Paramylon synthesis by Euglena gracilis photoheterotrophically grown under low O2 pressure

Special culture conditions for Euglena gracilis Z and ZR are described. They induce interactions between the chloroplast and mitochondrial metabolisms leading to paramylon synthesis. When grown in continuous light under pure nitrogen and in the presence of lactate as the sole carbon source, sugar synthesis occurs during the first 24 h of culture with the participation of both mitochondria (using lactate) and of chloroplasts (fixing CO₂ from lactate decarboxylation). The activities of ribulose bisphosphate carboxylase, phosphoenolpyruvate carboxylase, and phosphoenolpyruvate carboxykinase are very high and mitochondria and chloroplasts develop then a common network of vesicles in which paramylon grains can be seen. Electron micrographs demonstrate membrane continuity between the two types of organelles. Occasionally the mitochondrial matrix and the chloroplast stroma are separated by only a unit membrane.Abbreviations Chl chlorophyll - OAA oxaloacetic acid - PEP phosphoenolpyruvate - RuBP ribulose bisphosphate - DTT 1,4-dithiothreitol - PVP polyvinylpyrrolidone     

C4 syndrome of the species in theDichotomiflora group of the genusPanicum (Gramineae)

Leaf anatomy, pattern of post-illumination CO₂ burst (PIB) and activity of three C₄-acid decarboxylating enzymes in C₄ photosynthesis were investigated with the leaves of five species in theDichotomiflora group of the genusPanicum. All species had mestome sheaths, exhibited the sharp pattern of PIB in less than 30 sec of darkness and were classified as NAD-malie enzyme species biochemically. However, they clearly fell into two groups according to the difference in chloroplast location in bundle sheath cells (BSC).P. coloratum var.makarikariense, P. lanipes andP. stapfianum had centripetal chloroplasts, whereasP. laevifolium andP. longijubatum had centrifugal chloroplasts, whereas cv. Kabulabula and cv. Solai had centrifugal chlorplasts. The results indicate that theDichotomiflora group had the two leaf anatomical variations of NAD-malic enzyme species. In addition, the results onP. coloratum suggest that this species may be divided into two separate species by chloroplast location in BSC. The ultrastructural features of leaves ofP. dichtomiflorum, NAD-malic enzyme species with centrifugal chloroplasts, were also investigated. Chloroplasts in BSC had well-developed grana, and numerous large mitochondria with extensively developed internal membrane structure were restricted to the area between the chloroplsts and the vacuole in BSC.     

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

Summary The ultrastructure of chloroplasts from two genera of coenocytic green algae,Codium andCaulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO₂-dependent and ferricyanide-dependent O₂ evolution was measured under the same conditions of osmotic strength and detergent concentration.The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O₂ evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO₂-dependent O₂ evolution in both species, but this was partially restored in theCodium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when theCodium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen.Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as inCodium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar,Caulerpa chloroplasts could not regain their capacity for CO₂-dependent O₂ evolution.Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids.Caulerpa chloroplasts were less sensitive to detergent than those ofCodium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.     

Analysis of chloroplast and mitochondrial DNA in asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens

Organellar DNA of asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens were analysed by DNA hybridization methods using the spinach chloroplast probepSBD, wheat mitochondrial genenad5 and petunia mitochondrial geneorf25. Eight of the 12 asymmetric hybrid plants hadS. tuberosum chloroplast DNA and the remaining fourS. brevidens chloroplast DNA. A novel mitochondrial hybridization pattern was present in eight out of the 17 hybrids tested. In six hybrids, novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Isolation of a cDNA encoding mitochondrial citrate synthase from Arabidopsis thaliana

We isolated a cDNA clone from Arabidopsis thaliana encoding the TCA cycle enzyme, citrate synthase. The plant enzyme displays 48% and 44% amino acid residue similarity with the pig, and yeast polypeptides, respectively. Many proteins, including citrate synthase, which are destined to reside in organelles such as mitochondria and chloroplasts, are the products of the nucleocytoplasmic protein synthesizing machinery and are imported post-translationally to the site of function. We present preliminary investigations toward the establishment of an in vitro plant mitochondrial import system allowing for future studies to dissect this process in plants where the cell must differentiate between mitochondria and chloroplast and direct their polypeptides appropriately.     

Cybrids of Nicotiana tabacum and Petunia hybrida have an intergeneric mixture of chloroplasts from P. hybrida and mitochondria identical or similar to N. tabacum

Summary The mitochondrial genomes of cybrids of Nicotiana tabacum containing chloroplasts of Petunia hybrida were characterized by restriction endonuclease digestion and agarose gel electrophoresis. Cybrids that displayed normal growth and development contained mitochondrial DNA indistinguishable from N. tabacum mitochondrial DNA. Cybrids that displayed abnormal growth and development contained mitochondrial DNA that differed from N. tabacum either by possessing a few additional fragments, by lacking a few fragments, or both. In spite of these differences, the mitochondrial DNA of cybrids showing abnormal growth and development was much more similar to N. tabacum than to P. hybrida mitochondrial DNA. In those cybrids that contained P. hybrida chloroplasts and N. tabacum mitochondria, cotransfer of cytoplasmic organelles did not occur. Although P. hybrida chloroplasts can interact compatibly with the N. tabacum nucleus, no cybrids were found in which P. hybrida mitochondria coexisted with the N. tabacum nucleus.     

Immunological comparison of the pyruvate dehydrogenase complexes from pea mitochondria and chloroplasts

The pyruvate dehydrogenase complex (PDC) in pea (Pisum sativum L., cv. Little Marvel) was studied immunologically using antibodies to specific subunits of mammalian PDC. Pea mitochondria and chloroplasts were both found to contain PDC, but distinct differences were noted in the subunit relative molecular mass (M_r) values of the individual enzymes in the mitochondrial and chloroplast PDC complexes. In particular, the mitochondrial E3 enzyme (dihydrolipoamide dehydrogenase; EC 1.8.1.4) has a high subunit M_r value of 67 000, while the chloroplast E3 enzyme has a subunit M_r value of 52 000, similar in size to the prokaryotic, yeast ad mammalian E3 enzymes. In addition, component X (not previously noted in plant PDC) was also found to be present in two distinct forms in pea mitochondrial and chloroplast complexes. As in the case of E3, mitochondrial component X has a higher subunit M_r value (67 000) than component X from chloroplasts (48 000), which is similar in size to its mammalian counterpart. The subunit M_r value of E2 (dihydrolipoamide acetyltransferase; EC 2.3.1.12) in both mitochondria and chloroplasts (50 000) is lower than that of mammalian E2 (74 000) but similar to that of yeast E2 (58 000), and is consistent with the presence of only a single lipoyl domain. Neither mitochondria nor chloroplasts showed any appreciable cross-reactivity with antiserum to mammalian E1 (pyruvate dehydrogenase; EC 1.2.4.1). However, mitochondria cross-reacted strongly with antiserum to yeast E1, giving a single band (M_r 41 000) which is thought to be E1_a. Chloroplasts showed no cross-reactivity with yeast E1, indicating that the mitochondrial E1_a subunit and its chloroplast equivalent are antigenically distinct polypeptides.Abbreviations E1 pyruvate dehydrogenase - E2 dihydrolipoamide acetyltransferase - E3 dihydrolipoamide dehydrogenase - M_r relative molecular mass - PDC pyruvate dehydrogenase multienzyme complex - SDS sodium dodecyl sulphate The financial support of the Agricultural and Food Research Council is gratefully acknowledged. We thank Steve Hill (Department of Botany, University of Edinburgh, UK) for advice on mitochondrial isolation, and James Neagle (Department of Biochemistry, University of Glasgow) and Ailsa Carmichael for helpful discussion.     

Peculiar properties of the PsaF photosystem I protein from the green alga Chlamydomonas reinhardtii: presequence independent import of the PsaF protein into both chloroplasts and mitochondria

It has previously been shown that presequences of nuclear-encoded chloroplast proteins from the green alga Chlamydomonas reinhardtii contain a region that may form an amphiphilic -helix, a structure characteristic of mitochondrial presequences. We have tested two precursors of chloroplast proteins (the PsaF and PsaK photosystem I subunits) from C. reinhardtii for the ability to be imported into spinach leaf mitochondria in vitro. Both precursors bound to spinach mitochondria. The PsaF protein was converted into a protease-protected form with high efficiency in a membrane potential-dependent manner, indicating that the protein had been imported, whereas the PsaK protein was not protease protected. The protease protection of PsaF was not inhibited by a synthetic peptide derived from the presequence of the N. plumbaginifolia mitochondrial F₁ subunit. Furthermore, if the presequence of PsaF was truncated or deleted by in vitro mutagenesis, the protein was still protease-protected with approximately the same efficiency as the full-length precursor. These results indicate that PsaF can be imported by spinach mitochondria in a presequence-independent manner. However, even in the absence of the presequence, this process was membrane potential-dependent. Interestingly, the presequence-truncated PsaF proteins were also protease-protected upon incubation with C. reinhardtii chloroplasts. Our results indicate that the C. reinhardtii chloroplast PsaF protein has peculiar properties and may be imported not only into chloroplasts but also into higher-plant mitochondria. This finding indicates that additional control mechanisms in the cytosol that are independent of the presequence are required to achieve sorting between chloroplasts and mitochondria in vivo.Abbreviations cTP chloroplast transit peptide - mTP mitochondrial targeting peptide - Rubisco ribulose-1,5-bisphosphate carboxylase/oxygenase - pF₁(1,25) a synthetic peptide derived from the first 25 residues of the Nicotiana plumbaginifolia mitochondrial ATP synthase F₁ subunit - PsaF(2–30) and PsaF(2–61) mutant proteins lacking regions corresponding to residues 2–30 and 2–61 in the PsaF precursor protein, respectively     

Cytoplasmic inheritance of mitochondria and chloroplasts in the anisogamous brown alga Mutimo cylindricus (Phaeophyceae)

Based on the morphology of gametes, sexual reproduction in brown algae is usually classified into three types: isogamy, anisogamy, and oogamy. In isogamy, chloroplasts and chloroplast DNA (chlDNA) in the sporophyte cells are inherited biparentally, while mitochondria (or mitochondrial DNA, mtDNA) is inherited maternally. In oogamy, chloroplasts and mitochondria are inherited maternally. However, the patterns of mitochondrial and chloroplast inheritance in anisogamy have not been clarified. Here, we examined derivation of mtDNA and chlDNA in the zygotes through strain-specific PCR analysis using primers based on single nucleotide polymorphism in the anisogamous brown alga Mutimo cylindricus. In 20-day-old sporophytes after fertilization, mtDNA and chlDNA derived from female gametes were detected, thus confirming the maternal inheritance of both organelles. Additionally, the behavior of mitochondria and chloroplasts in the zygotes was analyzed by examining the consecutive serial sections using transmission electron microscopy. Male mitochondria were isolated or compartmentalized by a double-membrane and then completely digested into a multivesicular structure 2 h after fertilization. Meanwhile, male chloroplasts with eyespots were observed even in 4-day-old, seven-celled sporophytes. The final fate of male chloroplasts could not be traced. Organelle DNA copy number was also examined in female and male gametes. The DNA copy number per chloroplast and mitochondria in male gametes was lower compared with female organelles. The degree of difference is bigger in mtDNA. Thus, changes in different morphology and DNA amount indicate that maternal inheritance of mitochondria and chloroplasts in this species may be based on different processes and timing after fertilization.

     

Changes in Chloroplast Ultrastructure in Pssu-ipt Tobacco During Plant Ontogeny

Changes in chloroplast ultrastructure and total content of endogenous cytokinins (CK) were studied during different phases of plant development in transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1). Permanent overproduction of CK was found in both rooted (SE) and grafted (G) Pssu-ipt plants in all phases of plant development with the peak in vegetative and flowering phase in the latter ones. No such a correlation was observed in SE on the contrary to control non-transgenic plants (SR1) and grafts (SRG), which showed also CK increase at juvenile and flowering phases. No significant differences in parameters of chloroplast ultrastructure, such as length of chloroplast, starch content, granum width, and number of thylakoids per granum, were proved between chloroplasts from young mature leaves of control and transgenic tobacco during plant ontogeny. Nevertheless, several anomalies in the ultrastructure of cell organelles were found in Pssu-ipt tobacco. Amoeboid shape of chloroplasts was often observed in connection with tubular clusters resembling peripheral reticulum. The distinct crystalline structures located in chloroplasts might be formed by LHC protein aggregates. Smaller crystals of unknown composition were found also in mitochondria. Numerous crystalline cores were present in peroxisomes. The alterations might be the result of imbalance of phytohormone content, degradation effect of CK overproduction, or the example of acclimation to permanent stress.     

C4-Dicarboxylic acid metabolism in bundle-sheath chloroplasts,mitochondria and strands of Eriochloa borumensis Hack., a phosphoenolpyruvate-carboxykinase type C4 species

C₄-acid metabolism by isolated bundlesheath chloroplasts, mitochondria and strands of Eriochloa borumensis Hack., a phosphoennolpyruvate-carboxykinase (PEP-CK) species, was investigated. Aspartate, oxaloacetate (OAA) and malate were decarboxylated by strands with several-fold stimulation upon illumination. There was strictly light-dependent decarboxylation of OAA and malate by the chloroplasts, but the chloroplasts did not decarboxylate aspartate in light or dark. PEP was a primary product of OAA or malate decarboxylation by the chloroplasts and its formation was inhibited by 3-(3,4-dichlorophenyl)-1, 1-dimethylurea or NH₄Cl. There was very little conversion of PEP to pyruvate by bundle-sheath chloroplasts, mitochondria or strands. Decarboxylation of the three C₄-acids by mitochondria was light-independent. Pyruvate was the only product of mitochondrial metabolism of C₄-acids, and was apparently transaminated in the cytoplasm since PEP and alanine were primarily exported out of the bundle-sheath strands. Light-dependent C₄-acid decarboxylation by the chloroplasts is suggested to be through the PEP-CK, while the mitochondrial C₄-acid decarboxylation may proceed through the NAD-malic enzyme (NAD-ME) system. In vivo both aspartate and malate are considered as transport metobolites from mesophyll to bundle-sheath cells in PEP-CK species. Aspartate would be metabolized by the mitochondria to OAA. Part of the OAA may be converted to malate and decarboxylated through NAD-ME, and part may be transported to the chloroplasts for decarboxylation through PEP-CK localized in the chloroplasts. Malate transported from mesophyll cells may serve as carboxyl donor to chloroplasts through the chloroplastic NAD-malate dehydrogenase and PEP-CK. Bundle-sheath strands and chloroplasts fixed ¹⁴CO₂ at high rates and exhibited C₄-acid-dependent O₂ evolution in the light. Studies with 3-mercaptopicolinic acid, a specific inhibitor of PEP-CK, have indicated that most (about 70%) of the OAA formed from aspartate is decarboxylated through the chloroplastic PEP-CK and the remaining (about 30%) OAA through the mitochondrial NAD-ME. Pyruvate stimulation of aspartate decarboxylation is discussed; a pyruvate-alanine shuttle and an aspartate-alanine shuttle are proposed between the mesophyll and bundle-sheath cells during aspartate decarboxylation through the PEP-CK and NAD-ME system respectively.Abbreviations CK carboxykinase - -Kg -ketoglutarate - ME malic enzyme - 3-MPA 3-mercaptopicolinic acid - OAA oxaloacetate - PEP phosphoenolpyruvate - R5P ribose-5-phosphate     

The three-dimensional structure of theCyphomandra betacea chloroplast peripheral reticulum

Summary The peripheral reticulum (PR) inCyphomandra betacea chloroplasts originates as vesicles budding from the inner membrane of the chloroplast envelope which elongate to form tubules then aggregate or branch to form discrete PR units. Individual PR units of many coiled tubules may be connected with other units by narrow tubules. Serial sectioning revealed the discrete units to be approximately 650–1,000 nm wide, 400–500 nm high and 500–600 nm deep and to possess a compact morphology. TheCyphomandra PR structure is compared to the morphologies of chloroplast reticula reported for other plant species. A scheme to group PR from different species into 3 distinct morphological categories is outlined and discussed.     

Relationship of the tubular system with other organelles in chlorride cells of the channel catfish,Ictalurus punctatus

The morphology of chloride cells in the channel catfish, Ictalurus punctatus, has been studied by transmission electron microscopy. The chloride cell possesses abundant tubules, mitochondria, and granules. The employment of a special membrane stain in conjunction with a two- or tridimensional analysis reveals a complex interjoining and interlocking ring system of tubules. Tubular sides constituting the complex rings frequently lack granules. The tubular rings join with tubulous mitochondrial profiles and other cytoplasmic components. © 1995 Wiley-Liss, Inc.     

Intracellular localization of serine acetyltransferase in spinach leaves

Intact chloroplasts isolated from spinach leaves by a combination of differential and Percoll density gradient centrifugation and free of mitochondrial and peroxisomal contamination contained about 35% of the total leaf serine acetyltransferase (EC 2.3.1.30) activity. No appreciable activity of the enzyme could be detected in the gradient fractions containing broken chloroplasts, mitochondria, and peroxisomes. L-cysteine added to the incubation mixture at 1 mM almost completely inhibited serine acetyltransferase activity, both of leaf and chloroplast extracts. D-cysteine was much less inhibitory. L-cystine up to 5 mM and O-acetyl-L-serine up to 10 mM had no effect on the enzyme activity. When measured at pH 8.4, the enzyme extracted from the leaves had a K _m for L-serine of 2.4, the enzyme from the chloroplasts a K _m of 2.8 mM.Abbreviations NAS N-acetyl-L-serine - NADP-GPD NADP-dependent glyceraldehyde-3-phosphate dehydrogenase - OAS O-acetyl-L-serine - OASSase O-acetyl-L-serine sulfhydrylase - 3-PGA D-3-phosphoglycerate - SATase serine acetyltransferase     

NaCl对齿肋赤藓叶肉细胞超微结构的影响

齿肋赤藓(Syntrichia caninervis)是古尔班通古特沙漠苔藓结皮层中的优势物种,对荒漠生态系统的稳定性及功能多样性具有十分重要的意义。利用透射电镜技术对不同浓度Na Cl胁迫下齿肋赤藓叶肉细胞超微结构进行了观察。结果表明:齿肋赤藓叶肉细胞在未胁迫(0 mmol/L)处理下排列疏松,各种细胞结构完整,叶绿体基质排列均匀且叶绿体内含少量淀粉粒和脂质球。在轻度盐Na Cl胁迫(100 mmol/L)下,齿肋赤藓叶肉细胞结构依然保持完整,叶绿体基质均匀,叶肉细胞超微结构仅有较小变化。在中度盐Na Cl胁迫(200、300 mmol/L)下,齿肋赤藓叶肉细胞发生质壁分离,出现晶体结构,且中央大液泡发生破裂;叶绿体由梭形变成椭球形或圆球状,出现空泡化并伴随有轻微的解体;叶绿体类囊体肿胀,脂质球数量增加。在高度Na Cl胁迫(400、500 mmol/L)下,齿肋赤藓细胞的质壁分离加剧,叶肉细胞出现大量泡状结构和膜片层,叶肉细胞死亡;叶绿体片层结构消失,空泡化加重,脂质球数量增加且体积变大,叶绿体内外膜消失,叶绿体大部分解体,在叶肉细胞中几乎看不到叶绿体的存在。上述结果表明,叶绿体膜结构的损伤与盐胁迫下叶肉细胞死亡有密切关系。     

Photosynthesis, Photorespiration and Respiration of Chloroplasts From Acetabularia mediterrania

A chloroplast fraction isolated from Acetabularia mediterrania carries on photosynthesis at rates essentially equal to those of whole cells. Electron and phase contrast microscopy reveals that the chloroplasts are intact and well preserved. Preparations contain no identifiable peroxisomes, but some cytoplasmic and mitochondrial contamination is present. Photosynthesis and CO₂ production in light by chloroplast preparations are in many respects similar to that of bean leaves, although the measured rates are somewhat lower. Respiration and photosynthesis of chloroplast preparations and whole cells of Acetabularia is essentially similar except that cells have a strong dark-type respiration which continues in light and is CO₂ dependent, the substrate being mainly recent photosynthate. The data suggest that chloroplasts are the site of photorespiration.     

NaCl胁迫对宁夏枸杞叶和幼根显微及超微结构的影响

以宁夏枸杞为材料,采用超薄切片技术制备样品,应用光学显微镜和透射电镜分析了不同浓度NaCl胁迫条件下宁夏枸杞叶和幼根显微及超微结构的变化.结果表明:随着NaCl胁迫的加重,(1)叶片上表皮细胞增厚,栅栏组织细胞出现缩短现象,排列疏松且紊乱;幼根的初生结构无明显变化.(2)叶片栅栏组织中叶绿体不再紧靠在细胞膜上,叶绿体双...     

Density gradient and differential centrifugation methods for chloroplast purification and enzyme localization in leaf tissue

Intact chloroplasts, isolated by differential-centrifugation and sucrose density-gradient methods, have been used to study the degree of apparent artifactual adsorption of citrate synthase (EC 4.1.3.7) to the organelles. Unfractionated homogenates layered directly on to sucrose density gradients gave elution profiles showing definite citrate synthase activity in the intact and broken plastid regions, along with the major mitochondrial peak. Nonreversible triose-phosphate dehydrogenase (EC 1.2.1.9), a cytosolic marker, showed no activity in any particulate region of the gradient. Crude chloroplast pellets and twice washed (resedimented and resuspended) chloroplasts layered on to the gradient gave progressively reduced citrate synthase activity in the plastid regions. In addition, the peak in the mitochondrial region of the gradient was virtually eliminated when washed chloroplasts were fractionated on the gradient. Differences in protein binding behavior on the chloroplasts may necessitate the inclusion of a washing step in chloroplast purification procedures. Moreover, repeated sedimentation and resuspension can also be a useful procedure to reduce mitochondrial contamination of chloroplast preparations.Work supported by the Rubber Research Institute of Malaysia     

Plant Cell Membranes as Biochemical Targets of the Phytotoxin Helminthosporol

Helminthosporol is one of the natural sesquiterpenoid toxins isolated and identified in the culture medium of the phytopathogenic ascomycete fungus Cochliobolus sativus. The effect of this phytotoxin was investigated on enzymatic activities, electron and ion transport in mitochondria, chloroplasts, and microsomes of plant. The results indicate that helminthosporol drastically affects the membrane permeability of these organelles to protons and subtrate anions, inhibiting the mitochondrial oxidative phosphorylation, the photophosphorylation in chloroplasts, and the proton pumping across the cell plasma membrane. The 1,3--glucan synthase activity, involved in defense mechanisms of plant cells against stress and damage, e.g., during pathogen attack, was also strongly inhibited by the toxin.     

Temperature-sensitive formation of chloroplast protrusions and stromules in mesophyll cells of Arabidopsis thaliana

Summary. In leaf mesophyll cells of transgenic Arabidopsis thaliana plants expressing GFP in the chloroplast, stromules (stroma-filled tubules) with a length of up to 20 μm and a diameter of about 400–600 nm are observed in cells with spaces between the chloroplasts. They appear extremely dynamic, occasionally branched or polymorphic. In order to investigate the effect of temperature on chloroplasts, we have constructed a special temperature-controlled chamber for usage with a light microscope (LM-TCC). This LM-TCC enables presetting of the temperature for investigation directly at the microscope stage with an accuracy of ±0.1 °C in a temperature range of 0 °C to +60 °C. With the LM-TCC a temperature-dependent appearance of chloroplast protrusions has been found. These structures have a considerably smaller length-to-diameter ratio than typical stromules and reach a length of 3–5 μm. At 5–15 °C (low temperatures), almost no chloroplast protrusions are observed, but they appear with increasing temperatures. At 35–45 °C (high temperatures), numerous chloroplast protrusions with a beaklike appearance extend from a single chloroplast. Interaction of stromules with other organelles has also been investigated by transmission electron microscopy. At 20 °C, transverse sections of stromules are frequently observed with a diameter of about 450 nm. A close membrane-to-membrane contact of stromules with the nucleus and mitochondria has been visualised. Golgi stacks and microbodies are found in the spatial vicinity of stromules. At 5 °C, virtually no chloroplast protrusions or stromules are observed. At 35 °C, chloroplast protrusions are present as broader thylakoid-free stroma-filled areas, resulting in an irregular chloroplast appearance. Correspondence and reprints: Department of Physiology and Cell Physiology of Alpine Plants, Institute of Botany, University of Innsbruck, Sternwartestrasse 15, 6020 Innsbruck, Austria.