addressed several questions about instruments, methods and applications based on Chl a fluorescence. Here, additional Chl a fluorescence-related topics are discussed again in a question and answer format. Examples are the effect of connectivity on photochemical quenching, the correction of F _V /F _M values for PSI fluorescence, the energy partitioning concept, the interpretation of the complementary area, probing the donor side of PSII, the assignment of bands of 77 K fluorescence emission spectra to fluorescence emitters, the relationship between prompt and delayed fluorescence, potential problems when sampling tree canopies, the use of fluorescence parameters in QTL studies, the use of Chl a fluorescence in biosensor applications and the application of neural network approaches for the analysis of fluorescence measurements. The answers draw on knowledge from different Chl a fluorescence analysis domains, yielding in several cases new insights.     

Why is chlorophyll <Emphasis Type="Italic">b</Emphasis> only used in light-harvesting systems?

Chlorophylls (Chl) are important pigments in plants that are used to absorb photons and release electrons. There are several types of Chls but terrestrial plants only possess two of these: Chls a and b. The two pigments form light-harvesting Chl a/b-binding protein complexes (LHC), which absorb most of the light. The peak wavelengths of the absorption spectra of Chls a and b differ by c. 20 nm, and the ratio between them (the a/b ratio) is an important determinant of the light absorption efficiency of photosynthesis (i.e., the antenna size). Here, we investigated why Chl b is used in LHCs rather than other light-absorbing pigments that can be used for photosynthesis by considering the solar radiation spectrum under field conditions. We found that direct and diffuse solar radiation (PAR_dir and PAR_diff, respectively) have different spectral distributions, showing maximum spectral photon flux densities (SPFD) at c. 680 and 460 nm, respectively, during the daytime. The spectral absorbance spectra of Chls a and b functioned complementary to each other, and the absorbance peaks of Chl b were nested within those of Chl a. The absorption peak in the short wavelength region of Chl b in the proteinaceous environment occurred at c. 460 nm, making it suitable for absorbing the PAR_diff, but not suitable for avoiding the high spectral irradiance (SIR) waveband of PAR_dir. In contrast, Chl a effectively avoided the high SPFD and/or high SIR waveband. The absorption spectra of photosynthetic complexes were negatively correlated with SPFD spectra, but LHCs with low a/b ratios were more positively correlated with SIR spectra. These findings indicate that the spectra of the photosynthetic pigments and constructed photosystems and antenna proteins significantly align with the terrestrial solar spectra to allow the safe and efficient use of solar radiation.     

Potential Outdoor Cultivation of Green Microalgae Based on Response to Changing Temperatures and by Combining with Air Temperature Occurrence

In this study, our working hypothesis was to examine whether temperature alters biomass and metabolite production by microalgae according to strain. We also addressed whether it is possible to choose a strain suitable for growing in each season of a given region. A factorial experiment revealed a significant interaction between chlorophylls a and b (Chl a and Chl b), carotenoid/Chl (a?+?b) ratio, biomass and total lipid productivity of six green microalgae (four Chlorella spp., Chlorella sorokiniana and Neochloris oleoabundans) after 15 days at four temperatures. At 39/35 °C, two Chlorella sp. strains (IPR7115 and IPR7117) showed higher total carotenoids/Chl (a?+?b) (0.578 and 0.830), respectively. N. oleoabundans had the highest Chl a (8210 μg L^?1) and Chl b (1909 μg L^?1) at 19/15 °C and highest maximum dry biomass (2900 mg L^?1), specific growth rate (0.538 day^?1) and total lipids (1003 mg L^?1) at 15/8 °C. We applied a method to infer the growth of these six green microalgae in outdoor ponds, as based on their response to changing temperatures and by combining with historical data on day/night air temperature occurrence for a given region. We conclude that the use of regionalized maps based on air temperature is a good strategy for predicting microalgal cultivation in outdoor ponds based on their features and tolerance to changing temperature.     

Effects of heat stress on photosynthetic electron transport in a marine cyanobacterium <Emphasis Type="Italic">Arthrospira</Emphasis> sp.

Arthrospira (Spirulina) is widely used as human health food and animal feed. In cultures grown outdoors in open ponds, Arthrospira cells are subjected to various environmental stresses, such as high temperature. A better understanding of the effects of high temperature on photosynthesis may help optimize the productivity of Arthrospira cultures. In this study, the effects of heat stress on photosynthetic rate, chlorophyll a fluorescence transients, and photosystem (PS) II, PSI activities in a marine cyanobacterium Arthrospira sp. were examined. Arthrospira cells grown at 25 °C were treated for 30 min at 25 (control), 30, 34, 37, or 40 °C in the dark. Heat stress (30–37 °C) enhanced net photosynthetic O₂ evolution rate. Heat stress caused over-reduction PSII acceptor side, damage of donor side of PSII, decrease in the energetic connectivity of PSII units, and decrease in the performance of PSII. When the temperature changed from 25 to 37 °C, PSII activity decreased, while PSI activity increased, the enhancement of photosynthetic O₂ evolution was synchronized with the increase in PSI activity. When temperature was further increased to 40 °C, it induced a decrease in photosynthetic O₂ evolution rate and a more severe decrease in PSII activity, but an increase in PSI activity. These results suggest that PSI activity was the decisive factor determining the change of photosynthetic O₂ evolution when Arthrospira was exposed to a temperature from 25 to 37 °C, but then, PSII activity became the decisive factor adjusting the change of photosynthetic O₂ evolution when the temperature was increased to 40 °C.     

<Superscript>15</Superscript>N photo-CIDNP MAS NMR analysis of reaction centers of <Emphasis Type="Italic">Chloracidobacterium thermophilum</Emphasis>

Photochemically induced dynamic nuclear polarization (photo-CIDNP) has been observed in the homodimeric, type-1 photochemical reaction centers (RCs) of the acidobacterium, Chloracidobacterium (Cab.) thermophilum, by ¹⁵N magic-angle spinning (MAS) solid-state NMR under continuous white-light illumination. Three light-induced emissive (negative) signals are detected. In the RCs of Cab. thermophilum, three types of (bacterio)chlorophylls have previously been identified: bacteriochlorophyll a (BChl a), chlorophyll a (Chl a), and Zn-bacteriochlorophyll a′ (Zn-BChl a′) (Tsukatani et al. in J Biol Chem 287:5720–5732, 2012). Based upon experimental and quantum chemical ¹⁵N NMR data, we assign the observed signals to a Chl a cofactor. We exclude Zn-BChl because of its measured spectroscopic properties. We conclude that Chl a is the primary electron acceptor, which implies that the primary donor is most likely Zn-BChl a′. Chl a and 8¹-OH Chl a have been shown to be the primary electron acceptors in green sulfur bacteria and heliobacteria, respectively, and thus a Chl a molecule serves this role in all known homodimeric type-1 RCs.     

<Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 CruA (sll0147) encodes lycopene cyclase and requires bound chlorophyll <Emphasis Type="Italic">a</Emphasis> for activity

The genome of the model cyanobacterium, Synechococcus sp. PCC 7002, encodes two paralogs of CruA-type lycopene cyclases, SynPCC7002_A2153 and SynPCC7002_A0043, which are denoted cruA and cruP, respectively. Unlike the wild-type strain, a cruA deletion mutant is light-sensitive, grows slowly, and accumulates lycopene, γ-carotene, and 1-OH-lycopene; however, this strain still produces β-carotene and other carotenoids derived from it. Expression of cruA from Synechocystis sp. PCC 6803 (cruA ₆₈₀₃) in Escherichia coli strains that synthesize either lycopene or γ-carotene did not lead to the synthesis of either γ-carotene or β-carotene, respectively. However, expression of this orthologous cruA ₆₈₀₃ gene (sll0147) in the Synechococcus sp. PCC 7002 cruA deletion mutant produced strains with phenotypic properties identical to the wild type. CruA₆₈₀₃ was purified from Synechococcus sp. PCC 7002 by affinity chromatography, and the purified protein was pale yellow-green due to the presence of bound chlorophyll (Chl) a and β-carotene. Native polyacrylamide gel electrophoresis of the partly purified protein in the presence of lithium dodecylsulfate at 4 °C confirmed that the protein was yellow-green in color. When purified CruA₆₈₀₃ was assayed in vitro with either lycopene or γ-carotene as substrate, β-carotene was synthesized. These data establish that CruA₆₈₀₃ is a lycopene cyclase and that it requires a bound Chl a molecule for activity. Possible binding sites for Chl a and the potential regulatory role of the Chl a in coordination of Chl and carotenoid biosynthesis are discussed.     

Chlorophylls <Emphasis Type="Italic">d</Emphasis> and <Emphasis Type="Italic">f</Emphasis> and their role in primary photosynthetic processes of cyanobacteria

The finding of unique Chl d- and Chl f-containing cyanobacteria in the last decade was a discovery in the area of biology of oxygenic photosynthetic organisms. Chl b, Chl c, and Chl f are considered to be accessory pigments found in antennae systems of photosynthetic organisms. They absorb energy and transfer it to the photosynthetic reaction center (RC), but do not participate in electron transport by the photosynthetic electron transport chain. However, Chl d as well as Chl a can operate not only in the light-harvesting complex, but also in the photosynthetic RC. The long-wavelength (Q_y) Chl d and Chl f absorption band is shifted to longer wavelength (to 750 nm) compared to Chl a, which suggests the possibility for oxygenic photosynthesis in this spectral range. Such expansion of the photosynthetically active light range is important for the survival of cyanobacteria when the intensity of light not exceeding 700 nm is attenuated due to absorption by Chl a and other pigments. At the same time, energy storage efficiency in photosystem 2 for cyanobacteria containing Chl d and Chl f is not lower than that of cyanobacteria containing Chl a. Despite great interest in these unique chlorophylls, many questions related to functioning of such pigments in primary photosynthetic processes are still not elucidated. This review describes the latest advances in the field of Chl d and Chl f research and their role in primary photosynthetic processes of cyanobacteria.     

Parasitism Rate of <Emphasis Type="Italic">Habrobracon hebetor</Emphasis> to <Emphasis Type="Italic">Ephestia kuehniella</Emphasis> Larvae: Residual Effect of Deltamethrin,Fenvalerate and Azadirachtin

Sublethal concentrations of chemical insecticides may cause changes in some behavioral characteristics of natural enemies such as functional responses. The residual effect of three synthetic insecticides including deltamethrin, fenvalerate and azadirachtin were studied on functional response of Habrobracon hebetor Say to Ephestia kuehniella Zeller larvae. Seven host densities (2, 4, 8, 16, 32, 64 and 96) were used during a 24 h period. The resulting data were appropriately fit to Type II functional response models in all treatments: (1) control (0.0916 h^?1; and T _h  = 0.2011 h); (2) deltamethrin (a = 0.0839 h^?1; and T _h  = 0.3560 h); (3) fenvalerate (a = 0.0808 h^?1 and T _h  = 0.3623 h); and (4) azadirachtin (a = 0.0900 h^?1 and T _h  = 0.2042 h). Maximum theoretical parasitism rate (T/T _h ) was 119.34 estimated for control wasps. There was no significant difference between the values of attack rates (a and a + D _a ) in all treatments while the handling time was statistically affected in female wasps treated with fenvalerate. Our findings will be useful in safe application of these insecticides in pest management programmes.     

Acclimation of shade-tolerant and light-resistant <Emphasis Type="Italic">Tradescantia</Emphasis> species to growth light: chlorophyll <Emphasis Type="Italic">a</Emphasis> fluorescence,electron transport,and xanthophyll content

In this study, we have compared the photosynthetic characteristics of two contrasting species of Tradescantia plants, T. fluminensis (shade-tolerant species), and T. sillamontana (light-resistant species), grown under the low light (LL, 50–125 µmol photons m^?2 s^?1) or high light (HL, 875–1000 µmol photons m^?2 s^?1) conditions during their entire growth period. For monitoring the functional state of photosynthetic apparatus (PSA), we measured chlorophyll (Chl) a emission fluorescence spectra and kinetics of light-induced changes in the heights of fluorescence peaks at 685 and 740 nm (F ₆₈₅ and F ₇₄₀). We also compared the light-induced oxidation of P₇₀₀ and assayed the composition of carotenoids in Tradescantia leaves grown under the LL and HL conditions. The analyses of slow induction of Chl a fluorescence (SIF) uncovered different traits in the LL- and HL-grown plants of ecologically contrasting Tradescantia species, which may have potential ecophysiological significance with respect to their tolerance to HL stress. The fluorometry and EPR studies of induction events in chloroplasts in situ demonstrated that acclimation of both Tradescantia species to HL conditions promoted faster responses of their PSA as compared to LL-grown plants. Acclimation of both species to HL also caused marked changes in the leaf anatomy and carotenoid composition (an increase in Violaxanthin?+?Antheraxantin?+?Zeaxanthin and Lutein pools), suggesting enhanced photoprotective capacity of the carotenoids in the plants grown in nature under high irradiance. Collectively, the results of the present work suggest that the mechanisms of long-term PSA photoprotection in Tradescantia are based predominantly on the light-induced remodeling of pigment-protein complexes in chloroplasts.     

Uphill energy transfer in photosystem I from <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>. Time-resolved fluorescence measurements at 77 K

Energetic properties of chlorophylls in photosynthetic complexes are strongly modulated by their interaction with the protein matrix and by inter-pigment coupling. This spectral tuning is especially striking in photosystem I (PSI) complexes that contain low-energy chlorophylls emitting above 700 nm. Such low-energy chlorophylls have been observed in cyanobacterial PSI, algal and plant PSI–LHCI complexes, and individual light-harvesting complex I (LHCI) proteins. However, there has been no direct evidence of their presence in algal PSI core complexes lacking LHCI. In order to determine the lowest-energy states of chlorophylls and their dynamics in algal PSI antenna systems, we performed time-resolved fluorescence measurements at 77 K for PSI core and PSI–LHCI complexes isolated from the green alga Chlamydomonas reinhardtii. The pool of low-energy chlorophylls observed in PSI cores is generally smaller and less red-shifted than that observed in PSI–LHCI complexes. Excitation energy equilibration between bulk and low-energy chlorophylls in the PSI–LHCI complexes at 77 K leads to population of excited states that are less red-shifted (by ~?12 nm) than at room temperature. On the other hand, analysis of the detection wavelength dependence of the effective trapping time of bulk excitations in the PSI core at 77 K provided evidence for an energy threshold at ~?675 nm, above which trapping slows down. Based on these observations, we postulate that excitation energy transfer from bulk to low-energy chlorophylls and from bulk to reaction center chlorophylls are thermally activated uphill processes that likely occur via higher excitonic states of energy accepting chlorophylls.     

Sucrose dependent mineral phosphate solubilization in <Emphasis Type="Italic">Enterobacter asburiae</Emphasis> PSI3 by heterologous overexpression of periplasmic invertases

Enterobacter asburiae PSI3 solubilizes mineral phosphates in the presence of glucose by the secretion of gluconic acid generated by the action of a periplasmic pyrroloquinoline quinone dependent glucose dehydrogenase. In order to achieve mineral phosphate solubilization phenotype in the presence of sucrose, plasmids pCNK4 and pCNK5 containing genes encoding the invertase enzyme of Zymomonas mobilis (invB) and of Saccharomyces cerevisiae (suc2) under constitutive promoters were constructed with malE signal sequence (in case of invB alone as the suc2 is secreted natively). When introduced into E. asburiae PSI3, E. a. (pCNK4) and E. a. (pCNK5) transformants secreted 21.65 ± 0.94 and 22 ± 1.3 mM gluconic acid, respectively, in the presence of 75 mM sucrose and they also solubilized 180 ± 4.3 and 438 ± 7.3 µM P from the rock phosphate. In the presence of a mixture of 50 mM sucrose and 25 mM glucose, E. a. (pCNK5) secreted 34 ± 2.3 mM gluconic acid and released 479 ± 8.1 µM P. Moreover, in the presence of a mixture of eight sugars (10 mM each) in the medium, E. a. (pCNK5) released 414 ± 5.3 µM P in the buffered medium. Thus, this study demonstrates incorporation of periplasmic invertase imparted P solubilization ability to E. asburiae PSI3 in the presence of sucrose and mixture of sugars.     

Exogenous 5-aminolevulinic acid alleviates the detrimental effects of UV-B stress on lettuce (<Emphasis Type="Italic">Lactuca sativa</Emphasis> L) seedlings

One of the abiotic stress factors affecting plant metabolism is ultraviolet-B (UV-B) radiation. 5-Aminolevulinic acid (ALA), a key precursor of porphyrin biosynthesis, promotes plant growth and crop yields. To investigate the alleviating effects of exogenous ALA on the damages caused by UV-B exposure, two different concentrations [10 ppm (ALA1) and 25 ppm (ALA2)] of ALA were applied to lettuce seedlings for 24 h and then they were exposed to 3.3 W m^?2 UV-B. Results showed that UV-B treatment significantly decreased chlorophyll a and b (Chl a and b) concentration, enhanced the activity of antioxidant enzymes, total phenolic concentration, soluble sugar contents, expression of phenylalanine ammonia lyase (PAL) and γ-tocopherol methyltransferase (γ-TMT) genes, the concentration of malondialdehyde (MDA), hydrogen peroxide (H₂O₂), and the rate of superoxide radical (\({\text{O}}_{2}^{ - }\)) generation in the lettuce seedlings when compared to the control. Pre-treatment with exogenous ALA significantly enhanced UV-B stress tolerance in lettuce seedlings by decreasing the reactive oxygen species. On the other hand, ALA application caused more increases in the PAL and γ-TMT gene expression, antioxidant enzymes activities, Chl a and b concentration, total phenolic content, antioxidant capacity and the concentrations of soluble sugars. Obtained results indicated that UV-B radiation exerts an adverse effect on lettuce seedlings, and some of the negative effects of UV-B radiation can be alleviated by exogenous ALA.     

Use of the atLEAF+ chlorophyll meter for a nondestructive estimate of chlorophyll content

At present, chlorophyll meters are widely used for a quick and nondestructive estimate of chlorophyll (Chl) contents in plant leaves. Chl meters allow to estimate the Chl content in relative units - the Chl index (CI). However, using such meters, one can face a problem of converting CI into absolute values of the pigment content and comparing data acquired with different devices and for different plant species. Many Chl meters (SPAD-502, CL-01, CCM-200) demonstrated a high degree of correlation between the CI and the absolute pigment content. A number of formulas have been deduced for different plant species to convert the CI into the absolute value of the photosynthetic pigment content. However, such data have not been yet acquired for the atLEAF+ Chl meter. The purpose of the present study was to assess the applicability of the atLEAF+ Chl meter for estimating the Chl content. A significant species-specific exponential relationships between the atLEAF value (corresponding to CI) and extractable Chl a, Chl b, Chl (a+b) for Calamus dioicus and Cleistanthus sp. were shown. The correlations between the atLEAF values and the content of Chl a, Chl b, and Chl (a+b) per unit of leaf area was stronger than that per unit of dry leaf mass. The atLEAF value- Chl b correlation was weaker than that of atLEAF value-Chl a and atLEAF value-Chl (a+b) correlations. The influence of light conditions (Chl a/b ratio) on the atLEAF value has been also shown. The obtained results indicated that the atLEAF+ Chl meter is a cheap and convenient tool for a quick nondestructive estimate of the Chl content, if properly calibrated, and can be used for this purpose along with other Chl meters.     

Changes in photosynthesis of alpine plant <Emphasis Type="Italic">Saussurea superba</Emphasis> during leaf expansion

The native alpine plant Saussurea superba is widely distributed in Qinghai–Tibetan Plateau regions. The leaves of S. superba grow in whorled rosettes, and are horizontally oriented to maximize sunlight exposure. Experiments were conducted in an alpine Kobresia humilis meadow near Haibei Alpine Meadow Ecosystem Research Station (37°29′–37°45′N, 101°12′–101°33′E; alt. 3200 m). Leaf growth, photosynthetic pigments and chlorophyll fluorescence parameters were measured in expanding leaves of S. superba. The results indicate that leaf area increased progressively from inner younger leaves to outside fully expanded ones, and then slightly decreased in nearly senescent leaves, due to early unfavorable environmental conditions, deviating from the ordinary growth pattern. The specific leaf area decreased before leaves were fully expanded, and the leaf thickness was largest in mature leaves. There were no significant changes in the content of chlorophylls (Chl) and carotenoids (Car), but the ratios of Chl a/b and Car/Chl declined after full expansion of the leaves. The variation of Chl a/b coincided well with changes in photochemical quenching (q _P) and the fraction of open PSII reaction centers (q _L). The maximum quantum efficiency of PSII photochemistry after 5 min dark relaxation (F _(v)/F _(m)) continuously increased from younger leaves to fully mature leaves, suggesting that mature leaves could recover more quickly from photoinhibition than younger leaves. The light-harvesting capacity was relatively steady during leaf expansion, as indicated by the maximum quantum efficiency of open PSII centers (\(F_{\text{v}}^{{\prime }}\)/\(F_{\text{m}}^{{\prime }}\)). UV-absorbing compounds could effectively screen harmful solar radiation, and are a main protection way on the photosynthetic apparatus. The decline of q _P and q _L during maturation, together with limitation of quantum efficiency of PSII reaction centers (L _(PFD)), shows a decrease of oxidation state of Q_A in PSII reaction centers under natural sunlight. Furthermore, light-induced (Φ _NPQ) and non-light-induced quenching (Φ _NO) were consistent with variation of L _(PFD). It is concluded that the leaves of S. superba could be classified into four functional groups: young, fully expanded, mature, and senescent. Quick recovery from photoinhibition was correlated with protection by screening pigments, and high level of light energy trapping was correlated with preservation of photosynthetic pigments. Increasing of Φ _NPQ and Φ _NO during leaves maturation indicates that both thermal dissipation of excessive excitation energy in safety and potential threat to photosynthetic apparatus were strengthened due to the declination of q _P and q _L, and enhancement of L _(PFD).     

Comparative analyses of physiological assays and chlorophyll <Emphasis Type="Italic">a</Emphasis> variable fluorescence parameters: investigating the importance of phosphorus availability in oligotrophic and eutrophic freshwater systems

Diverse measurements of nutrient status indicators were used to test the severity of physiological phosphorus (P) limitation of phytoplankton among lake systems ranging from oligotrophic to eutrophic, based on P and chlorophyll a (Chl a) concentrations. Metabolic assays and particulate nutrient ratios were used to estimate nutrient status at sites located in Lake Erie, Lake Ontario and Lake Huron. Variable fluorescence ratios (F _v/F _m), relative electron transport rates and their response to irradiance were measured by the pulse-amplitude-modulated fluorometer. Under summer stratified conditions, P deficiency was strongest in the oligotrophic sites and nitrogen (N) status indicators and Chl a variable parameters revealed no severe N deficiency. Nutrient amendment assays showed positive associations with P additions and Chl a fluorescence parameters at P-deficient sites. In the most oligotrophic sites, N additions revealed a modest increase only detected by the Chl a fluorescence parameters. Phytoplankton communities were also associated with nutrient status, where chrysophytes and cryptophytes were important in P-deficient sites and cyanobacteria, phyrrophyta, and diatoms were prevalent in nutrient-rich sites. The results confirmed that Chl a fluorescence parameters can reveal P deficiency and indicate its severity among the range of trophic status in aquatic systems.     

The identification of IsiA proteins binding chlorophyll <Emphasis Type="Italic">d</Emphasis> in the cyanobacterium <Emphasis Type="Italic">Acaryochloris marina</Emphasis>

The bioavailable iron in many aquatic ecosystems is extremely low, and limits the growth and photosynthetic activity of phytoplankton. In response to iron limitation, a group of chlorophyll-binding proteins known as iron stress-induced proteins are induced and serve as accessory light-harvesting components for photosystems under iron limitation. In the present study, we investigated physiological features of Acaryochloris marina in response to iron-deficient conditions. The growth doubling time under iron-deficient conditions was prolonged to ~3.4 days compared with 1.9 days under normal culture conditions, accompanied with dramatically decreased chlorophyll content. The isolation of chlorophyll-binding protein complexes using sucrose density gradient centrifugation shows six main green bands and three main fluorescence components of 712, 728, and 748 nm from the iron-deficient culture. The fluorescence components of 712 and 728 nm co-exist in the samples collected from iron-deficient and iron-replete cultures and are attributed to Chl d-binding accessory chlorophyll-binding antenna proteins and also from photosystem II. A new chlorophyll-binding protein complex with its main fluorescence peak at 748 nm was observed and enriched in the heaviest fraction from the samples collected from the iron-deficient culture only. Combining western blotting analysis using antibodies of CP47 (PSII), PsaC (PSI) and IsiA and proteomic analysis on an excised protein band at ~37 kDa, the heaviest fraction (?F6) isolated from iron-deficient culture contained Chl d-bound PSI–IsiA supercomplexes. The PSII-antenna supercomplexes isolated from iron-replete conditions showed two fluorescence peaks of 712 and 728 nm, which can be assigned as 6-transmembrane helix chlorophyll-binding antenna and photosystem II fluorescence, respectively, which is supported by protein analysis of the fractions (F5 and F6).     

Effects of light intensity on leaf photosynthetic characteristics,chloroplast structure,and alkaloid content of <Emphasis Type="Italic">Mahonia bodinieri</Emphasis> (Gagnep.) Laferr.

The variation of light intensity has obvious effects on leaf external morphology, internal anatomy, and physiological characteristics; it even induces changes in secondary metabolite production. The effects of different irradiance levels on biomass, gas exchange parameters, and photosynthetic pigment contents in Mahonia bodinieri (Gagnep.) Laferr. were analyzed here. Combined analyses of physiology, cytology, and HPLC were used to study the differences in leaf morphology, structure, physiological characters, and alkaloid content in response to different irradiances. The results indicated that the highest foliar biomass was observed under I ₅₀ (50 % of full sunlight) followed by I ₃₀ (30 % of full sunlight), the highest net photosynthetic rate, stomatal conductance, transpiration rate values were observed under I ₃₀ followed by I ₅₀, and lower values occurred in I ₁₀ (10 % of full sunlight) and I ₁₀₀ (full sunlight). With increased light intensity, total leaf area and the contents of chlorophyll a (Chl a), chlorophyll b (Chl b), and chlorophyll (Chl a+b) per unit leaf area were clearly reduced, whereas leaf mass per area, carotenoid content, leaf thickness, thickness of palisade and spongy parenchyma, and stomatal density were all significantly increased. Electron microscopic observation revealed that the number of grana, stroma lamellae and the number of starch grains in chloroplasts were decreased, the number of plastoglobuli was increased when irradiance levels increased. The estimated total yield of alkaloids in a single plant was higher under I ₃₀ and I ₅₀ than under I ₁₀ or I ₁₀₀ as a result of the higher biomass of the plants. Therefore, I ₃₀ and I ₅₀ were not only beneficial to increase biomass, but also suitable for the synthesis and accumulation of the major secondary metabolites (alkaloids). Our findings provide valuable data for the determination and regulation of irradiance levels during artificial cultivation of M. bodinieri.     

Cloning of gonadal aromatase gene <Emphasis Type="Italic">Cyp19a</Emphasis> in an endemic fish (<Emphasis Type="Italic">Schizothorax kozlovi</Emphasis>) of the Upper Yangtze River,and temperature effects on its expression

In order to elucidate the role of Cyp19a in sex differentiation of Schizothorax kozlovi, the full length cDNA of Cyp19a was cloned from the mature ovary of S. kozlovi by using rapid amplification of cDNA ends method, and then its relative mRNA expression levels among tissues and temperature groups were determined by using quantitative real-time PCR. The complete Cyp19a cDNA of 1795 bp of S. kozlovi was obtained, which encoded 517 amino acids and belonged to gonadal aromatase. Its deduced amino acid sequence had the above 70 % identity compared with gonadal aromatase genes of teleost fishes, but only 62–67 % when compared with brain aromatase genes of fishes. It was expressed only in heart and gonad, but no expression in other tissues, presenting relatively high tissue specificity. It also exhibited sex-specific expression pattern in gonads, but no sex differences in heart. Comparing with the Cyp19a expression levels at 12 days post hatching (dph), significant temperature effects were revealed in low temperature group (10 °C) at 18 dph, and in high temperature group (26 °C) at 40 dph. It suggested that gonadal aromatase Cyp19a gene may play important roles on the feminization or masculinization of S. kozlovi affected by temperature during the early developmental stage.     

A morphophysiological analysis of the effects of drought and shade on <Emphasis Type="Italic">Catalpa bungei</Emphasis> plantlets

The interactive effects of shade and drought on the morphological and physiological traits of Catalpa bungei plantlets were assessed. Seedling growth, biomass, biomass allocation, leaf morphology, chlorophyll (Chl) content and gas-exchange parameters were measured in plants raised for 3 months under three light levels [80% (HI), 50% (MI), 30% (LI)] and two water levels [moisture (M) and drought (D)]. The results showed that shade greatly decreased growth, biomass, leaf area (LA) and Chl a/b; increased specific leaf area (SLA) and Chl content; and reduced photosynthetic rate (P _n). Drought reduced the growth, biomass, LA, SLA, Chl a/b, P _n, stomatal conductance (G _s), transpiration rate (T _r) and intercellular carbon dioxide concentration (C _i) and increased the Chl content. Stomatal closure was an early physiological response to water stress. Light, water and their interaction significantly affected plant traits and their bivariate relationships. The phenotypic plasticity index of light (0.47) was much higher than that of water (0.21), indicating that light was the main driver of the variations observed. Under drought stress, growth, biomass, leaf and stem biomass allocation significantly decreased in the HI and MI environments, whereas no significant difference was observed in growth or biomass parameters under the LI condition. Furthermore, no significant difference was observed in P _n, G _s, or T _r under the LI condition under water stress. Our results showed that shade did not alter the negative effects caused by drought stress in MI but did alleviate the negative effects of the LI condition. In summary, the effect of drought on C. bungei plantlets depends on the irradiance conditions.