Excitation transfer and quenching in photosystem II, enlightened by carotenoid triplet state in leaves.

Accumulation of carotenoid (Car) triplet states was investigated by singlet-triplet annihilation, measured as chlorophyll (Chl) fluorescence quenching in sunflower and lettuce leaves. The leaves were illuminated by Xe flashes of 4 µs length at half-height and 525-565 or 410-490 nm spectral band, maximum intensity 2 mol quanta m-2 s-1, flash photon dose up to 10 µmol m-2 or 4-10 PSII excitations. Superimposed upon the non-photochemically unquenched Fmd state, fluorescence was strongly quenched near the flash maximum (minimum yield Fe), but returned to the Fmd level after 30-50 µs. The fraction of PSII containing a 3Car in equilibrium with singlet excitation was calculated as Te = (Fmd-Fe)/Fmd. Light dependence of Te was a rectangular hyperbola, whose initial slope and plateau were determined by the quantum yields of triplet formation and annihilation and by the triplet lifetime. The intrinsic lifetime was 9 µs, but it was strongly shortened by the presence of O2. The triplet yield was 0.66 without nonphotochemical quenching (NPQ) but approached zero when NP-Quenched fluorescence approached 0.2 Fmd. The results show that in the Fmd state a light-adapted charge-separated PSIIL state is formed (Sipka et al., The Plant Cell 33:1286-1302, 2021) in which Pheo-P680+ radical pair formation is hindered, and excitation is terminated in the antenna by 3Car formation. The results confirm that there is no excitonic connectivity between PSII units. In the PSIIL state each PSII is individually turned into the NPQ state, where excess excitation is quenched in the antenna without 3Car formation.

Evidence for a Role for NAD(P)H Dehydrogenase in Concentration of CO2 in the Bundle Sheath Cell of Zea mays.

Prior studies with Nicotiana and Arabidopsis described failed assembly of the chloroplastic NDH [NAD(P)H dehydrogenase] supercomplex by serial mutation of several subunit genes. We examined the properties of Zea mays leaves containing Mu and Ds insertions into nuclear gene exons encoding the critical o- and n-subunits of NDH, respectively. In vivo reduction of plastoquinone in the dark was sharply diminished in maize homozygous mutant compared to normal leaves but not to the extreme degree observed for the corresponding lesions in Arabidopsis. The net carbon assimilation rate (A) at high irradiance and saturating CO2 levels was reduced by one-half due to NDH mutation in maize although no genotypic effect was evident at very low CO2 levels. Simultaneous assessment of chlorophyll fluorescence and A in maize at low (2% by volume) and high (21%) O2 levels indicated the presence of a small, yet detectable, O2-dependent component of total linear photosynthetic electron transport in 21% O2 This O2-dependent component decreased with increasing CO2 level indicative of photorespiration. Photorespiration was generally elevated in maize mutant compared to normal leaves. Quantification of the proportion of total electron transport supporting photorespiration enabled estimation of the bundle sheath cell CO2 concentration (Cb) using a simple kinetic model of ribulose bisphosphate carboxylase/oxygenase function. The A versus Cb relationships overlapped for normal and mutant lines consistent with occurrence of strictly CO2-limited photosynthesis in the mutant bundle sheath cell. The results are discussed in terms of a previously reported CO2 concentration model [Laisk A, Edwards GE (2000) Photosynth Res 66: 199-224].

Fluorescence F 0 of photosystems II and I in developing C3 and C 4 leaves, and implications on regulation of excitation balance.

This work addresses the question of occurrence and function of photosystem II (PSII) in bundle sheath (BS) cells of leaves possessing NADP-malic enzyme-type C4 photosynthesis (Zea mays). Although no requirement for PSII activity in the BS has been established, several component proteins of PSII have been detected in BS cells of developing maize leaves exhibiting O2-insensitive photosynthesis. We used the basal fluorescence emissions of PSI (F 0I) and PSII (F 0II) as quantitative indicators of the respective relative photosystem densities. Chl fluorescence induction was measured simultaneously at 680 and 750 nm. In mature leaves, the F m(680)/F 0(680) ratio was 10.5 but less in immature leaves. We propose that the lower ratio was caused by the presence of a distinct non-variable component, F c, emitting at 680 and 750 nm. After F c was subtracted, the fluorescence of PSI (F 0I) was detected as a non-variable component at 750 nm and was undetectably low at 680 nm. Contents of Chls a and b were measured in addition to Chl fluorescence. The Chl b/(a + b) was relatively stable in developing sunflower leaves (0.25-0.26), but in maize it increased from 0.09 to 0.21 with leaf tissue age. In sunflower, the F 0I/(F 0I + F 0II) was 0.39 ± 0.01 independent of leaf age, but in maize, this parameter was 0.65 in young tissue of very low Chl content (20-50 mg m(-2)) falling to a stable level of 0.53 ± 0.01 at Chl contents >100 mg m(-2). The values of F 0I/(F 0I + F 0II) showed that in sunflower, excitation was partitioned between PSII and PSI in a ratio of 2:1, but the same ratio was 1:1 in the C4 plant. The latter is consistent with a PSII:PSI ratio of 2:1 in maize mesophyll cells and PSI only in BS cells (2:1:1 distribution). We suggest, moreover, that redox mediation of Chl synthesis, rather than protein accumulation, regulates photosystem assembly to ensure optimum excitation balance between functional PSII and PSI. Indeed, the apparent necessity for two Chls (a and b) may reside in their targeted functions in influencing accumulation of PSI and PSII, respectively, as opposed to their spectral differences.

Light-harvesting complex B7 shifts the irradiance response of photosynthetic light-harvesting regulation in leaves of Arabidopsis thaliana.

The nuclear LHCB7 gene is common in higher plants, encodes a transcript that is well expressed in a subset of leaf mesophyll cells, and is associated with a protein product that is homologous to pigment-binding components of the photosystem (PS) II peripheral antenna complex. We compared the physiological properties of wild type and LHCB7-deficient leaves [DNA insertion, Arabidopsis thaliana (At) ecotype Columbia] in terms of pigment content, CO2 exchange, in vivo transmittance at 810 nm, and chlorophyll fluorescence. The latter two techniques are functional indicators for PSI and PSII, respectively. Key features of the mutant phenotype were confirmed using antisense technology and a hemizygote of two independent AtLHCB7 DNA insertion lines. Growth, leaf pigment composition, white light absorptance, and levels of AtLHCB1-6 were not significantly different in the mutant compared to wild type. Likewise, neither intrinsic PSII light capture efficiency nor partitioning of absorbed radiation to PSII was affected by the mutation. The absence of AtLHCB7 is associated with lower rates of light-saturated photosynthesis and a diminished irradiance threshold for induction of photoprotective non-photochemical quenching. Overall, the pattern of change in light utilization parameters and plastoquinol level indicated that loss of AtLHCB7 expression led to slower Rubisco turnover characterized by pH-dependent balancing of electron transport to reduced carbon assimilation capacity (photosynthetic control). No effect of AtLHCB7 genotype on xanthophyll de-epoxidation state was detected suggesting that factors in addition to lumenal pH influence zeaxanthin accumulation.

Regulatory mechanisms underlying C4 photosynthesis.

C4 photosynthesis is an adaptation that evolved to alleviate the detrimental effects of photorespiration as a result of the gradual decline in atmospheric carbon dioxide levels. In most C4 plants, two cell types, bundle sheath and mesophyll, cooperate in carbon fixation, and, in so doing, are able to alleviate photorespiratory losses. Although much of the biochemistry is well characterized, little is known about the genetic mechanisms underlying the cell-type specificity driving C4 . However, several studies have shown that regulation acts at multiple levels, including transcriptional, post-transcriptional, post-translational and epigenetic. One example of such a regulatory mechanism is the cell-specific accumulation of major photorespiratory transcripts/proteins in bundle sheath cells, where ribulose-1,5-bisphosphate carboxylase/oxygenase is localized. Although many of the genes are expressed in the bundle sheath, some are expressed in both cell types, implicating post-transcriptional control mechanisms. Recently, ultra-high-throughput sequencing techniques and sophisticated mass spectrometry instrumentation have provided new opportunities to further our understanding of C4 regulation. Computational pipelines are being developed to accommodate the mass of data associated with these techniques. Finally, we discuss a readily transformable C4 grass--Setaria viridis--that has great potential to serve as a model for the genetic dissection of C4 photosynthesis in the grasses.

Conservation without borders: building communication and action across disciplinary boundaries for effective conservation.

Interdisciplinary approaches to conservation research and environmental management continue to garner interest among practitioners, academics, and students. Yet, cases of practitioners and researchers from different disciplines successfully working in concert towards an integrated conservation approach are rare. What is preventing practitioners of multiple disciplines from harmoniously working together? Why are practitioners and academics struggling to apply their graduate training to real world conservation? What is preventing the benefits of cooperation and partnerships between different disciplines addressing conservation from being realized? This special issue "Conservation without Borders: Building Communication and Action across Disciplinary Boundaries for Effective Conservation" asks readers to consider the numerous interpretations and implications of the phrase "Conservation without Borders" and to reflect on how different academic and disciplinary lenses can contribute to a more integrated approach to tackling conservation challenges. The articles that comprise this special issue offer readers insights into the ways in which different disciplines view conservation work and interdisciplinary approaches to environmental problems. Bringing these perspectives and approaches together in one place is a step towards improving communication across disciplines for the purpose of achieving more successful biodiversity conservation.

Fast cyclic electron transport around photosystem I in leaves under far-red light: a proton-uncoupled pathway?

Fast cyclic electron transport (CET) around photosystem I (PS I) was observed in sunflower (Helianthus annuus L.) leaves under intense far-red light (FRL) of up to 200 mumol quanta m(-2) s(-1). The electron transport rate (ETR) through PS I was found from the FRL-dark transmittance change at 810 and 950 nm, which was deconvoluted into redox states and pool sizes of P700, plastocyanin (PC) and cytochrome f (Cyt f). PC and P700 were in redox equilibrium with K(e) = 35 (ΔE(m) = 90 mV). PS II ETR was based on O(2) evolution. CET [(PS I ETR) - (PS II ETR)] increased to 50-70 mumol e(-) m(-2) s(-1) when linear electron transport (LET) under FRL was limited to 5 mumol e(-) m(-2) s(-1) in a gas phase containing 20-40 mumol CO(2) mol(-1) and 20 mumol O(2) mol(-1). Under these conditions, pulse-saturated fluorescence yield F(m) was non-photochemically quenched; however, F(m) was similarly quenched when LET was driven by low green or white light, which energetically precluded the possibility for active CET. We suggest that under FRL, CET is rather not coupled to transmembrane proton translocation than the CET-coupled protons are short-circuited via proton channels regulated to open at high ΔpH. A kinetic analysis of CET electron donors and acceptors suggests the CET pathway is that of the reversed Q-cycle: Fd -> (FNR) -> Cyt c(n) -> Cyt b(h) -> Cyt b(l) -> Rieske FeS -> Cyt f -> PC -> P700 ->-> Fd. CET is activated when PQH(2) oxidation is opposed by high ΔpH, and ferredoxin (Fd) is reduced due to low availability of e(-) acceptors. The physiological significance of CET may be photoprotective, as CET may be regarded as a mechanism of energy dissipation under stress conditions.

Seeing (and doing) conservation through cultural lenses.

In this paper, we first discuss various vantage points gained through the authors' experience of approaching conservation through a "cultural lens." We then draw out more general concerns that many anthropologists hold with respect to conservation, summarizing and commenting on the work of the Conservation and Community Working Group within the Anthropology and Environment Section of the American Anthropological Association. Here we focus on both critiques and contributions the discipline of anthropology makes with regard to conservation, and show how anthropologists are moving beyond conservation critiques to engage actively with conservation practice and policy. We conclude with reflections on the possibilities for enhancing transdisciplinary dialogue and practice through reflexive questioning, the adoption of disciplinary humility, and the realization that "cross-border" collaboration among conservation scholars and practitioners can strengthen the political will necessary to stem the growing commoditization and ensuing degradation of the earth's ecosystems.

High glycolate oxidase activity is required for survival of maize in normal air.

A mutant in the maize (Zea mays) Glycolate Oxidase1 (GO1) gene was characterized to investigate the role of photorespiration in C4 photosynthesis. An Activator-induced allele of GO1 conditioned a seedling lethal phenotype when homozygous and had 5% to 10% of wild-type GO activity. Growth of seedlings in high CO2 (1%-5%) was sufficient to rescue the mutant phenotype. Upon transfer to normal air, the go1 mutant became necrotic within 7 d and plants died within 15 d. Providing [1-14C]glycolate to leaf tissue of go1 mutants in darkness confirmed that the substrate is inefficiently converted to 14CO2, but both wild-type and GO-deficient mutant seedlings metabolized [1-14C]glycine similarly to produce [14C]serine and 14CO2 in a 1:1 ratio, suggesting that the photorespiratory pathway is otherwise normal in the mutant. The net CO2 assimilation rate in wild-type leaves was only slightly inhibited in 50% O2 in high light but decreased rapidly and linearly with time in leaves with low GO. When go1 mutants were shifted from high CO2 to air in light, they accumulated glycolate linearly for 6 h to levels 7-fold higher than wild type and 11-fold higher after 25 h. These studies show that C4 photosynthesis in maize is dependent on photorespiration throughout seedling development and support the view that the carbon oxidation pathway evolved to prevent accumulation of toxic glycolate.

Phylogeny-directed structural analysis of the Arabidopsis PsbS protein.

Plant psbS proteins are essential for regulated thermal dissipation of excess light referred to as non-photochemical quenching of chlorophyll fluorescence yield (NPQ). Amino acid sequences derived from 65 psbS genes from 44 species were aligned to reveal extensive conservation consistent with of motifs that underlie intrinsic aspects of the NPQ mechanism. Site-directed mutagenesis was employed to block presumptive zeaxanthin or chlorophyll-binding sites in Arabidopsis psbS by disrupting ion-bonding between two pairs of non-adjacent glutamate and arginine residues. Transgenic Arabidopsis lines synthesizing only the altered psbS forms exhibited severely impaired NPQ capacity. In addition, the phylogenetic depth of the psbS database permitted identification of cryptic sites of adaptive evolution. Instances of localized positive selection were rare and largely limited to the family Poaceae (grasses). Specifically, adaptive evolution was detected in a hydrophilic stroma-exposed region and was correlated with the presence of the C4 pathway of carbon fixation.

Laser triangulation for liquid film thickness measurements through multiple interfaces.

Laser triangulation is used to measure the thickness of a liquid film in a test section consisting of a quartz viewing window, a water layer, and a hydrophobic membrane. The triangulation sensor acquires measurements to the bounding surfaces of the film while peering through multiple interfaces. This allows the difference between the two measurements to constitute the local film thickness. A refraction model is developed and applied to the analysis of data collected from the experiment. For verification, an empirical method is also developed and compared to the analytical approach. The measurement technique is intended to assess the stability of liquid films for use as gas-liquid contactors.

PsbS genotype in relation to coordinated function of PS II and PS I in Arabidopsis leaves.

Application of multiple probes to systems that carry specific mutations provides a powerful means for studying how known regulators of light utilization interact in vivo. Two lines of Arabidopsis thaliana were studied, each carrying a unique lesion in the nuclear psbS gene encoding a 22-kDa pigment-binding protein (PS II-S) essential for full expression of photoprotective, rapid-phase, nonphotochemical quenching of chlorophyll fluorescence (NPQ). The PS II-S protein is absent in line npq4-1 due to deletion of psbS. Line npq4-9 expresses normal levels of PS II-S but carries a single amino acid substitution that lowers NPQ capacity by about 50%. A prior report [Peterson RB and Havir EA (2001) Planta 214: 142-152] described an altered pattern of redox states of the acceptor side of Photosystem II (PS II) and donor side of Photosystem I (PS I) for npq4-9 suggesting that interphotosystem electron transport may be restricted by a higher transthylakoid DeltapH in this line. In vivo steady state fluorescence and absorbance measurements (820 nm) confirmed these earlier observations for line npq4-9 but not for npq4-1. Thus, the prior results cannot be correlated simply to a loss of NPQ capacity. Likewise, the kinetics of the 820-nm absorbance change did not indicate a substantial effect of psbS genotype on electron flow from plastoquinol to PS I. A simple model is proposed to relate linear electron transport rate (measured gasometrically) to a parameter (based on fluorescence) that provides a relative measure of the density of excitation available for photochemistry in PS II. Surprisingly, analyses using this model suggested that the in vivo midpoint potential of the primary quinone acceptor in PS II (Q(A)) is lowered in both psbS mutant lines. This heretofore-unsuspected role for PS II-S is discussed with regard to: (1) numerous prior reports indicating plasticity of the redox potential of Q(A) and (2) the basis for the contrasting regulation of quantum yields of PS I and II in npq4-1 and npq4-9.

Control of cytochrome b6f at low and high light intensity and cyclic electron transport in leaves.

The light-dependent control of photosynthetic electron transport from plastoquinol (PQH(2)) through the cytochrome b(6)f complex (Cyt b(6)f) to plastocyanin (PC) and P700 (the donor pigment of Photosystem I, PSI) was investigated in laboratory-grown Helianthus annuus L., Nicotiana tabaccum L., and naturally-grown Solidago virgaurea L., Betula pendula Roth, and Tilia cordata P. Mill. leaves. Steady-state illumination was interrupted (light-dark transient) or a high-intensity 10 ms light pulse was applied to reduce PQ and oxidise PC and P700 (pulse-dark transient) and the following re-reduction of P700(+) and PC(+) was recorded as leaf transmission measured differentially at 810-950 nm. The signal was deconvoluted into PC(+) and P700(+) components by oxidative (far-red) titration (V. Oja et al., Photosynth. Res. 78 (2003) 1-15) and the PSI density was determined by reductive titration using single-turnover flashes (V. Oja et al., Biochim. Biophys. Acta 1658 (2004) 225-234). These innovations allowed the definition of the full light response curves of electron transport rate through Cyt b(6)f to the PSI donors. A significant down-regulation of Cyt b(6)f maximum turnover rate was discovered at low light intensities, which relaxed at medium light intensities, and strengthened again at saturating irradiances. We explain the low-light regulation of Cyt b(6)f in terms of inactivation of carbon reduction cycle enzymes which increases flux resistance. Cyclic electron transport around PSI was measured as the difference between PSI electron transport (determined from the light-dark transient) and PSII electron transport determined from chlorophyll fluorescence. Cyclic e(-) transport was not detected at limiting light intensities. At saturating light the cyclic electron transport was present in some, but not all, leaves. We explain variations in the magnitude of cyclic electron flow around PSI as resulting from the variable rate of non-photosynthetic ATP-consuming processes in the chloroplast, not as a principle process that corrects imbalances in ATP/NADPH stoichiometry during photosynthesis.

The multiphasic nature of nonphotochemical quenching: implications for assessment of photosynthetic electron transport based on chlorophyll fluorescence.

Defining a quantitative relationship between chlorophyll a fluorescence yield and Photosystem II (PS II) function is important to photosynthesis research. Prior work [Peterson and Havir (2003) Photosynth Res 75: 57-70] indicated an apparent effect of psbS genotype on the in vivo rate constant for photochemistry in PS II (k(P0)). The nuclear psbS gene encodes a 22-kDa pigment-binding antenna protein (PS II-S) essential for photoprotective nonphotochemical quenching (NPQ) in PS II. Ten Arabidopsis thaliana lines were chosen for study, encompassing effects on PS II-S expression level and/or structure due to single-site amino acid substitution. Short-term (i.e. seconds) irradiance-dependent changes in steady state fluorescence yields F(o) and F(m)(open and closed centers, respectively) were evaluated for compliance with the reversible radical pair (RRP) model of PS II. All lines (including normal Nicotiana tabacum and Zea mays) deviated from the RRP scheme in the same way indicating that psbS genotype per se does not alter interactions between the antenna and reaction center and thereby affect k(P0). Rather, observed departures from RRP model behavior are consistent with overestimation of F(m) due to perturbing effects of the saturating multiple turnover flash employed in its measurement. Reversal of direct quenching of singlet states by plastoquinone during the flash could occur but by itself cannot account for the anomalous covariation in F(o) and F(m). Reduction of the PS II acceptor side apparently either amplifies the rate constant for fluorescence or suppresses that of xanthophyll-dependent thermal deactivation (q(E)). A procedure was devised that considers F(o) when correcting maximal fluorescence values for measurement bias. A high degree of consistency in assessment of PS II quantum yield based on corrected fluorescence parameters and simultaneous CO(2) exchange measurements was noted under both steady state and transient conditions (360 mul CO(2)l(-1), 1% O(2)).

Deciphering the 820 nm signal: redox state of donor side and quantum yield of Photosystem I in leaves.

By recording leaf transmittance at 820 nm and quantifying the photon flux density of far red light (FRL) absorbed by long-wavelength chlorophylls of Photosystem I (PS I), the oxidation kinetics of electron carriers on the PS I donor side was mathematically analyzed in sunflower (Helianthus annuus L.), tobacco (Nicotiana tabacum L.) and birch (Betula pendula Roth.) leaves. PS I donor side carriers were first oxidized under FRL, electrons were then allowed to accumulate on the PS I donor side during dark intervals of increasing length. After each dark interval the electrons were removed (titrated) by FRL. The kinetics of the 820 nm signal during the oxidation of the PS I donor side was modeled assuming redox equilibrium among the PS I donor pigment (P700), plastocyanin (PC), and cytochrome f plus Rieske FeS (Cyt f + FeS) pools, considering that the 820 nm signal originates from P700(+) and PC(+). The analysis yielded the pool sizes of P700, PC and (Cyt f + FeS) and associated redox equilibrium constants. PS I density varied between 0.6 and 1.4 mumol m(-2). PS II density (measured as O(2) evolution from a saturating single-turnover flash) ranged from 0.64 to 2.14 mumol m(-2). The average electron storage capacity was 1.96 (range 1.25 to 2.4) and 1.16 (range 0.6 to 1.7) for PC and (Cyt f + FeS), respectively, per P700. The best-fit electrochemical midpoint potential differences were 80 mV for the P700/PC and 25 mV for the PC/Cyt f equilibria at 22 degrees C. An algorithm relating the measured 820 nm signal to the redox states of individual PS I donor side electron carriers in leaves is presented. Applying this algorithm to the analysis of steady-state light response curves of net CO(2) fixation rate and 820 nm signal shows that the quantum yield of PS I decreases by about half due to acceptor side reduction at limiting light intensities before the donor side becomes oxidized at saturating intensities. Footnote:

Contrasting modes of regulation of PS II light utilization with changing irradiance in normal and psbS mutant leaves of Arabidopsis thaliana.

Complementary techniques of chlorophyll a fluorescence, steady state CO(2) exchange, and O(2) release during a multiple turnover flash were applied to compare responses to irradiance for leaves of wild type and psbS mutants. The latter included variants in which the psbS gene was deleted (npq4-1) or possessed a single point mutation (npq4-9). Nonphotochemical quenching (NPQ) was reduced by up to 80 and 50%, respectively, in these lines at high irradiance. Analysis of changes in steady-state fluorescence yields and quantum yield of linear electron transport in the context of the reversible radical pair model of Photosystem II (PS II) indicated that NPQ occurs by nonradiative deactivation of chlorophyll singlet states in normal leaves. In contrast, application of the same criteria together with the observed irreversibility of NPQ and decline in density of functional PS II reaction centers following excessive illumination indicated a change in reaction center properties for the psbS deletion phenotype (Npq4-1(-)). Specifically, PS II reaction centers in Npq4-1(-) convert to a photochemically inactive, yet strongly quenching, form in intense light. The possibility of formation of a carotenoid or chlorophyll cation quencher in the reaction center is discussed. Results for the point mutant phenotype (Npq4-9(-)) were intermediate to those of wild-type and Npq4-1(-). Furthermore, wild-type leaves exhibited a significant reversible increase in the PS II in vivo rate constant for photochemistry (k(P0)) in saturating compared to limiting light. Changes in k(P0) could not be accounted for in terms of a classic phosphorylation-dependent (state transition) mechanism. Changes in k(P0) may arise from alternate pigment-protein conformations that alter the way excitons equilibrate among PS II chromophores. The lack of similar irradiance-dependent changes in k(P0) for the psbS mutants suggests a role for the PS II-S protein in the regulation of exciton distribution.

Use of Temperature-Sensitive Gel for the Concentration of Escherichia Coli in Milk.

Temperature-sensitive gels (cross-linked, partially hydrolyzed polyacrylamide gels) with the property to absorb solutes less than 1 nm in diameter at 4°C and collapse at higher temperatures (190°C) were used to concentrate Escherichia coli from 2% low-fat milk. Milk samples seeded with bacteria were reduced eight-fold in volume with average recoveries of 40-66% at an optimal pH of 5.5. Repeated use of the gel did not affect its efficiency.