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P Horton

Publications and source records attributed to P Horton.

At least 19 recordsLinked to original sources
PubMed2001

Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. 1. Isolated chloroplasts.

Nonphotochemical quenching of chlorophyll fluorescence in plants is indicative of a process that dissipates excess excitation energy from the light-harvesting antenna of photosystem II. The major fraction of quenching is obligatorily dependent upon the thylakoid DeltapH and is regulated by the de-epoxidation state of the xanthophyll cycle carotenoids associated with the light-harvesting complexes. Basic principles of enzyme kinetics have been used to investigate this process in isolated chloroplasts. The extent of quenching was titrated against the estimated thylakoid lumen pH, and a sigmoidal relationship was obtained with a Hill coefficient of 4.5 and a pK of 4.7. Upon de-epoxidation, these parameters changed to 1.6 and 5.7, respectively. Antimycin A suppressed quenching, increasing the Hill coefficient and reducing the pK. The rate of induction of quenching fitted second-order kinetics with respect to illumination time, and the rate constant was dependent upon the DeltapH, the de-epoxidation state, the presence of antimycin, and also the presence of dibucaine, a quenching enhancer. All these data are consistent with the notion that quenching is caused by a conformational transition in a chloroplast thylakoid protein; this transition shows cooperativity with respect to proton binding, and is controlled by de-epoxidation state and various exogenous reagents.

Chlorophyll↗
PubMed2001

Kinetic analysis of nonphotochemical quenching of chlorophyll fluorescence. 2. Isolated light-harvesting complexes.

The chlorophyll fluorescence yield of purified photosystem II light-harvesting complexes can be lowered by manipulation of experimental conditions. In several important respects, this quenching resembles the nonphotochemical quenching observed in isolated chloroplasts and leaves, therefore providing a model system for investigating the underlying mechanism. A methodology based on the principles of enzyme kinetic analysis has already been applied to isolated chloroplasts, and this same experimental approach was used here with purified LHCIIb, CP26, and CP29. It was found that the kinetics of the decrease in fluorescence yield robustly fitted a second-order kinetic model with respect to time after induction of quenching. The second-order rate constant was dependent upon the complex that was analyzed, the detergent concentration, the solution pH, and the presence of exogenous xanthophyll cycle carotenoids. In contrast, the formation of an absorbance change at 683 nm that accompanies quenching displayed first-order kinetics. The reversal of quenching also displayed second-order kinetics. These data show that quenching results from a binary reaction, possibly arising between two chlorophyll molecules. On the basis of these data, a model for the regulation of nonphotochemical quenching based upon the allosteric control of the conformation of light-harvesting complexes by protonation and xanthophyll binding is presented.

Chlorophyll↗
PubMed2001

Configuration and dynamics of xanthophylls in light-harvesting antennae of higher plants. Spectroscopic analysis of isolated light-harvesting complex of photosystem II and thylakoid membranes.

Resonance Raman excitation spectroscopy combined with ultra low temperature absorption spectral analysis of the major xanthophylls of higher plants in isolated antenna and intact thylakoid membranes was used to identify carotenoid absorption regions and study their molecular configuration. The major electronic transitions of the light-harvesting complex of photosystem II (LHCIIb) xanthophylls have been identified for both the monomeric and trimeric states of the complex. One long wavelength state of lutein with a 0-0 transition at 510 nm was detected in LHCIIb trimers. The short wavelength 0-0 transitions of lutein and neoxanthin were located at 495 and 486 nm, respectively. In monomeric LHCIIb, both luteins absorb around 495 nm, but slight differences in their protein environments give rise to a broadening of this band. The resonance Raman spectra of violaxanthin and zeaxanthin in intact thylakoid membranes was determined. The broad 0-0 absorption transition for zeaxanthin was found to be located in the 503-511 nm region. Violaxanthin exhibited heterogeneity, having two populations with one absorbing at 497 nm (0-0), 460 nm (0-1), and 429 nm (0-2), and the other major pool absorbing at 488 nm (0-0), 452 nm (0-1), and 423 nm (0-2). The origin of this heterogeneity is discussed. The configuration of zeaxanthin and violaxanthin in thylakoid membranes was different from that of free pigments, and both xanthophylls (notably, zeaxanthin) were found to be well coordinated within the antenna proteins in vivo, arguing against the possibility of their free diffusion in the membrane and supporting our recent biochemical evidence of their association with intact oligomeric light-harvesting complexes (Ruban, A. V., Lee, P. J., Wentworth, M., Young, A. J., and Horton, P. (1999) J. Biol. Chem. 274, 10458-10465).

Carotenoids↗
PubMed2001

Increasing rice photosynthesis by manipulation of the acclimation and adaptation to light.

There are three important considerations in assessing the interaction of crop plants with light: (a) how does the plant respond to the light environment both in the short-term (regulation) and in the long-term (acclimation), (b) under what conditions are these responses inadequate, leading to photoinhibition, and (c) are the responses optimally adapted for maximum agricultural yield? Despite a wealth of knowledge about these processes in model plant species, it is impossible to predict how significant they are in influencing the yield of rice. Therefore, in collaboration with IRRI, we have undertaken a study of photoinhibition and photoacclimation of rice under field conditions. The results of this study are presented, along with an assessment of the implications for improvement of rice yield.

Adaptation, Physiological↗
PubMed2001

Acclimation of Arabidopsis thaliana to the light environment: the existence of separate low light and high light responses.

The capacity for photosynthetic acclimation in Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta was assessed during growth over a broad range of irradiance. Discontinuities in the response to growth irradiance were revealed for the light- and CO2-saturated rate of photosynthesis (Pmax) and the ratio of chlorophyll a to chlorophyll b (Chl a/b). Three separate phases in the response of Pmax and Chl a/b to growth light were evident, with increases at low and high irradiance ranges and a plateau at intermediate irradiance. By measuring all chlorophyll-containing components of the thylakoid membrane that contribute to Chl a/b we reveal that distinct strategies for growth at low and high irradiance underlie the discontinuous response. These strategies include, in addition to changes in the major light-harvesting complexes of photosystem II (LHCII), large shifts in the amounts of both reaction centres as well as significant changes in the levels of minor LHCII and LHCI components.

Acclimatization↗
PubMed2001

Crystal structure of a bifunctional transformylase and cyclohydrolase enzyme in purine biosynthesis.

ATIC, the product of the purH gene, is a 64 kDa bifunctional enzyme that possesses the final two activities in de novo purine biosynthesis, AICAR transformylase and IMP cyclohydrolase. The crystal structure of avian ATIC has been determined to 1.75 A resolution by the MAD method using a Se-methionine modified enzyme. ATIC forms an intertwined dimer with an extensive interface of approximately 5,000 A(2) per monomer. Each monomer is composed of two novel, separate functional domains. The N-terminal domain (up to residue 199) is responsible for the IMPCH activity, whereas the AICAR Tfase activity resides in the C-terminal domain (200-593). The active sites of the IMPCH and AICAR Tfase domains are approximately 50 A apart, with no structural evidence of a tunnel connecting the two active sites. The crystal structure of ATIC provides a framework to probe both catalytic mechanisms and to design specific inhibitors for use in cancer chemotherapy and inflammation.

Animals↗
PubMed2001

Tsukuba BB: a branch and bound algorithm for local multiple alignment of DNA and protein sequences.

In this paper we present a branch and bound algorithm for local gapless multiple sequence alignment (motif alignment) and its implementation. The algorithm uses both score-based bounding and a novel bounding technique based on the "consistency" of the alignment. A sequence order independent search tree is used in conjunction with a technique for avoiding redundant calculations inherent in the structure of the tree. This is the first program to exploit the fact that the motif alignment problem is easier for short motifs. Indeed, for a short fixed motif width, the running time of the algorithm is asymptotically linear in the size of the input. We tested the performance of the program on a dataset of 300 E. coli promoter sequences and a dataset of 85 lipocalin protein sequences. For a motif width of 4, the optimal alignment of the entire set of sequences can be found. For the more natural motif width of 6, the program can align 21 sequences of length 100, more than twice the number of sequences which can be aligned by the best previous exact algorithm. The algorithm can relax the constraint of requiring each sequence to be aligned, and align 105 of the 300 promoter sequences with a motif width of 6. For the lipocalin dataset, we introduce a technique for reducing the effective alphabet size with a minimal loss of useful information. With this technique, we show that the program can find meaningful motifs in a reasonable amount of time by optimizing the score over three motif positions.

Algorithms↗
PubMed2001

Antisense inhibition of the photosynthetic antenna proteins CP29 and CP26: implications for the mechanism of protective energy dissipation.

The specific roles of the chlorophyll a/b binding proteins CP29 and CP26 in light harvesting and energy dissipation within the photosynthetic apparatus have been investigated. Arabidopsis was transformed with antisense constructs against the genes encoding the CP29 or CP26 apoprotein, which gave rise to several transgenic lines with remarkably low amounts of the antisense target proteins. The decrease in the level of CP24 protein in the CP29 antisense lines indicates a physical interaction between these complexes. Analysis of chlorophyll fluorescence showed that removal of the proteins affected photosystem II function, probably as a result of changes in the organization of the light-harvesting antenna. However, whole plant measurements showed that overall photosynthetic rates were similar to those in the wild type. Both antisense lines were capable of the qE type of nonphotochemical fluorescence quenching, although there were minor changes in the capacity for quenching and in its induction kinetics. High-light-induced violaxanthin deepoxidation to zeaxanthin was not affected, although the pool size of these pigments was decreased slightly. We conclude that CP29 and CP26 are unlikely to be sites for nonphotochemical quenching.

Amino Acid Sequence↗
PubMed2000

Allosteric regulation of the light-harvesting system of photosystem II.

Non-photochemical quenching of chlorophyll fluorescence (NPQ) is symptomatic of the regulation of energy dissipation by the light-harvesting antenna of photosystem II (PS II). The kinetics of NPQ in both leaves and isolated chloroplasts are determined by the transthylakoid delta pH and the de-epoxidation state of the xanthophyll cycle. In order to understand the mechanism and regulation of NPQ we have adopted the approaches commonly used in the study of enzyme-catalysed reactions. Steady-state measurements suggest allosteric regulation of NPQ, involving control by the xanthophyll cycle carotenoids of a protonation-dependent conformational change that transforms the PS II antenna from an unquenched to a quenched state. The features of this model were confirmed using isolated light-harvesting proteins. Analysis of the rate of induction of quenching both in vitro and in vivo indicated a bimolecular second-order reaction; it is suggested that quenching arises from the reaction between two fluorescent domains, possibly within a single protein subunit. A universal model for this transition is presented based on simple thermodynamic principles governing reaction kinetics.

Allosteric Regulation↗
PubMed2000

Pigment binding site properties of two photosystem II antenna proteins. A resonance raman investigation.

Two light-harvesting proteins associated with photosystem II of higher plants, namely the major antenna complex LHCIIb and the minor Lhcb4 protein (CP29), have been investigated by resonance Raman spectroscopy. One of the two chlorophylls b and up to five of the six chlorophylls a present in Lhcb4 are shown to adopt similar binding conformations to the (presumably) corresponding molecules in LHCIIb, whereas at least two chlorophylls in the former protein assume unique conformations relative to the bulk complex. The overall conformation of bound xanthophyll molecules is identical in the two antenna proteins, although some small differences are apparent. The pigment binding properties of these two LHCs are discussed, with particular reference to possible structural motifs within this extended family of proteins.

Binding Sites↗
PubMed2000

Chlorophyll fluorescence quenching in isolated light harvesting complexes induced by zeaxanthin.

Non-photochemical quenching of chlorophyll fluorescence in plants occurs in the light harvesting antenna of photosystem II and is regulated by the xanthophyll cycle. A new in vitro model for this process has been developed. Purified light harvesting complexes above the detergent critical micelle concentration have a stable high fluorescence yield but a rapidly inducible fluorescence quenching occurs upon addition of zeaxanthin. Violaxanthin was without effect, lutein and antheraxanthin induced a marginal response, whereas the violaxanthin analogue, auroxanthin, induced strong quenching. Quenching was not caused by aggregation of the complexes but was accompanied by a spectral broadening and red shift, indicating a zeaxanthin-dependent alteration in the chlorophyll environment.

Chlorophyll↗
PubMed2000

Prospects for crop improvement through the genetic manipulation of photosynthesis: morphological and biochemical aspects of light capture.

The prospects for genetic manipulation of photosynthesis are assessed with an emphasis on the biochemical and morphological aspects of light capture. The connection between different parts of the photosynthetic process is considered together with the influence of environmental factors, development and acclimation, and metabolic regulation. The sites of real and potential photosynthetic losses are identified, using tropical rice as a case study. The important interaction between photosynthetic capacity, acclimation to the light environment, nitrogen accumulation and canopy architecture are discussed. The possibility of genetic intervention to increase both biomass accumulation and improve nitrogen economy simultaneously are considered. Finally, the numerous procedures for genetic manipulation of light harvesting are also discussed, with a view of improving radiation-use efficiency in crops.

Biomass↗
PubMed1999

Determination of the stoichiometry and strength of binding of xanthophylls to the photosystem II light harvesting complexes.

Xanthophylls have a crucial role in the structure and function of the light harvesting complexes of photosystem II (LHCII) in plants. The binding of xanthophylls to LHCII has been investigated, particularly with respect to the xanthophyll cycle carotenoids violaxanthin and zeaxanthin. It was found that most of the violaxanthin pool was loosely bound to the major complex and could be removed by mild detergent treatment. Gentle solubilization of photosystem II particles and thylakoids allowed the isolation of complexes, including a newly described oligomeric preparation, enriched in trimers, that retained all of the in vivo violaxanthin pool. It was estimated that each LHCII monomer can bind at least one violaxanthin. The extent to which different pigments can be removed from LHCII indicated that the relative strength of binding was chlorophyll b > neoxanthin > chlorophyll a > lutein > zeaxanthin > violaxanthin. The xanthophyll binding sites are of two types: internal sites binding lutein and peripheral sites binding neoxanthin and violaxanthin. In CP29, a minor LHCII, both a lutein site and the neoxanthin site can be occupied by violaxanthin. Upon activation of the violaxanthin de-epoxidase, the highest de-epoxidation state was found for the main LHCII component and the lowest for CP29, suggesting that only violaxanthin loosely bound to LHCII is available for de-epoxidation.

Binding Sites↗
PubMed1999

Unusual carotenoid composition and a new type of xanthophyll cycle in plants.

The capture of photons by the photosynthetic apparatus is the first step in photosynthesis in all autotrophic higher plants. This light capture is dominated by pigment-containing proteins known as light-harvesting complexes (LHCs). The xanthophyll-carotenoid complement of these LHCs (neoxanthin, violaxanthin, and lutein) is highly conserved, with no deletions and few, uncommon additions. We report that neoxanthin, considered an integral component of LHCs, is stoichiometrically replaced by lutein-5,6-epoxide in the parasitic angiosperm Cuscuta reflexa, without compromising the structural integrity of the LHCs. Lutein-5,6-epoxide differs from neoxanthin in that it is involved in a light-driven deepoxidation cycle similar to the deepoxidation of violaxanthin in the xanthophyll cycle, which is implicated in protection against photodamage. The absence of neoxanthin and its replacement by lutein-5,6-epoxide changes our understanding of the structure-function relationship in LHCs, has implications for biosynthetic pathways involving neoxanthin (such as the plant hormone abscisic acid), and identifies one of the early steps associated with the evolution of heterotrophy from autotrophy in plants.

Journal Article↗
PubMed1999

Acclimation of Arabidopsis thaliana to the light environment: the role of photoreceptors.

The regulation by light of the composition of the photosynthetic apparatus was investigated in photomorphogenic mutants of Arabidopsis thaliana (L.) Heynh. cv. Landsberg erecta. Leaf chlorophyll, photosynthesis, photosystem II function, and ribulose-1, 5-bisphosphate carboxylase-oxygenase and photosystem II contents were determined for plants grown under high- or low-irradiance growth regimes. Although certain mutant lines had altered chloroplast composition compared to the wild type, all photoreceptor mutants tested were capable of light-dependent changes in chloroplast composition and photosynthetic function, indicating that photoreceptors do not play a central role in the regulation of acclimation at the level of the chloroplast. However, the clear acclimation defect in a det1 signal transduction mutant indicates that photoreceptor-controlled responses either share regulatory components with acclimation, or are important in the expression of components which in turn regulate acclimation. We suggest that the COP/DET/FUS regulatory cluster is a focus for multiple signal transduction pathways, including some of the metabolic signals which form the basis for the acclimatory response.

Arabidopsis↗
PubMed1999

Spectroscopic characterization of the spinach Lhcb4 protein (CP29), a minor light-harvesting complex of photosystem II.

A spectroscopic characterization is presented of the minor photosystem II chlorophyll a/b-binding protein CP29 (or the Lhcb4 protein) from spinach, prepared by a modified form of a published protocol [Henrysson, T., Schroder, W. P., Spangfort, M. & Akerlund, H.-E. (1989) Biochim. Biophys. Acta 977, 301-308]. The isolation procedure represents a quicker, cheaper means of isolating this minor antenna protein to an equally high level of purity to that published previously. The pigment-binding protein shows similarities to other related light-harvesting complexes (LHCs), including the bulk complex LHCIIb but more particularly another minor antenna protein CP26 (Lhcb5). It is also, in the main, similar to other preparations of CP29, although some significant differences are discussed. In common with CP26, the protein binds about six chlorophyll a and two chlorophyll b molecules. Two chlorophyll b absorption bands are present at 638 and 650 nm and they are somewhat more pronounced than in a recent report [Giuffra, E., Zucchelli, G., Sandonà, D., Croce, R., Cugini, D., Garlaschi, F.M., Bassi, R. & Jennings, R.C. (1997) Biochem. 36, 12984-12993]. The bands give rise to positive and negative linear dichroism, respectively; both show negative CD bands (cf. bands with similar properties at 637 and 650 nm in CP26). Chlorophyll a absorption is dominated by a large contribution at 674 nm which also shows similarities to the major band in LHCIIb and CP26, while (as for CP26) a reduction in absorption around 670 nm is observed relative to the bulk complex. Principal differences from LHCIIb and CP26, and from other CP29 preparations, occur in the carotenoid region.

Circular Dichroism↗
PubMed1999

The xanthophyll cycle modulates the kinetics of nonphotochemical energy dissipation in isolated light-harvesting complexes, intact chloroplasts, and leaves of spinach

We analyzed the kinetics of nonphotochemical quenching of chlorophyll fluorescence (qN) in spinach (Spinacia oleracea) leaves, chloroplasts, and purified light-harvesting complexes. The characteristic biphasic pattern of fluorescence quenching in dark-adapted leaves, which was removed by preillumination, was evidence of light activation of qN, a process correlated with the de-epoxidation state of the xanthophyll cycle carotenoids. Chloroplasts isolated from dark-adapted and light-activated leaves confirmed the nature of light activation: faster and greater quenching at a subsaturating transthylakoid pH gradient. The light-harvesting chlorophyll a/b-binding complexes of photosystem II were isolated from dark-adapted and light-activated leaves. When isolated from light-activated leaves, these complexes showed an increase in the rate of quenching in vitro compared with samples prepared from dark-adapted leaves. In all cases, the quenching kinetics were fitted to a single component hyperbolic function. For leaves, chloroplasts, and light-harvesting complexes, the presence of zeaxanthin was associated with an increased rate constant for the induction of quenching. We discuss the significance of these observations in terms of the mechanism and control of qN.

Journal Article↗