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P Horton

Publications and source records attributed to P Horton.

At least 19 recordsLinked to original sources
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↗
PubMed1999

Interactions between senescence and leaf orientation determine in situ patterns of photosynthesis and photoinhibition in field-grown rice

Photosynthesis and photoinhibition in field-grown rice (Oryza sativa L.) were examined in relation to leaf age and orientation. Two varieties (IR72 and IR65598-112-2 [BSI206]) were grown in the field in the Philippines during the dry season under highly irrigated, well-fertilized conditions. Flag leaves were examined 60 and 100 d after transplanting. Because of the upright nature of 60-d-old rice leaves, patterns of photosynthesis were determined by solar movements: light falling on the exposed surface in the morning, a low incident angle of irradiance at midday, and light striking the opposite side of the leaf blade in the afternoon. There was an early morning burst of CO2 assimilation and high levels of saturation of photosystem II electron transfer as incident irradiance reached a maximum level. However, by midday the photochemical efficiency increased again almost to maximum. Leaves that were 100 d old possessed a more horizontal orientation and were found to suffer greater levels of photoinhibition than younger leaves, and this was accompanied by increases in the de-epoxidation state of the xanthophyll cycle. Older leaves had significantly lower chlorophyll content but only slightly diminished photosynthesis capacity.

Journal Article↗
PubMed1999

Chloroplast acclimation in leaves of Guzmania monostachia in response to high light.

Acclimation of leaves to high light (HL; 650 micromol m(-2) s(-1)) was investigated in the long-lived epiphytic bromeliad Guzmania monostachia and compared with plants maintained under low light (LL; 50 micromol m(-2) s(-1)). Despite a 60% decrease in total chlorophyll in HL-grown plants, the chlorophyll a/b ratio remained stable. Additionally, chloroplasts from HL-grown plants had a much lower thylakoid content and reduced granal stacking. Immunofluorescent labeling techniques were used to quantify the level of photosynthetic polypeptides. HL-grown plants had 30% to 40% of the content observed in LL-grown plants for the light-harvesting complex associated with photosystems I and II, the 33-kD photosystem II polypeptide, and Rubisco. These results were verified using conventional biochemical techniques, which revealed a comparable 60% decrease in Rubisco and total soluble protein. When expressed on a chlorophyll basis, the amount of protein and Rubisco was constant for HL- and LL-grown plants. Acclimation to HL involves a tightly coordinated adjustment of photosynthesis, indicating a highly regulated decrease in the number of photosynthetic units manifested at the level of the content of light-harvesting and electron transport components, the amount of Rubisco, and the induction of Crassulacean acid metabolism. This response occurs in mature leaves and may represent a strategy that is optimal for the resource-limited epiphytic niche.

Chlorophyll↗
PubMed1998

The relationship between the binding of dicyclohexylcarbodiimide and quenching of chlorophyll fluorescence in the light-harvesting proteins of photosystem II.

The relationship between the binding of dicyclohexylcarbodiimide (DCCD) to isolated light-harvesting proteins of photosystem II and the inhibition of chlorophyll fluorescence quenching by DCCD have been investigated. For a range of different complexes an approximately linear relationship was obtained between the efficiency of DCCD binding and the DCCD-dependent reversal of fluorescence quenching. The most efficient labeling was found for the minor light-harvesting complexes, CP29 and CP26. In the case of the former, five different preparations were compared including two reconstituted complexes in which a putative DCCD-binding site had been mutagenized. Again, an approximately linear relationship between DCCD binding and the extent of reversal of fluorescence quenching was found. However, the binding of DCCD was found to occur at least an order of magnitude faster than the change in fluorescence. The results are discussed in terms of the multiplicity of DCCD-binding sites and the influence of protein structure on both the binding of DCCD and the fluorescence quenching mechanism.

Binding Sites↗
PubMed1998

Ultrafast evolution of the excited states in the chlorophyll a/b complex CP29 from green plants studied by energy-selective pump-probe spectroscopy.

The energy transfer process in the minor light-harvesting antenna complex CP29 of green plants was probed in multicolor transient absorption experiments at 77 K using selective subpicosecond excitation pulses at 640 and 650 nm. Energy flow from each of the chlorophyll (Chl) b molecules of the complex could thus be studied separately. The analysis of our data showed that the "blue" Chl b (absorption around 640 nm) transfers excitation to a "red" Chl a with a time constant of 350 +/- 100 fs, while the 'red' Chl b (absorption at 650 nm) transfers on a picosecond time scale (2.2 +/- 0.5 ps) toward a "blue" Chl a. Furthermore, both fast (280 +/- 50 fs) and slow (10-13 ps) equilibration processes among the Chl a molecules were observed, with rates and associated spectra very similar to those of the major antenna complex, LHC-II. Based on the protein sequence homology between CP29 and LHC-II, a basic modelling of the observed kinetics was performed using the LHC-II structure and the Förster theory of energy transfer. Thus, an assignment for the spectral properties and orientation of the two Chl's b, as well as for their closest Chl a neighbors, is put forward, and a comparison is made with the previous assignments and models for LHC-II and CP29.

Chlorophyll↗
PubMed1998

Styrene oxide adducts in an oligodeoxynucleotide containing the human N-ras codon 12: minor groove structures of the R(12,1)- and S(12,1)-alpha-(N2-guanyl) stereoisomers determined by 1H nuclear magnetic resonance.

The R- and S-alpha-(N2-guanyl)styrene oxide (SO) adducts at X5 in d(G1G2C3A4X5G6T7G8G9T10G11).d(C12A13C14C15A16C17C18T19G20C21C22 ), encompassing codon 12 of the human N-ras protooncogene (underlined), were examined using 1H NMR spectroscopy. These were the R(12,1) and S(12,1) adducts, indicating the location of the R or S adduct at the first position of codon 12. These differed from the R- and S(12, 2)-alpha-SO adducts [Zegar, I. S., Setayesh, F. R., DeCorte, B. L., Harris, C. M., Harris, T. M., and Stone, M. P. (1996) Biochemistry 35, 4334-4348] in that the base pair 5' to the lesion was changed from G.C to A.T, while the base pair 3' to the lesion was changed from T.A to G.C. Comparison of the R- and S(12,1) adducts with the R- and S(12,2) adducts allowed the effects of flanking bases on the conformations of the alpha-SO adducts to be examined. This change in flanking base affected the R-SO lesion. The R(12,1) adduct structure was disordered at the adduct site, and a refined structure could not be obtained. NOE and chemical shift data suggested that the styrenyl moiety was oriented in the minor groove and in the 3'-direction from the site of adduction. In contrast, this change in flanking base did not affect the S-SO lesion. The S(12,1) adduct yielded a refined structure, with the styrenyl moiety edgewise in the minor groove and oriented in the 5'-direction relative to the site of adduction. A total of 232 interproton distances, including 13 styrene-DNA distances, were obtained. A total of 12 NOE-restrained molecular dynamics calculations converged with pairwise root-mean-square deviation of 1.10 A. The sixth-root residual index between calculated and experimental NOE intensities was 8.0 x 10(-)2 A. The styrene aromatic protons appeared as three resonances, suggesting rapid rotation. The possibility of a hydrogen bond between the styrene hydroxyl and C18 O2 in the S(12,1) adduct could not be confirmed. This work illustrates the dual roles of stereochemistry and sequence in modulating the properties of guanine N2 alpha-SO adducts.

Codon↗
PubMed1997

Carotenoid-dependent oligomerization of the major chlorophyll a/b light harvesting complex of photosystem II of plants.

Under many environmental conditions, plants are exposed to levels of sunlight in excess of those required for photosynthesis. Then, a regulated increase in the rate of nonradiative dissipation of excess excitation energy in the thylakoid membrane correlates with the conversion of the carotenoid violaxanthin into zeaxanthin and provides protection from the damaging effects of excessive irradiation. The hypothesis that these carotenoids specifically control the oligomerization of the light harvesting complexes of photosystem II was tested by investigating the effects of violaxanthin and zeaxanthin on the behavior of the major complex, LHCIIb, on sucrose gradients; it was found that zeaxanthin stimulated the formation of LHCIIb aggregates with reduced chlorophyll fluorescence yield whereas violaxanthin caused the inhibition of such aggregation and an elevation of fluorescence. Measurements of 77 K fluorescence indicated that zeaxanthin was not exerting an additional direct quenching of chlorophyll fluorescence. These effects can explain the physiological control of the light harvesting system by the xanthophyll cycle.

Biopolymers↗
PubMed1997

Better prediction of protein cellular localization sites with the k nearest neighbors classifier.

We have compared four classifiers on the problem of predicting the cellular localization sites of proteins in yeast and E. coli. A set of sequence derived features, such as regions of high hydrophobicity, were used for each classifier. The methods compared were a structured probabilistic model specifically designed for the localization problem, the k nearest neighbors classifier, the binary decision tree classifier, and the naïve Bayes classifier. The result of tests using stratified cross validation shows the k nearest neighbors classifier to perform better than the other methods. In the case of yeast this difference was statistically significant using a cross-validated paired t test. The result is an accuracy of approximately 60% for 10 yeast classes and 86% for 8 E. coli classes. The best previously reported accuracies for these datasets were 55% and 81% respectively.

Algorithms↗
PubMed1996

Identification of proton-active residues in a higher plant light-harvesting complex.

The thermal dissipation of absorbed light energy by the light-harvesting apparatus of higher plants is important in protecting the photosynthetic machinery from the effects of excess illumination. A major mechanism for such photoprotection, known as trans-thylakoid delta pH-dependent chlorophyll fluorescence quenching (qE), is induced by acidification of the lumen, is correlated with the interconversion of xanthophyll pigments, and is manifested as quenching of chloropyll fluorescence. The mechanistic basis for qE remains unknown. The reagent N, N'-dicyclohexylcarbodiimide (DCCD) specifically inhibits qE and covalently binds to two minor light-harvesting pigment-protein complexes (LHCII), LHCIIa and LHCIIc. It is shown that DCCD treatment of isolated LHCIIc complexes reverses acid-induced chlorophyll fluorescence quenching in an in vitro system. Fingerprinting of [14C]DCCD-labeled LHCIIc demonstrates that there are two DCCD-sensitive amino acid residues on this complex, and these are shown to be glutamate residues, each of which is located near the lumen. In view of the effects of DCCD on the pattern of proton release from photosystem II during photosynthesis, we propose a model for the mechanism of the induction of qE--that these residues from part of a proton pathway, the lumen pH being sensed via its effects on the rate of proton release. One possibility is that the resulting changes in the protonation state of these carboxyl side chains may modulate the structural and energetic organization of LHCII.

Amino Acid Sequence↗
PubMed1996

Dynamic properties of the minor chlorophyll a/b binding proteins of photosystem II, an in vitro model for photoprotective energy dissipation in the photosynthetic membrane of green plants.

Excess light energy absorbed by the chloroplast membranes of green plants is dissipated by nonradiative de-excitation in order to protect against photodamage. This is observed as the nonphotochemical quenching of chlorophyll fluorescence, which has been suggested to result from an alteration in the structure and function of the chlorophyll a/b light-harvesting complexes of photosystem II (LHCII) due to the combined effects of protonation and the de-epoxidation of bound violaxanthin to form zeaxanthin. In agreement with this hypothesis, it is shown that the light-harvesting chlorophyll a/b proteins purified from spinach leaves exhibit pH-stimulated quenching of chlorophyll fluorescence; this quenching shares all the key features observed for the nonphotochemical quenching of chlorophyll fluorescence in vivo. In the case of the two minor complexes, LHCIIa (CP29) and LHCIIc (CP26), quenching is much greater than in the bulk complex LHCIIb and is strongly inhibited by the reagent dicyclohexylcarbodiimide. The carotenoids violaxanthin and zeaxanthin cause strong inhibition and stimulation of quenching, respectively, in these complexes. The results of this study are consistent with the suggestion that the minor light-harvesting complexes play a crucial role in photoprotective energy dissipation in the photosynthetic membrane of green plants. Moreover, for the first time, a system using isolated LHCIIa and LHCIIc for the study of the regulation of light harvesting is described.

Carotenoids↗
PubMed1996

A probabilistic classification system for predicting the cellular localization sites of proteins.

We have defined a simple model of classification which combines human provided expert knowledge with probabilistic reasoning. We have developed software to implement this model and have applied it to the problem of classifying proteins into their various cellular localization sites based on their amino acid sequences. Since our system requires no hand tuning to learn training data, we can now evaluate the prediction accuracy of protein localization sites by a more objective cross-validation method than earlier studies using production rule type expert systems. 336 E. coli proteins were classified into 8 classes with an accuracy of 81% while 1484 yeast proteins were classified into 10 classes with an accuracy of 55%. Additionally we report empirical results using three different strategies for handling continuously valued variables in our probabilistic reasoning system.

Cells↗