Singlet oxygen scavenging activity of tocopherol and plastochromanol in Arabidopsis thaliana: relevance to photooxidative stress

In the present study, singlet oxygen (¹O₂) scavenging activity of tocopherol and plastochromanol was examined in tocopherol cyclase‐deficient mutant (vte1) of Arabidopsis thaliana lacking both tocopherol and plastochromanol. It is demonstrated here that suppression of tocopherol and plastochromanol synthesis in chloroplasts isolated from vte1 Arabidopsis plants enhanced ¹O₂ formation under high light illumination as monitored by electron paramagnetic resonance spin‐trapping spectroscopy. The exposure of vte1 Arabidopsis plants to high light resulted in the formation of secondary lipid peroxidation product malondialdehyde as determined by high‐pressure liquid chromatography. Furthermore, it is shown here that the imaging of ultra‐weak photon emission known to reflect oxidation of lipids was unambiguously higher in vte1 Arabidopsis plants. Our results indicate that tocopherol and plastochromanol act as efficient ¹O₂ scavengers and protect effectively lipids against photooxidative damage in Arabidopsis plants.     

Hydroxy‐plastochromanol and plastoquinone‐C as singlet oxygen products during photo‐oxidative stress in Arabidopsis

In the present study, we have shown that hydroxy‐plastochromanol and plastoquinone‐C, the hydroxy derivatives of plastochromanol and plastoquinone‐9, respectively, are specifically formed from the parent compounds upon action of singlet oxygen and can be regarded as stable, specific, natural products of singlet oxygen action during photo‐oxidative stress in vivo. The presented data indicate that plastoquinone‐C formation dominates mainly during relatively short periods of high light stress where efficient production of singlet oxygen takes place, whereas hydroxy‐plastochromanol is rather formed under conditions of long‐term, less pronounced generation of singlet oxygen. An interesting observation was that hydroxy‐plastochromanol is formed even at very low light conditions (5–10 μmol photons m^?2 s^?1), indicating that singlet oxygen is generated not only during high light stress but also its formation by photosystem II is inseparably connected with the functioning of this photosystem even at the lowest light intensities.     

The plastoquinone pool outside the thylakoid membrane serves in plant photoprotection as a reservoir of singlet oxygen scavengers

The Arabidopsis vte1 mutant is devoid of tocopherol and plastochromanol (PC‐8). When exposed to excess light energy, vte1 produced more singlet oxygen (¹O₂) and suffered from extensive oxidative damage compared with the wild type. Here, we show that overexpressing the solanesyl diphosphate synthase 1 (SPS1) gene in vte1 induced a marked accumulation of total plastoquinone (PQ‐9) and rendered the vte1 SPS1oex plants tolerant to photooxidative stress, indicating that PQ‐9 can replace tocopherol and PC‐8 in photoprotection. High total PQ‐9 levels were associated with a noticeable decrease in ¹O₂ production and higher levels of Hydroxyplastoquinone (PQ‐C), a ¹O₂‐specific PQ‐9 oxidation product. The extra PQ‐9 molecules in the vte1 SPS1oex plants were stored in the plastoglobules and the chloroplast envelopes, rather than in the thylakoid membranes, whereas PQ‐C was found almost exclusively in the thylakoid membranes. Upon exposure of wild‐type plants to high light, the thylakoid PQ‐9 pool decreased, whereas the extrathylakoid pool remained unchanged. In vte1 and vte1 SPS1oex plants, the PQ‐9 losses in high light were strongly amplified, affecting also the extrathylakoid pool, and PQ‐C was found in high amounts in the thylakoids. We conclude that the thylakoid PQ‐9 pool acts as a ¹O₂ scavenger and is replenished from the extrathylakoid stock.     

New prenyllipid metabolites identified in Arabidopsis during photo‐oxidative stress

In the present study, we have identified new prenyllipid metabolites formed during high light stress in Arabidopsis thaliana, whose origin and function remained unknown so far. It was found that plastoquinone‐C accumulates mainly in the reduced form under high light conditions, as well as during short‐term excess light illumination both in the wild‐type and tocopherol biosynthetic vte1 mutant, suggesting that plastoquinone‐C, a singlet oxygen‐derived prenyllipid, is reduced in chloroplasts by photosystem II or enzymatically, outside thylakoids. Plastoquinone‐B, a fatty acid ester of plastoquinone‐C, was identified for the first time in Arabidopsis in high light grown wild‐type plants and during short‐time, excess light illumination of the wild‐type plants and the vte1 mutant. The gene expression analysis showed that vte2 gene is most pronouncedly up‐regulated among the prenyllipid biosynthetic genes under high light and induction of its expression is mainly caused by an increased level of singlet oxygen, as was demonstrated in experiments with D₂O‐treated plants under excess light conditions.     

Tocochromanols, plastoquinol, and other biological prenyllipids as singlet oxygen quenchers-determination of singlet oxygen quenching rate constants and oxidation products

Singlet oxygen quenching rate constants for tocopherol and tocotrienol homologues have been determined in organic solvents of different polarities, as well as for other biological prenyllipids such as plastoquinol, ubiquinol, and alpha-tocopherolquinol. The obtained results showed that the quenching activity of tocochromanols was mainly due to the chromanol ring of the molecule and the activity increased with the number of the methyl groups in the ring and solvent polarity. Among prenylquinols, alpha-tocopherolquinol was the most active scavenger of singlet oxygen followed by ubiquinol and plastoquinol. The oxidation products of tocopherols were identified as 8a-hydroperoxy-tocopherones which are converted to the corresponding tocopherolquinones under acidic conditions. The primary oxidation products of prenylquinols, containing unsaturated side chains, were the corresponding prenylquinones that were further oxidized to hydroxyl side-chain derivatives. In the case of plastochromanol, the gamma-tocotrienol homologue found in some seed oils, mainly the hydroxyl derivatives were formed, although 8a-hydroperoxy-gamma-tocopherones were also formed to a minor extent, both from plastochromanol and from its hydroxyl, side-chain derivatives. The obtained results were discussed in terms of the activity of different prenyllipids as singlet oxygen scavengers in vivo.     

Mutations of the ER to plastid lipid transporters TGD1, 2, 3 and 4 and the ER oleate desaturase FAD2 suppress the low temperature‐induced phenotype of Arabidopsis tocopherol‐deficient mutant vte2

Previous studies with the tocopherol‐deficient Arabidopsis thaliana vte2 mutant demonstrated an important role for tocopherols in the development of transfer cell walls and maintenance of photoassimilate export capacity during low‐temperature (LT) adaptation. To further understand the processes linking tocopherol deficiency and the vte2 LT phenotypes, a genetic screen was performed for sve mutations (suppressor of the vte2 low temperature‐induced phenotype). The three strongest sve loci had differing impacts on LT‐induced sugar accumulation, photoassimilate export reduction and vascular‐specific callose deposition in vte2. sve1 completely suppressed all vte2 LT phenotypes and is a new allele of fad2, the endoplasmic reticulum‐localized oleate desaturase. sve2 showed partial suppression, and is a new allele of trigalactosyldiacylglycerol1 (tgd1), a component of the ER‐to‐plastid lipid ATP‐binding cassette (ABC) transporter. Introduction of tgd2, tgd3 and tgd4 mutations into the vte2 background similarly suppressed the vte2 LT phenotypes, indicating a key role for ER‐to‐plastid lipid transport in the vte2 LT phenotype. sve7 partially suppressed all vte2 LT phenotypes by affecting fatty acid and lipid metabolism at low temperatures only. Detailed analyses of acyl lipid composition indicated that all suppressors alleviated the increase in the level of linoleic acid esterified to phosphatidylcholine (PC‐18:2) in LT‐treated vte2, and this alleviation significantly correlated with their extent of suppression of photoassimilate export. Identification and characterization of the sve loci showed that the PC‐18:2 change is an early and key component in vte2 LT‐induced responses, and highlighted the interaction of tocopherols with non‐plastid lipid metabolism.     

Light,temperature and tocopherol status influence foliar vascular anatomy and leaf function in Arabidopsis thaliana

This study addressed whether the winter annual Arabidopsis thaliana can adjust foliar phloem and xylem anatomy both differentially and in parallel. In plants acclimated to hot vs cool temperature, foliar minor vein xylem‐to‐phloem ratio was greater, whereas xylem and phloem responded concomitantly to growth light intensity. Across all growth conditions, xylem anatomy correlated with transpiration rate, while phloem anatomy correlated with photosynthetic capacity for two plant lines (wild‐type Col‐0 and tocopherol‐deficient vte1 mutant) irrespective of tocopherol status. A high foliar vein density (VD) was associated with greater numbers and cross‐sectional areas of both xylem and phloem cells per vein as well as higher rates of both photosynthesis and transpiration under high vs low light intensities. Under hot vs cool temperature, high foliar VD was associated with a higher xylem‐to‐phloem ratio and greater relative rates of transpiration to photosynthesis. Tocopherol status affected development of foliar vasculature as dependent on growth environment. The most notable impact of tocopherol deficiency was seen under hot growth temperature, where the vte1 mutant exhibited greater numbers of tracheary elements (TEs) per vein, a greater ratio of TEs to sieve elements, with smaller individual sizes of TEs, and resulting similar total areas of TEs per vein and transpiration rates compared with Col‐0 wild‐type. These findings illustrate the plasticity of foliar vascular anatomy acclimation to growth environment resulting from independent adjustments of the vasculature's components.     

Physiological and antioxidant responses of two accessions of Arabidopsis thaliana in different light and temperature conditions

During their lifetime, plants need to adapt to a changing environment, including light and temperature. To understand how these factors influence plant growth, we investigated the physiological and antioxidant responses of two Arabidopsis accessions, Shahdara (Sha) from the Shahdara valley (Tajikistan, Central Asia) in a mountainous area and Lovvik‐5 (Lov‐5) from northern Sweden to different light and temperature conditions. These accessions originate from different latitudes and have different life strategies, both of which are known to be influenced by light and temperature. We showed that both accessions grew better in high‐light and at a lower temperature (16°C) than in low light and at 23°C. Interestingly, Sha had a lower chlorophyll content but more efficient non‐photochemical quenching than Lov‐5. Sha, also showed a higher expression of vitamin E biosynthetic genes. We did not observe any difference in the antioxidant prenyllipid level under these conditions. Our results suggest that the mechanisms that keep the plastoquinone (PQ)‐pool in more oxidized state could play a role in the adaptation of these accessions to their local climatic conditions.     

Vitamin E protects against photoinhibition and photooxidative stress in Arabidopsis thaliana

Vitamin E is considered a major antioxidant in biomembranes, but little evidence exists for this function in plants under photooxidative stress. Leaf discs of two vitamin E mutants, a tocopherol cyclase mutant (vte1) and a homogentisate phytyl transferase mutant (vte2), were exposed to high light stress at low temperature, which resulted in bleaching and lipid photodestruction. However, this was not observed in whole plants exposed to long-term high light stress, unless the stress conditions were extreme (very low temperature and very high light), suggesting compensatory mechanisms for vitamin E deficiency under physiological conditions. We identified two such mechanisms: nonphotochemical energy dissipation (NPQ) in photosystem II (PSII) and synthesis of zeaxanthin. Inhibition of NPQ in the double mutant vte1 npq4 led to a marked photoinhibition of PSII, suggesting protection of PSII by tocopherols. vte1 plants accumulated more zeaxanthin in high light than the wild type, and inhibiting zeaxanthin synthesis in the vte1 npq1 double mutant resulted in PSII photoinhibition accompanied by extensive oxidation of lipids and pigments. The single mutants npq1, npq4, vte2, and vte1 showed little sensitivity to the stress treatments. We conclude that, in cooperation with the xanthophyll cycle, vitamin E fulfills at least two different functions in chloroplasts at the two major sites of singlet oxygen production: preserving PSII from photoinactivation and protecting membrane lipids from photooxidation.     

Obligatory short‐day plant,Perilla frutescens var. crispa can flower in response to low‐intensity light stress under long‐day conditions

An obligatory short‐day plant, Perilla frutescens var. crispa was induced to flower under long‐day conditions when grown under low‐intensity light (30 µmol m^?2 s^?1). Plant size was smaller under lower light intensity, indicating that the low‐intensity light acted as a stress factor. The phenomenon is categorized as stress‐induced flowering. Low‐intensity light treatment for 4 weeks induced 100% flowering. The plants responded to low‐intensity light immediately after the cotyledons expanded, and the flowering response decreased with increasing plant age. The induced plants produced fertile seeds, and the progeny developed normally. The plants that flowered under low‐intensity light had greener leaves. This greening was because of the decrease in anthocyanin content, and there was a negative correlation between the anthocyanin content and percent flowering. Treatment with L‐2‐aminooxy‐3‐phenylpropionic acid, an inhibitor of phenylalanine ammonia‐lyase (PAL), did not induce flowering under non‐inductive light conditions and inhibited flowering under inductive low‐intensity light conditions. The metabolic pathway regulated by PAL may be involved in the flowering induced by low‐intensity light.     

Light acclimation of Chlamydomonas acidophila accumulating in the hypolimnion of an acidic lake (pH 2.6)

1. The unicellular green alga Chlamydomonas acidophila accumulates in a thin phytoplankton layer in the hypolimnion (deep chlorophyll maximum, DCM) of an extremely acidic lake (Lake 111, pH 2.6, Lusatia, Germany), in which the underwater light spectrum is distorted and red‐shifted. 2. Chlamydomonas acidophila exhibited a significantly higher absorption efficiency and a higher cellular chlorophyll b content when incubated in the red shifted underwater light of Lake 111 than in a typical, blue‐green dominated, light spectrum. 3. Chlamydomonas acidophila has excellent low light acclimation properties (increased chlorophyll b content, increased oxygen yield and a low light saturation point for photosynthesis) that support survival of the species in the low light climate of the DCM. 4. In situ acclimation to the DCM under low light and temperature decreased maximum photosynthetic rate in autotrophic C. acidophila cultures, whereas the presence of glucose under these conditions enhanced photosynthetic efficiency and capacity. 5. The adaptive abilities of C. acidophila to light and temperature shown in this study, in combination with the absence of potent competitors because of low lake pH, most probably enable the unusual dominance of the green alga in the DCM of Lake 111.     

Plastoquinol as a singlet oxygen scavenger in photosystem II

It has been found that in Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to α-tocopherol. In relation to chlorophyll, after 18 h of growth under low light with the inhibitor, the content of α-tocopherol was 22.2 mol/1000 mol chlorophyll and that of total plastoquinone (oxidized and reduced) was 19 mol/1000 mol chlorophyll, while after 2 h of high-light stress the corresponding amounts dropped to 6.4 and 6.2 mol/1000 mol chlorophyll for α-tocopherol and total plastoquinone, respectively. The degradation of both prenyllipids was partially reversed by diphenylamine, a singlet oxygen scavenger. It was concluded that plastoquinol, as well as α-tocopherol is decomposed under high-light stress as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The levels of both α-tocopherol and of the reduced plastoquinone are not affected significantly in the absence of the inhibitor due to a high turnover rate of both prenyllipids, i.e., their degradation is compensated by fast biosynthesis. The calculated turnover rates under high-light conditions were twofold higher for total plastoquinone (0.23 nmol/h/ml of cell culture) than for α-tocopherol (0.11 nmol/h/ml). We have also found that the level of α-tocopherolquinone, an oxidation product of α-tocopherol, increases as the α-tocopherol is consumed. The same correlation was also observed for γ-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low-light growth conditions, the synthesis of plastoquinone-C, a hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. The presented data also suggest that the two plastoquinones may have different biosynthetic pathways in C. reinhardtii.     

Chemical quenching of singlet oxygen by plastoquinols and their oxidation products in Arabidopsis

Prenylquinols (tocochromanols and plastoquinols) serve as efficient physical and chemical quenchers of singlet oxygen (¹O₂) formed during high light stress in higher plants. Although quenching of ¹O₂ by prenylquinols has been previously studied, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is limited in vivo. In the present study, the role of plastoquinol‐9 (PQH₂‐9) in chemical quenching of ¹O₂ was studied in Arabidopsis thaliana lines overexpressing the SOLANESYL DIPHOSPHATE SYNTHASE 1 gene (SPS1oex) involved in PQH₂‐9 and plastochromanol‐8 biosynthesis. In this work, direct evidence for chemical quenching of ¹O₂ by plastoquinols and their oxidation products is presented, which is obtained by microscopic techniques in vivo. Chemical quenching of ¹O₂ was associated with consumption of PQH₂‐9 and formation of its various oxidized forms. Oxidation of PQH₂‐9 by ¹O₂ leads to plastoquinone‐9 (PQ‐9), which is subsequently oxidized to hydroxyplastoquinone‐9 [PQ(OH)‐9]. We provide here evidence that oxidation of PQ(OH)‐9 by ¹O₂ results in the formation of trihydroxyplastoquinone‐9 [PQ(OH)₃‐9]. It is concluded here that PQH₂‐9 serves as an efficient ¹O₂ chemical quencher in Arabidopsis, and PQ(OH)₃‐9 can be considered as a natural product of ¹O₂ reaction with PQ(OH)‐9. The understanding of the mechanisms underlying ¹O₂ chemical quenching provides information on the role of plastoquinols and their oxidation products in the response of plants to photooxidative stress.     

STUDIES ON THE AUTECOLOGY OF THE MARINE DIATOM THALASSIOSIRA NORDENSKIÖLDIICLEVE. 1. THE INFLUENCE OF DAYLENGTH,LIGHT INTENSITY,AND TEMPERATURE ON GROWTH1

The marine diatom Thalassiosira nordenskiöldii Clave was grown at 48 different combinations of daylength (9:15, 12:12, 15:9 LD), light intensity (0.011, 0.027, 0.066, 0.100 ly/min [g cal/cm²/min]), and temperature (0, 5, 10, 15 C). Growth occurred at all combinations of light and temperature except at 15 C at the highest light level. Maximum growth (K = 1.8 doublings/day) occurred at 10 C under the 15:9 LD cycle. At 15 C the maximum rate was 1.7 doublings/day but occurred at the shortest day-length (9:15 LD). The maxima at 5 and 0 C were 1.32 and 0.67 doubling/day, respectively. At 0 C growth was similar over a wide range of light intensities (K = 0.6–0.65), with, maximum growth being attained at a much lower light intensity than at 5 C. Above 5 C there was a decrease in the light intensity at which maximum growth occurred and excessive light became inhibitory to growth. At 15 C the light intensity at which maximum growth occurred was greater with shorter day-lengths. The temperature optimum was 10 C at 15:9 and 15 C at 9:15 LD. The chlorophyll a content of the cells was greatest under low light intensities and short daylengths, while temperature had a variable effect. The response of Thalassiosira in the laboratory contrasts with, its apparent preference for low temperatures in nature (0–5 C). The experiments suggest that the termination of the bloom of Thalassiosira in Narragansett Bay and elsewhere is not solely temperature dependent.     

Plastoquinol as a singlet oxygen scavenger in photosystem II

It has been found that in Chlamydomonas reinhardtii cells, under high-light stress, the level of reduced plastoquinone considerably increases while in the presence of pyrazolate, an inhibitor of plastoquinone and tocopherol biosynthesis, the content of reduced plastoquinone quickly decreases, similarly to alpha-tocopherol. In relation to chlorophyll, after 18 h of growth under low light with the inhibitor, the content of alpha-tocopherol was 22.2 mol/1000 mol chlorophyll and that of total plastoquinone (oxidized and reduced) was 19 mol/1000 mol chlorophyll, while after 2 h of high-light stress the corresponding amounts dropped to 6.4 and 6.2 mol/1000 mol chlorophyll for alpha-tocopherol and total plastoquinone, respectively. The degradation of both prenyllipids was partially reversed by diphenylamine, a singlet oxygen scavenger. It was concluded that plastoquinol, as well as alpha-tocopherol is decomposed under high-light stress as a result of a scavenging reaction of singlet oxygen generated in photosystem II. The levels of both alpha-tocopherol and of the reduced plastoquinone are not affected significantly in the absence of the inhibitor due to a high turnover rate of both prenyllipids, i.e., their degradation is compensated by fast biosynthesis. The calculated turnover rates under high-light conditions were twofold higher for total plastoquinone (0.23 nmol/h/ml of cell culture) than for alpha-tocopherol (0.11 nmol/h/ml). We have also found that the level of alpha-tocopherolquinone, an oxidation product of alpha-tocopherol, increases as the alpha-tocopherol is consumed. The same correlation was also observed for gamma-tocopherol and its quinone form. Moreover, in the presence of pyrazolate under low-light growth conditions, the synthesis of plastoquinone-C, a hydroxylated plastoquinone derivative, was stimulated in contrast to plastoquinone, indicating for the first time a functional role for plastoquinone-C. The presented data also suggest that the two plastoquinones may have different biosynthetic pathways in C. reinhardtii.     

Tocopherols play a crucial role in low-temperature adaptation and Phloem loading in Arabidopsis

To test whether tocopherols (vitamin E) are essential in the protection against oxidative stress in plants, a series of Arabidopsis thaliana vitamin E (vte) biosynthetic mutants that accumulate different types and levels of tocopherols and pathway intermediates were analyzed under abiotic stress. Surprisingly subtle differences were observed between the tocopherol-deficient vte2 mutant and the wild type during high-light, salinity, and drought stresses. However, vte2, and to a lesser extent vte1, exhibited dramatic phenotypes under low temperature (i.e., increased anthocyanin levels and reduced growth and seed production). That these changes were independent of light level and occurred in the absence of photoinhibition or lipid peroxidation suggests that the mechanisms involved are independent of tocopherol functions in photoprotection. Compared with the wild type, vte1 and vte2 had reduced rates of photoassimilate export as early as 6 h into low-temperature treatment, increased soluble sugar levels by 60 h, and increased starch and reduced photosynthetic electron transport rate by 14 d. The rapid reduction in photoassimilate export in vte2 coincides with callose deposition exclusively in phloem parenchyma transfer cell walls adjacent to the companion cell/sieve element complex. Together, these results indicate that tocopherols have a more limited role in photoprotection than previously assumed but play crucial roles in low-temperature adaptation and phloem loading.     

Diurnal rhythm of emergence from pupae in parasitic wasp Trichogramma evanescens Westw. (Insecta: Hymenoptera)

Abstract

Pupal eclosion of Trichogramma evanescens Westw. was studied in different conditions of light‐darkness and temperature fluctuations. The results revealed that under natural light cycles Trichogramma exhibits a distinct rhythm of emergence from pupae. Maximum emergence takes place in the morning. This rhythm persists in constant dim red light and temperature, so it is endogenous in nature. The rhythm can be entrained by artificial 24‐h temperature cycles or by day‐night cycles of light with a very low intensity of illumination (<0.01 lux). Nevertheless a single pulse of bright light or of high temperature is not able to reset the rhythm. The emergence rhythm was also absent if the culture was grown in constant darkness and temperature.     

Genome‐wide association mapping for phenotypic plasticity in rice

Phenotypic plasticity of plants in response to environmental changes is important for adapting to changing climate. Less attention has been paid to exploring the advantages of phenotypic plasticity in resource‐rich environments to enhance the productivity of agricultural crops. Here, we examined genetic variation for phenotypic plasticity in indica rice (Oryza sativa L.) across two diverse panels: (1) a Phenomics of Rice Adaptation and Yield (PRAY) population comprising 301 accessions; and (2) a Multi‐parent Advanced Generation Inter‐Cross (MAGIC) indica population comprising 151 accessions. Altered planting density was used as a proxy for elevated atmospheric CO₂ response. Low planting density significantly increased panicle weight per plant compared with normal density, and the magnitude of the increase ranged from 1.10 to 2.78 times among accessions for the PRAY population and from 1.05 to 2.45 times for the MAGIC population. Genome‐wide‐association studies validate three E nvironmental R esponsiveness (ER) candidate alleles (qER1–3) that were associated with relative response of panicle weight to low density. Two of these alleles were tested in 13 genotypes to clarify their biomass responses during vegetative growth under elevated CO₂ in Japan. Our study provides evidence for polymorphisms that control rice phenotypic plasticity in environments that are rich in resources such as light and CO₂.     

Spatial and temporal variation in Nothofagus pumilio growth at tree line along its latitudinal range (35°40'–55° S) in the Chilean Andes

Aim To identify the dominant spatial and temporal patterns of Nothofagus pumilio radial growth over its entire latitudinal range in Chile, and to find how these patterns relate to temperature and precipitation variation from instrumental records. Location This study comprises 48 tree line or high elevation N. pumilio sites in the Chilean Andes between 35° 36′ and 55° S. Nothofagus pumilio is a deciduous tree species that dominates the upper tree line of the Chilean and Argentinean Andes in this latitudinal range. Methods At each of the sampled sites, two cores from 15 to 40 living trees were collected using increment borers. Cores were processed, tree rings were measured and cross‐dated, using standard dendrochronological procedures. Radii from nearby sites were grouped into 13 study regions. A composite tree‐ring width chronology was developed for each region in order to capture and integrate the common growth patterns. For the identification of the dominant patterns of growth, as well as temperature and precipitation variation, we used principal components (PCs) analysis. Correlation analysis was used for the study of the relationship of N. pumilio tree‐ring growth with temperature and precipitation records. Results Nothofagus pumilio tree line elevation is 1600 m in the northernmost region and gradually decreases to 400 m in the southernmost region. Despite local differences along the transect, the decrease in tree line elevation is fairly constant, averaging c. 60 m per degree of latitude (111 km). Tree growth at the northernmost regions shows a positive correlation with annual precipitation (PC1‐prec) and negative correlation with mean annual temperature (PC2‐temp), under a Mediterranean‐type climate where water availability is a major limiting factor. Conversely, tree growth is positively correlated with mean annual temperature (PC1‐temp) in the southern portion of the gradient, under a relatively cooler climate with little seasonality in precipitation. Main conclusions Our findings indicate that temperature has a spatially larger control of N. pumilio growth than precipitation, as indicated by a significant (P < 0.05) either positive or negative correlation of tree growth and PC1‐temp and/or PC2‐temp for nine of the 13 regional chronologies (69.2% of the total), whereas precipitation is significantly correlated with only two chronologies (15.4% of the total). Temporal patterns of N. pumilio tree growth reflected in PC1‐growth for the period between 1778 and 1996 indicate an increasing trend with above the mean values after 1963, showing high loadings in the southern part of the gradient. This trend may be explained by a well‐documented increase in temperature in southern Patagonia. Ongoing and future research on N. pumilio growth patterns and their relationship to climate covering the Chilean and Argentinean Andes will improve the understanding of long‐term climate fluctuations of the last three to four centuries, and their relationship to global change at a wide range of spatial and temporal scales.     

Effect of light internsity on vesicle formation in Chlorobium

1. Chlorobium limicola forma sp. thiosulfatophilum was cultivated at 22 and 22000 lux. 2. The content of bchl d on a protein basis in the low light intensity cultures was about twice that of the high light intensity cultures. 3. After growth at 22 lux the red bchl d peak was at c. 743 nm, while at the higher intensity this peak was at c. 732 nm. 4. Electron microscopy of thin sections of Chlorobium revealed that vesicle size was greater at the low light intensity than at the high. 5. This was confirmed by sucrose density gradient centrifugation of differentially ¹⁴C-labelled vesicles from cultures grown at the two intensities. 6. The optimum temperature for growth was about 35°C. Incubation at the optimum temperature was particularly beneficial at high light intensity.Abbreviation bchl bacteriochlorophyll