Chloroplast ultrastructure in leaves of tobacco plants with the introduced gene for the acyl-lipid Δ9-desaturase from <Emphasis Type="Italic">Synechococcus vulcanus</Emphasis> at normal and low temperature

Ultrastructural changes in chloroplasts of tobacco plants (Nicotiana tabacum L.) with the introduced desC gene for the acyl-lipid Δ9-desaturase from the thermophilic cyanobacterium Synechococcus vulcanus were investigated during plant acclimation to cold. Control plants were transformed with an empty pGA482 binary vector. At optimum growth temperature, a decreased number of grana and thylakoids and an increased number of plastoglobules and their larger area were observed in transgenic plants when compared to control ones. In control plants, acclimation to cold (6 days at 10°C) resulted in the larger areas of chloroplasts and grana. These changes indicated starting cold-induced injuries manifested in swelling of the stroma and a slight decrease in the total number of thylakoids in the chloroplast. In contrast, transgenic plants responded to cold by reducing the chloroplast, granal, and plastoglobule areas. Meantime, the number of thylakoids per granum increased noticeably. The total number of thylakoids in the chloroplast increased from 123 to 203. It was concluded that expression of the acyl-lipid Δ9-desaturase gene in tobacco plants provided for the formation of the cell ultrastructure similar to one characteristic of cold-tolerant plants.     

A homolog of GuidedEntry of Tail-anchored proteins3 functions in membrane-specific protein targeting in chloroplasts of Arabidopsis

The chloroplasts and mitochondria of photosynthetic eukaryotes contain proteins that are closely related to cytosolic Guided Entry of Tail-anchored proteins3 (Get3). Get3 is a targeting factor that efficiently escorts tail-anchored (TA) proteins to the ER. Because other components of the cytosolic-targeting pathway appear to be absent in organelles, previous investigators have asserted that organellar Get3 homologs are unlikely to act as targeting factors. However, we show here both that the Arabidopsis thaliana chloroplast homolog designated as GET3B is structurally similar to cytosolic Get3 proteins and that it selectively binds a thylakoid-localized TA protein. Based on genetic interactions between a get3b mutation and mutations affecting the chloroplast signal recognition particle-targeting pathway, as well as changes in the abundance of photosynthesis-related proteins in mutant plants, we propose that GET3B acts primarily to direct proteins to the thylakoids. Furthermore, through molecular complementation experiments, we show that function of GET3B depends on its ability to hydrolyze ATP, and this is consistent with action as a targeting factor. We propose that GET3B and related organellar Get3 homologs play a role that is analogous to that of cytosolic Get3 proteins, and that GET3B acts as a targeting factor in the chloroplast stroma to deliver TA proteins in a membrane-specific manner.

A chloroplast homolog of a cytosolic protein involved in posttranslational targeting of proteins to the ER is a targeting factor in chloroplasts that directs proteins to the thylakoids.     

Differences in chlorophyll-protein complexes and composition of polypeptides between thylakoids from bundle sheaths and mesophyll cells in maize

Thylakoids from enzymatically separated bundle sheath and mesophyll tissue chloroplasts were examined for their chlorophyll-proteins by tube sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS-PAGE). Differences were found in distribution of chlorophyll among peaks. The chlorophyll-protein a peak (CPa), considered to be the photosystem II (PSII) reaction centre by many authors, was seen to be absent in bundle sheath thylakoid samples. The slab SDS-PAGE revealed the absence of the polypeptides present in PSII preparations of chloroplast subfractions having only PSII activity. This finding confirms Anderson's hypothesis of the structure of grana and stroma thylakoids.     

Phenotypically normal transgenic T-cyt tobacco plants as a model for the investigation of plant gene expression in response to phytohormonal stress

The tumour-inducing T-DNA gene 4 (T-cyt gene) of the nopaline Ti plasmid pTiC58 was cloned and introduced into tobacco cells by leaf disc transformation using Agrobacterium plasmid vectors. Tobacco shoots exposed to elevated cytokinin levels were unable to develop roots and lacked apical dominance. Using exogenously applied phytohormone manipulations we were able to regenerate morphologically normal transgenic tobacco plants which differed in endogenous cytokinin levels from normal untransformed plants. Although T-cyt gene mRNA levels, as revealed by dot-blot hybridization data, in these rooting plants were only about half those in primary transformed shoots the total amount of cytokinins was much lower than in crown gall tissue or cytokinin-type transformed shoots as reported by others. Nevertheless the cytokinin content in T-cyt plants was about 3 times greater than in control tobacco plants.Elevated cytokinin levels have been shown to change the expression of several plant genes, including some nuclear genes encoding chloroplast proteins. Our results show that the mRNA levels of chloroplast rbcL gene increase in cytokinin-type transgenic tobacco plants as compared with untransformed plants. Data obtained suggest that T-cyt transgenic plants are a good model for studying plant gene activity in different parts of the plant under endogenous cytokinin stress.     

Potato virus Y HC-Pro Reduces the ATPase Activity of NtMinD,Which Results in Enlarged Chloroplasts in HC-Pro Transgenic Tobacco

Potato virus Y (PVY) is an important plant virus and causes great losses every year. Viral infection often leads to abnormal chloroplasts. The first step of chloroplast division is the formation of FtsZ ring (Z-ring), and the placement of Z-ring is coordinated by the Min system in both bacteria and plants. In our lab, the helper-component proteinase (HC-Pro) of PVY was previously found to interact with the chloroplast division protein NtMinD through a yeast two-hybrid screening assay and a bimolecular fluorescence complementation (BiFC) assay in vivo. Here, we further investigated the biological significance of the NtMinD/HC-Pro interaction. We purified the NtMinD and HC-Pro proteins using a prokaryotic protein purification system and tested the effect of HC-Pro on the ATPase activity of NtMinD in vitro. We found that the ATPase activity of NtMinD was reduced in the presence of HC-Pro. In addition, another important chloroplast division related protein, NtMinE, was cloned from the cDNA of Nicotiana tabacum. And the NtMinD/NtMinE interaction site was mapped to the C-terminus of NtMinD, which overlaps the NtMinD/HC-Pro interaction site. Yeast three-hybrid assay demonstrated that HC-Pro competes with NtMinE for binding to NtMinD. HC-Pro was previously reported to accumulate in the chloroplasts of PVY-infected tobacco and we confirmed this result in our present work. The NtMinD/NtMinE interaction is very important in the regulation of chloroplast division. To demonstrate the influence of HC-Pro on chloroplast division, we generated HC-Pro transgenic tobacco with a transit peptide to retarget HC-Pro to the chloroplasts. The HC-Pro transgenic plants showed enlarged chloroplasts. Our present study demonstrated that the interaction between HC-Pro and NtMinD interfered with the function of NtMinD in chloroplast division, which results in enlarged chloroplasts in HC-Pro transgenic tobacco. The HC-Pro/NtMinD interaction may cause the formation of abnormal chloroplasts in PVY-infected plants.     

Too rigid to fold: Carotenoid-dependent decrease in thylakoid fluidity hampers the formation of chloroplast grana

In chloroplasts of land plants, the thylakoid network is organized into appressed regions called grana stacks and loosely arranged parallel stroma thylakoids. Many factors determining such intricate structural arrangements have been identified so far, including various thylakoid-embedded proteins, and polar lipids that build the thylakoid matrix. Although carotenoids are important components of proteins and the lipid phase of chloroplast membranes, their role in determining the thylakoid network structure remains elusive. We studied 2D and 3D thylakoid network organization in carotenoid-deficient mutants (ccr1-1, lut5-1, szl1-1, and szl1-1npq1-2) of Arabidopsis (Arabidopsis thaliana) to reveal the structural role of carotenoids in the formation and dynamics of the internal chloroplast membrane system. The most significant structural aberrations took place in chloroplasts of the szl1-1 and szl1-1npq1-2 plants. Increased lutein/carotene ratio in these mutants impaired the formation of grana, resulting in a significant decrease in the number of thylakoids used to build a particular stack. Further, combined biochemical and biophysical analyses revealed that hampered grana folding was related to decreased thylakoid membrane fluidity and significant changes in the amount, organization, and phosphorylation status of photosystem (PS) II (PSII) supercomplexes in the szl1-1 and szl1-1npq1-2 plants. Such changes resulted from a synergistic effect of lutein overaccumulation in the lipid matrix and a decreased level of carotenes bound with PS core complexes. Moreover, more rigid membrane in the lutein overaccumulating plants led to binding of Rubisco to the thylakoid surface, additionally providing steric hindrance for the dynamic changes in the level of membrane folding.

Increases in lutein/carotenoid ratios lead to decreased thylakoid fluidity and hamper grana folding due to carotenoid-dependent changes in both photosynthetic complexes and lipid matrix organization.     

Isolation and characterization of paracrystalline structures from transgenic P<Emphasis Type="Italic">ssu-ipt</Emphasis> tobacco

Distinct crystalloids were found in chloroplasts of transgenic Pssu-ipt tobacco (Nicotiana tabacum L. cv. Petit Havana SR1) overproducing endogenous cytokinins. They were present both in rooted (T) and grafted (TC) transgenic plants contrary to control tobacco (C). The fractions enriched by crystalloids were isolated from chloroplasts using a continuous or a discontinuous Percoll gradient. Chlorophyll (Chl) fluorescence emission spectra at 77 K indicated the presence of aggregates of light-harvesting complex proteins (LHC2) that was not connected to reaction centres of photosystem 2 both in isolated chloroplasts and in the fraction of 80 % Percoll gradient from both types of transgenic tobacco. Further analyses, i.e. pigment contents, polypeptide composition by SDS-PAGE, and immunoblotting support our hypothesis that crystalloids inside chloroplasts of transgenic tobacco are formed by LHC2 aggregates. Treatment with two distinct detergents, chosen with respect to their effects (i.e. β-dodecyl maltoside or Triton X-100), resulted in different degree of disintegration of Chl a/b proteins in transgenic plants compared to the control. Electron microscopic observations and immunogold labelling with specific LHC2 antibodies carried on the resin embedded leaf sections or free suspensions of chloroplasts showed that gold particles were bound preferentially on the outer surface of crystalloids. Three-dimensional reconstruction of chloroplasts and crystalloids proved that paracrystalline structures varied moderately in their size and took up a significant portion of total chloroplast volume.     

Heat shock increases thermotolerance of photosynthetic electron transport and the content of chloroplast membranes and lipids in wheat leaves

Preliminary heating of 15-16-day-old wheat (Triticum aestivum L.) plants for 3 h at 37–38°C (heat shock, HS) increased the tolerance of photosynthetic electron transport (determined as the reduction of 2,6-dichlorophenol indophenol by isolated chloroplasts) toward heating of leaves at 42–48°C in high light (100 klx). At the same time, HS did not affect the activity of the xanthophyll cycle reactions in the 30–48°C temperature range. HS exposure induced an increase in the thylakoid length, the number of grana, and the average number of thylakoids per granum. The volume of the thylakoid system increased 1.4-fold. Such indices as the total content of chlorophylls (a + b), the chlorophyll a/b ratio, as well as the contents of individual carotenoids, chloroplast membrane proteins, and the soluble leaf proteins remained unchanged. The de novo photosynthetic membrane formation was accompanied by the 1.5-fold increase in major chloroplast lipids. It was concluded that, in mature wheat chloroplasts, HS induced the formation of thylakoids characterized by a changed molecular structure and by increased lipid/protein and lipid/chlorophyll ratios.     

Overexpression of Camellia sinensis H1 histone gene confers abiotic stress tolerance in transgenic tobacco

Key message

Overexpression of CsHis in tobacco promoted chromatin condensation, but did not affect the phenotype. It also conferred tolerance to low-temperature, high-salinity, ABA, drought and oxidative stress in transgenic tobacco.

Abstract

H1 histone, as a major structural protein of higher-order chromatin, is associated with stress responses in plants. Here, we describe the functions of the Camellia sinensis H1 Histone gene (CsHis) to illustrate its roles in plant responses to stresses. Subcellular localization and prokaryotic expression assays showed that the CsHis protein is localized in the nucleus, and its molecular size is approximately 22.5 kD. The expression levels of CsHis in C. sinensis leaves under various conditions were investigated by qRT-PCR, and the results indicated that CsHis was strongly induced by various abiotic stresses such as low-temperature, high-salinity, ABA, drought and oxidative stress. Overexpression of CsHis in tobacco (Nicotiana tabacum) promoted chromatin condensation, while there were almost no changes in the growth and development of transgenic tobacco plants. Phylogenetic analysis showed that CsHis belongs to the H1C and H1D variants of H1 histones, which are stress-induced variants and not the key variants required for growth and development. Stress tolerance analysis indicated that the transgenic tobacco plants exhibited higher tolerance than the WT plants upon exposure to various abiotic stresses; the transgenic plants displayed reduced wilting and senescence and exhibited greater net photosynthetic rate (Pn), stomatal conductance (Gs) and maximal photochemical efficiency (Fv/Fm) values. All the above results suggest that CsHis is a stress-induced gene and that its overexpression improves the tolerance to various abiotic stresses in the transgenic tobacco plants, possibly through the maintenance of photosynthetic efficiency.     

Synthesis of proteins by chloroplasts from iron-deficient Euglena gracilis

Chloroplasts isolated from Euglena gracilis made iron deficient by growth on 0.5 μm iron show distinct qualitative and quantitative changes in their polypeptide composition in comparison with iron-sufficient (40 μm) chloroplasts. These changes were noted in the stromal, thylakoid, and envelope subfractions. Iron-deficient chloroplasts have a sedimentation behavior similar to that of iron-sufficient chloroplasts and also contain substantial amounts of ribulose-1,5-bisphosphate carboxylase. In addition, iron-deficient chloroplasts incorporate [³H]leucine into polypeptides at rates about one-third of those from control chloroplasts (40 μm Fe) on a per-microgram-chlorophyll basis. Incorporation of [³H]leucine into specific polypeptides, resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, shows relatively normal synthesis of the large subunit of ribulose-1,5-bisphosphate carboxylase and two of the three major chloroplast-derived polypeptides of the thylakoids. No incorporation was detected, however, into a polypeptide of ca. 33 kd which is synthesized by normal plastids. Iron-deficient chloroplasts also synthesize a stromal polypeptide of ca. 85 kd not seen in chloroplasts from normal cells. This evidence is consistent with a direct or indirect role for iron in the regulation of synthesis of specific proteins in the chloroplast.     

Artificial targeting of a nucleus-encoded RNA-binding protein AtRZ1a to chloroplasts affects flowering and ABA response of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Despite the fact that many nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts and play essential roles in RNA metabolism in chloroplasts, the question of whether artificial targeting of a nucleus-encoded RBP to chloroplasts affects chloroplast function and plant growth has never been addressed. In this study, a nuclear zinc finger-containing Arabidopsis RBP, designated AtRZ1a, which was previously shown to play a role in stress response, was artificially targeted to chloroplasts, and the growth and stress response of the transgenic plants were evaluated. Confocal analysis of the cellular localization of the cTP_AtRZ1a protein containing the N-terminal chloroplast transit peptide (cTP) from rubisco small subunit revealed that the cTP_AtRZ1a fusion protein is successfully targeted to chloroplasts. When grown under normal conditions, flowering of the transgenic plants was delayed, and the FLC expression was significantly upregulated in the transgenic plants. Artificial targeting of AtRZ1a to chloroplasts severely inhibited seedling growth of the plants in the presence of ABA by upregulating expression of ABA signaling-related genes ABI3 and ABI4. Taken together, these results suggest that artificial targeting of a nucleus-encoded AtRZ1a to chloroplasts affects the growth and development of Arabidopsis under normal or ABA treated conditions.     

Differential regulation of cytokinin oxidase genes and cytokinin-induced auxin biosynthesis by cellular cytokinin level in transgenic poplars

Key message

The present work with transgenic poplar lines producing varying levels of trans -zeatin suggests the existence of a switching threshold for triggering ckx gene expression or suppressing cytokinin-induced auxin.

Abstract

Cytokinins have an important role in growth and developmental processes of plants. Transgenic plants with varying levels of cellular cytokinin are convenient tools for studying its role in morphogenetic as well as molecular responses. In this work, the transgenic lines producing either high level of cellular trans-zeatin (HX lines) or moderate level (MX lines) were compared with regard to their cytokinin oxidase activities and cellular auxin content. The HX lines showed typical cytokinin phenotypes including leafy shoots and spontaneous shoot formation on hormone free medium. In contrast, the MX lines did not show any striking phenotypes. However, in leaf disk culture on hormone free medium, they regenerated roots and subsequently formed shoots from the roots. Determination of cellular IAA content revealed a significant increase in the level in MX lines but not in HX lines. Of nine cytokinin oxidase genes (ckx) examined by qPCR, five were activated in HX lines but not in MX lines. Among them, ckx4 appeared to play a key role in maintaining cellular cytokinin level since it showed more than 1,000-fold increase in HX lines and in the leaf disks of untransformed control exposed to exogenous cytokinins. Although low level of cellular cytokinin did not induce the expression of ckx genes, it appeared to trigger cellular IAA biosynthesis.     

Light regulation of photosynthetic membrane structure,organization, and function

The light environment during plant growth determines the structural and functional properties of higher plant chloroplasts, thus revealing a dynamically regulated developmental system. Pisum sativum plants growing under intermittent illumination showed chloroplasts with fully functional photosystem (PS) II and PSI reaction centers that lacked the peripheral chlorophyll (Chi) a/b and Chl a light-harvesting complexes (LHC), respectively. The results suggest a light flux differential threshold regulation in the biosynthesis of the photosystem core and peripheral antenna complexes. Sun-adapted species and plants growing under far-red-depleted illumination showed grana stacks composed of few (3–5) thylakoids connected with long intergrana (stroma) thylakoids. They had a PSII/PSI reaction center ratio in the range 1.3–1.9. Shade-adapted species and plants growing under far-red-enrichcd illumination showed large grana stacks composed of several thylakoids, often extending across the entire chloroplast body, and short intergrana stroma thylakoids. They had a higher PSII/PSI reaction center ratio, in the range of 2.2–4.0. Thus, the relative extent of grana and stroma thylakoid formation corresponds with the relative amounts of PSII and PSI in the chloroplast, respectively. The structural and functional adaptation of the photosynthetic membrane system in response to the quality of illumination involves mainly a control on the rate of PSII and PSI complex biosynthesis.     

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology.     

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.     

Further characterization of ribosome binding to thylakoid membranes

Previous work indicated more polysomes bound to pea (Pisum sativum cv Progress No. 9) thylakoids in light than in the dark, in vivo (LE Fish, AT Jagendorf 1982 Plant Physiol 69: 814-825). With isolated intact chloroplasts incubated in darkness, addition of MgATP had no effect but 24 to 74% more RNA was thylakoid-bound at pH 8.3 than at pH 7. Thus, the major effect of light on ribosome-binding in vivo may be due to higher stroma pH. In isolated pea chloroplasts, initiation inhibitors (pactamycin and kanamycin) decreased the extent of RNA binding, and elongation inhibitors (lincomycin and streptomycin) increased it. Thus, cycling of ribosomes is controlled by translation, initiation, and termination. Bound RNA accounted for 19 to 24% of the total chloroplast RNA and the incorporation of [³H]leucine into thylakoids was proportional to the amount of this bound RNA. These data support the concept that stroma ribosomes are recruited into thylakoid polysomes, which are active in synthesizing thylakoid proteins.     

Overexpression of tomato GDP-l-galactose phosphorylase gene in tobacco improves tolerance to chilling stress

Key message

The overexpression of tomato GDP- l -galactose phosphorylase gene enhanced tolerance to chilling stress and reduced photoinhibition of photosystems I and II in transgenic tobacco.

Abstract

Chilling stress is a crucial factor that limits the geographical distribution and yield of chilling-sensitive plants. Ascorbate (AsA) protects plants by scavenging reactive oxygen species and reduces photoinhibition by promoting the conversion of violaxanthin to zeaxanthin in the xanthophyll cycle to dissipate excess excitation energy. Possible mechanisms of AsA for plant photoprotection under chilling stress were investigated by isolating the tomato GDP-l-galactose phosphorylase gene (SlGGP) and producing transgenic tobacco plants with overexpression of SlGGP. The transgenic plants subjected to chilling stress accumulated less H₂O₂, demonstrated lower levels of ion leakage and malondialdehyde, and acquired higher net photosynthetic rate, higher maximum photochemical efficiency of PSII, and higher D1 protein content compared with the wild-type (WT) plants. The transgenic plants subjected to chilling stress also showed higher GDP-l-galactose phosphorylase activity, increased AsA content as well as ascorbate peroxidase and oxidizable P700 activities than WT plants. Thus, SlGGP overexpression is crucial in promoting AsA synthesis and alleviating photoinhibition of two photosystems.     

Enhanced salt tolerance of transgenic tobacco plants by co-expression of PcINO1 and McIMT1 is accompanied by increased level of myo-inositol and methylated inositol

Introgression and functional expression of either the PcINO1 (l-myo-inositol 1-phosphate synthase or MIPS coding gene from the wild halophytic rice, Porteresia coarctata) or McIMTI (inositol methyl transferase, IMTI coding gene from common ice plant Mesembryanthemum crystallinum) has earlier been shown to confer salt tolerance to transgenic tobacco plants (Sheveleva et al., Plant Physiol 115:1211–1219, 1997; Majee et al., J Biol Chem 279:28539–28552, 2004). In this communication, we show that transgenic tobacco plants co-expressing PcINO1 and McIMT1 gene either in cytosol or in chloroplasts accumulate higher amount of total inositol (free and methyl inositol) compared to non-transgenic plants. These transgenic plants were more competent in terms of growth potential and photosynthetic activity and were less prone to oxidative stress under salt stress. A positive correlation between the elevated level of total inositol and methylated inositol and the capability of the double transgenic plants to withstand a higher degree of salt stress compared to the plants expressing either PcINO1 or McIMT1 alone is inferred.     

Stromal low temperature compartment derived from the inner membrane of the chloroplast envelope

Leaf discs of four dicotyledonous species, when incubated at temperatures of 4 to 18°C (optimum at 12°C) for 30 or 60 minutes, responded by accumulations of membranes in the chloroplast stroma in the space between the inner membrane of the envelope and the thylakoids. The accumulated membranes, here referred to as the low temperature compartment, were frequently continuous with the envelope membrane and exhibited kinetics of formation consistent with a derivation from the envelope. Results were similar for expanding leaves of garden pea (Pisum sativum), soybean (Glycine max), spinach (Spinacia oleracea), and tobacco (Nicotiana tabacum). We suggest that the stromal low temperature compartment may be analogous to the compartment induced to form between the transitional endoplasmic reticulum and the Golgi apparatus at low temperatures. The findings provide evidence for the possibility of a vesicular transfer of membrane constituents between the inner membrane of the chloroplast envelope and the thylakoids of mature chloroplasts in expanding leaves.