SPIRE-a software tool for bicontinuous phase recognition: application for plastid cubic membranes.

Bicontinuous membranes in cell organelles epitomize nature's ability to create complex functional nanostructures. Like their synthetic counterparts, these membranes are characterized by continuous membrane sheets draped onto topologically complex saddle-shaped surfaces with a periodic network-like structure. Their structure sizes, (around 50-500 nm), and fluid nature make transmission electron microscopy (TEM) the analysis method of choice to decipher their nanostructural features. Here we present a tool, Surface Projection Image Recognition Environment (SPIRE), to identify bicontinuous structures from TEM sections through interactive identification by comparison to mathematical "nodal surface" models. The prolamellar body (PLB) of plant etioplasts is a bicontinuous membrane structure with a key physiological role in chloroplast biogenesis. However, the determination of its spatial structural features has been held back by the lack of tools enabling the identification and quantitative analysis of symmetric membrane conformations. Using our SPIRE tool, we achieved a robust identification of the bicontinuous diamond surface as the dominant PLB geometry in angiosperm etioplasts in contrast to earlier long-standing assertions in the literature. Our data also provide insights into membrane storage capacities of PLBs with different volume proportions and hint at the limited role of a plastid ribosome localization directly inside the PLB grid for its proper functioning. This represents an important step in understanding their as yet elusive structure-function relationship.

A Tetratricopeptide Repeat Protein Regulates Carotenoid Biosynthesis and Chromoplast Development in Monkeyflowers (Mimulus).

Little is known about the factors regulating carotenoid biosynthesis in flowers. Here, we characterized the REDUCED CAROTENOID PIGMENTATION2 (RCP2) locus from two monkeyflower (Mimulus) species, the bumblebee-pollinated species Mimulus lewisii and the hummingbird-pollinated species Mimulus verbenaceus We show that loss-of-function mutations of RCP2 cause drastic down-regulation of the entire carotenoid biosynthetic pathway. The causal gene underlying RCP2 encodes a tetratricopeptide repeat protein that is closely related to the Arabidopsis (Arabidopsis thaliana) REDUCED CHLOROPLAST COVERAGE proteins. RCP2 appears to regulate carotenoid biosynthesis independently of RCP1, a previously identified R2R3-MYB master regulator of carotenoid biosynthesis. We show that RCP2 is necessary and sufficient for chromoplast development and carotenoid accumulation in floral tissues. Simultaneous down-regulation of RCP2 and two closely related paralogs, RCP2-L1 and RCP2-L2, yielded plants with pale leaves deficient in chlorophyll and carotenoids and with reduced chloroplast compartment size. Finally, we demonstrate that M. verbenaceus is just as amenable to chemical mutagenesis and in planta transformation as the more extensively studied M. lewisii, making these two species an excellent platform for comparative developmental genetics studies of closely related species with dramatic phenotypic divergence.

Understanding the role of starch sheath layer in graviception of Alternanthera philoxeroides: a biophysical and microscopical study.

Plants' ability to sense and respond to gravity is a unique and fundamental process. When a plant organ is tilted, it adjusts its growth orientation relative to gravity direction, which is achieved by a curvature of the organ. In higher, multicellular plants, it is thought that the relative directional change of gravity is detected by starch-filled organelles that occur inside specialized cells called statocytes, and this is followed by signal conversion from physical information to physiological information within the statocytes. The classic starch statolith hypothesis, i.e., the starch accumulating amyloplasts movement along the gravity vector within gravity-sensing cells (statocytes) is the probable trigger of subsequent intracellular signaling, is widely accepted. Acharya Jagadish Chandra Bose through his pioneering research had investigated whether the fundamental reaction of geocurvature is contractile or expansive and whether the geo-sensing cells are diffusedly distributed in the organ or are present in the form of a definite layer. In this backdrop, a microscopy based experimental study was undertaken to understand the distribution pattern of the gravisensing layer, along the length (node-node) of the model plant Alternanthera philoxeroides and to study the microrheological property of the mobile starch-filled statocytes following inclination-induced graviception in the stem of the model plant. The study indicated a prominent difference in the pattern of distribution of the gravisensing layer along the length of the model plant. The study also indicated that upon changing the orientation of the plant from vertical position to horizontal position there was a characteristic change in orientation of the mobile starch granules within the statocytes. In the present study for the analysis of the microscopic images of the stem tissue cross sections, a specialized and modified microscopic illumination setup was developed in the laboratory in order to enhance the resolution and contrast of the starch granules.

TGD5 is required for normal morphogenesis of non-mesophyll plastids, but not mesophyll chloroplasts, in Arabidopsis.

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.

Plastid diversity and chromoplast biogenesis in differently coloured carrots: role of the DcOR3Leu gene.

MAIN CONCLUSION: Distinct plastid types and ultrastructural changes are associated with differences in carotenoid pigment profiles in differently coloured carrots, and a variant of the OR gene, DcOR3Leu is vital for chromoplast biogenesis. Accumulation of different types and amounts of carotenoids in carrots impart different colours to their taproots. In this study, the carotenoid pigment profiles, morphology, and ultrastructure of plastids in 25 carrot varieties with orange, red, yellow, or white taproots were investigated by ultra-high performance liquid chromatography as well as light and transmission electron microscopy. α-/ß-Carotene and lycopene were identified as colour-determining carotenoids in orange and red carrots, respectively. In contrast, lutein was identified as the colour-determining carotenoid in almost all tested yellow and white carrots. The latter contained only trace amounts of lutein as a unique detectable carotenoid. Striking differences in plastid types that coincided with distinct carotenoid profiles were observed among the differently coloured carrots. Microscopic analysis of the different carotenoid pigment-loaded plastids revealed abundant crystalloid chromoplasts in the orange and red carrots, whereas amyloplasts were dominant in most of the yellow and white carrots, except for the yellow carrot 'Yellow Stone', where yellow chromoplasts were observed. Plastoglobuli and crystal remnants, the carotenoid sequestering substructures, were identified in crystalloid chromoplasts. Crystal remnants were often associated with a characteristic undulated internal membrane in orange carrots or several undulated membranes in red carrots. No crystal remnants, but some plastoglobuli, were observed in the plastids of all tested yellow and white carrots. In addition, the presence of chromoplast in carrot taproots was found to be associated with DcOR3Leu, a natural variant of DcOR3, which was previously reported to be co-segregated with carotene content in carrots. Knocking out DcOR3Leu in the orange carrot 'Kurodagosun' depressed chromoplast biogenesis and led to the generation of yellow carrots. Our results support that DcOR3Leu is vital but insufficient for chromoplasts biogenesis in carrots, and add to the understanding of the formation of chromoplasts in carrots.

A transcriptomic, metabolomic and cellular approach to the physiological adaptation of tomato fruit to high temperature.

High temperatures can negatively influence plant growth and development. Besides yield, the effects of heat stress on fruit quality traits remain poorly characterised. In tomato, insights into how fruits regulate cellular metabolism in response to heat stress could contribute to the development of heat-tolerant varieties, without detrimental effects on quality. In the present study, the changes occurring in wild type tomato fruits after exposure to transient heat stress have been elucidated at the transcriptome, cellular and metabolite level. An impact on fruit quality was evident as nutritional attributes changed in response to heat stress. Fruit carotenogenesis was affected, predominantly at the stage of phytoene formation, although altered desaturation/isomerisation arose during the transient exposure to high temperatures. Plastidial isoprenoid compounds showed subtle alterations in their distribution within chromoplast sub-compartments. Metabolite profiling suggests limited effects on primary/intermediary metabolism but lipid remodelling was evident. The heat-induced molecular signatures included the accumulation of sucrose and triacylglycerols, and a decrease in the degree of membrane lipid unsaturation, which influenced the volatile profile. Collectively, these data provide valuable insights into the underlying biochemical and molecular adaptation of fruit to heat stress and will impact on our ability to develop future climate resilient tomato varieties.

Eye-like ocelloids are built from different endosymbiotically acquired components.

Multicellularity is often considered a prerequisite for morphological complexity, as seen in the camera-type eyes found in several groups of animals. A notable exception exists in single-celled eukaryotes called dinoflagellates, some of which have an eye-like 'ocelloid' consisting of subcellular analogues to a cornea, lens, iris, and retina. These planktonic cells are uncultivated and rarely encountered in environmental samples, obscuring the function and evolutionary origin of the ocelloid. Here we show, using a combination of electron microscopy, tomography, isolated-organelle genomics, and single-cell genomics, that ocelloids are built from pre-existing organelles, including a cornea-like layer made of mitochondria and a retinal body made of anastomosing plastids. We find that the retinal body forms the central core of a network of peridinin-type plastids, which in dinoflagellates and their relatives originated through an ancient endosymbiosis with a red alga. As such, the ocelloid is a chimaeric structure, incorporating organelles with different endosymbiotic histories. The anatomical complexity of single-celled organisms may be limited by the components available for differentiation, but the ocelloid shows that pre-existing organelles can be assembled into a structure so complex that it was initially mistaken for a multicellular eye. Although mitochondria and plastids are acknowledged chiefly for their metabolic roles, they can also be building blocks for greater structural complexity.

Inhibition of Carotenoid Biosynthesis by CRISPR/Cas9 Triggers Cell Wall Remodelling in Carrot.

Recent data indicate that modifications to carotenoid biosynthesis pathway in plants alter the expression of genes affecting chemical composition of the cell wall. Phytoene synthase (PSY) is a rate limiting factor of carotenoid biosynthesis and it may exhibit species-specific and organ-specific roles determined by the presence of psy paralogous genes, the importance of which often remains unrevealed. Thus, the aim of this work was to elaborate the roles of two psy paralogs in a model system and to reveal biochemical changes in the cell wall of psy knockout mutants. For this purpose, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (Cas9) proteins (CRISPR/Cas9) vectors were introduced to carotenoid-rich carrot (Daucus carota) callus cells in order to induce mutations in the psy1 and psy2 genes. Gene sequencing, expression analysis, and carotenoid content analysis revealed that the psy2 gene is critical for carotenoid biosynthesis in this model and its knockout blocks carotenogenesis. The psy2 knockout also decreased the expression of the psy1 paralog. Immunohistochemical staining of the psy2 mutant cells showed altered composition of arabinogalactan proteins, pectins, and extensins in the mutant cell walls. In particular, low-methylesterified pectins were abundantly present in the cell walls of carotenoid-rich callus in contrast to the carotenoid-free psy2 mutant. Transmission electron microscopy revealed altered plastid transition to amyloplasts instead of chromoplasts. The results demonstrate for the first time that the inhibited biosynthesis of carotenoids triggers the cell wall remodelling.

The Roots of Rye (Secale cereale L.) Are Capable of Synthesizing Benzoxazinoids.

According to current opinion, the first step of benzoxazinoids (BXs) synthesis, that is, the conversion of indole-3-glycerol phosphate to indole, occurs exclusively in the photosynthesising parts of plants. However, the results of our previous work and some other studies suggest that this process may also occur in the roots. In this study, we provide evidence that the first step of BXs synthesis does indeed occur in the roots of rye seedlings. We detected ScBx1 transcripts, BX1 enzyme, and six BXs (2-hydroxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-1,4-benzoxazin-3-one, (2R)-2-O-ß-d-glucopyranosyl-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one glucoside, 2,4-dihydroxy- 7-methoxy-1,4-benzoxazin-3-one, 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one glucoside, and 6-methoxy-2-benzoxazolinone) in the roots developed from seeds deprived of the coleoptile at 2 days after sowing (i.e., roots without contact with aerial parts). In roots regenerated in vitro, both ScBx1 transcripts and BX1 enzyme were detected at a low but still measurable levels. Thus, BXs are able to be synthesised in both the roots and above-ground parts of rye plants.

Carotenogenesis and chromoplast development during ripening of yellow, orange and red colored Physalis fruit.

MAIN CONCLUSION: Formation of specific ultrastructural chromoplastidal elements during ripening of fruits of three different colored Physalis spp. is closely related to their distinct carotenoid profiles. The accumulation of color-determining carotenoids within the chromoplasts of ripening yellow, orange, and red fruit of Physalis pubescens L., Physalis peruviana L., and Physalis alkekengi L., respectively, was monitored by high-performance liquid chromatography/diode array detector/tandem mass spectrometry (HPLC-DAD-MS/MS) as well as light and transmission electron microscopy. Both yellow and orange fruit gradually accumulated mainly ß-carotene and lutein esters at variable levels, explaining their different colors at full ripeness. Upon commencing ß-carotene biosynthesis, large crystals appeared in their chromoplasts, while large filaments protruding from plastoglobules were characteristic elements of chromoplasts of orange fruit. In contrast to yellow and orange fruit, fully ripe red fruit contained almost no ß-carotene, but esters of both ß-cryptoxanthin and zeaxanthin at very high levels. Tubule bundles and unusual disc-like crystallites were predominant carotenoid-bearing elements in red fruit. Our study supports the earlier hypothesis that the predominant carotenoid type might shape the ultrastructural carotenoid deposition form, which is considered important for color, stability and bioavailability of the contained carotenoids.

Effect of Low Temperature on Chlorophyll Biosynthesis and Chloroplast Biogenesis of Rice Seedlings during Greening.

Rice (Oryza sativa L.) frequently suffers in late spring from severe damage due to cold spells, which causes the block of chlorophyll biosynthesis during early rice seedling greening. However, the inhibitory mechanism by which this occurs is still unclear. To explore the responsive mechanism of rice seedlings to low temperatures during greening, the effects of chilling stress on chlorophyll biosynthesis and plastid development were studied in rice seedlings. Chlorophyll biosynthesis was obviously inhibited and chlorophyll accumulation declined under low temperatures during greening. The decrease in chlorophyll synthesis was due to the inhibited synthesis of δ-aminolevulinic acid (ALA) and the suppression of conversion from protochlorophyllide (Pchlide) into chlorophylls (Chls). Meanwhile, the activities of glutamate-1-semialdehyde transaminase (GSA-AT), Mg-chelatase, and protochlorophyllide oxidoreductase (POR) were downregulated under low temperatures. Further investigations showed that chloroplasts at 18 °C had loose granum lamellae, while the thylakoid and lamellar structures of grana could hardly develop at 12 °C after 48 h of greening. Additionally, photosystem II (PSII) and photosystem I (PSI) proteins obviously declined in the stressed seedlings, to the point that the PSII and PSI proteins could hardly be detected after 48 h of greening at 12 °C. Furthermore, the accumulation of reactive oxygen species (ROS) and malondialdehyde (MDA) and cell death were all induced by low temperature. Chilling stress had no effect on the development of epidermis cells, but the stomata were smaller under chilling stress than those at 28 °C. Taken together, our study promotes more comprehensive understanding in that chilling could inhibit chlorophyll biosynthesis and cause oxidative damages during greening.

Isolation of plastids and mitochondria from Chromera velia.

MAIN CONCLUSION: We present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles. Chromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTrackerTM green, while their intactness and membrane potential were confirmed by staining with MitoTrackerTM orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.

Vesicles Are Persistent Features of Different Plastids.

Peripheral vesicles in plastids have been observed repeatedly, primarily in proplastids and developing chloroplasts, in which they are suggested to function in thylakoid biogenesis. Previous observations of vesicles in mature chloroplasts have mainly concerned low temperature pretreated plants occasionally treated with inhibitors blocking vesicle fusion. Here, we show that such vesicle-like structures occur not only in chloroplasts and proplastids, but also in etioplasts, etio-chloroplasts, leucoplasts, chromoplasts and even transforming desiccoplasts without any specific pretreatment. Observations are made both in C3 and C4 species, in different cell types (meristematic, epidermis, mesophyll, bundle sheath and secretory cells) and different organs (roots, stems, leaves, floral parts and fruits). Until recently not much focus has been given to the idea that vesicle transport in chloroplasts could be mediated by proteins, but recent data suggest that the vesicle system of chloroplasts has similarities with the cytosolic coat protein complex II system. All current data taken together support the idea of an ongoing, active and protein-mediated vesicle transport not only in chloroplasts but also in other plastids, obviously occurring regardless of chemical modifications, temperature and plastid developmental stage.

Unique chromoplast organisation and carotenoid gene expression in carotenoid-rich carrot callus.

MAIN CONCLUSION: The new model orange callus line, similar to carrot root, was rich in carotenoids due to altered expression of some carotenogenesis-associated genes and possessed unique diversity of chromoplast ultrastructure. Callus induced from carrot root segments cultured in vitro is usually pale yellow (p-y) and poor in carotenoids. A unique, non-engineered callus line of dark orange (d-o) colour was developed in this work. The content of carotenoid pigments in d-o callus was at the same level as in an orange carrot storage root and nine-fold higher than in p-y callus. Carotenoids accumulated mainly in abundant crystalline chromoplasts that are also common in carrot root but not in p-y callus. Using transmission electron microscopy, other types of chromoplasts were also found in d-o callus, including membranous chromoplasts rarely identified in plants and not observed in carrot root until now. At the transcriptional level, most carotenogenesis-associated genes were upregulated in d-o callus in comparison to p-y callus, but their expression was downregulated or unchanged when compared to root tissue. Two pathway steps were critical and could explain the massive carotenoid accumulation in this tissue. The geranylgeranyl diphosphate synthase gene involved in the biosynthesis of carotenoid precursors was highly expressed, while the ß-carotene hydroxylase gene involved in ß-carotene conversion to downstream xanthophylls was highly repressed. Additionally, paralogues of these genes and phytoene synthase were differentially expressed, indicating their tissue-specific roles in carotenoid biosynthesis and metabolism. The established system may serve as a novel model for elucidating plastid biogenesis that coincides with carotenogenesis.

The Formation and Sequestration of Nonendogenous Ketocarotenoids in Transgenic Nicotiana glauca.

Ketolated and hydroxylated carotenoids are high-value compounds with industrial, food, and feed applications. Chemical synthesis is currently the production method of choice for these compounds, with no amenable plant sources readily available. In this study, the 4,4' ß-oxygenase (crtW) and 3,3' ß-hydroxylase (crtZ) genes from Brevundimonas sp. SD-212 were expressed under constitutive transcriptional control in Nicotiana glauca, which has an emerging potential as a biofuel and biorefining feedstock. The transgenic lines produced significant levels of nonendogenous carotenoids in all tissues. In leaf and flower, the carotenoids (∼0.5% dry weight) included 0.3% and 0.48%, respectively, of nonendogenous ketolated and hydroxylated carotenoids. These were 4-ketolutein, echinenone (and its 3-hydroxy derivatives), canthaxanthin, phoenicoxanthin, 4-ketozeaxanthin, and astaxanthin. Stable, homozygous genotypes expressing both transgenes inherited the chemotype. Subcellular fractionation of vegetative tissues and microscopic analysis revealed the presence of ketocarotenoids in thylakoid membranes, not predominantly in the photosynthetic complexes but in plastoglobules. Despite ketocarotenoid production and changes in cellular ultrastructure, intermediary metabolite levels were not dramatically affected. The study illustrates the utility of Brevundimonas sp. SD-212 CRTZ and CRTW to produce ketocarotenoids in a plant species that is being evaluated as a biorefining feedstock, the adaptation of the plastid to sequester nonendogenous carotenoids, and the robustness of plant metabolism to these changes.

The endosomal protein CHARGED MULTIVESICULAR BODY PROTEIN1 regulates the autophagic turnover of plastids in Arabidopsis.

Endosomal Sorting Complex Required for Transport (ESCRT)-III proteins mediate membrane remodeling and the release of endosomal intraluminal vesicles into multivesicular bodies. Here, we show that the ESCRT-III subunit paralogs CHARGED MULTIVESICULAR BODY PROTEIN1 (CHMP1A) and CHMP1B are required for autophagic degradation of plastid proteins in Arabidopsis thaliana. Similar to autophagy mutants, chmp1a chmp1b (chmp1) plants hyperaccumulated plastid components, including proteins involved in plastid division. The autophagy machinery directed the release of bodies containing plastid material into the cytoplasm, whereas CHMP1A and B were required for delivery of these bodies to the vacuole. Autophagy was upregulated in chmp1 as indicated by an increase in vacuolar green fluorescent protein (GFP) cleavage from the autophagic reporter GFP-ATG8. However, autophagic degradation of the stromal cargo RECA-GFP was drastically reduced in the chmp1 plants upon starvation, suggesting that CHMP1 mediates the efficient delivery of autophagic plastid cargo to the vacuole. Consistent with the compromised degradation of plastid proteins, chmp1 plastids show severe morphological defects and aberrant division. We propose that CHMP1 plays a direct role in the autophagic turnover of plastid constituents.

The cellular machineries responsible for the division of endosymbiotic organelles.

Chloroplasts (plastids) and mitochondria evolved from endosymbiotic bacteria. These organelles perform vital functions in photosynthetic eukaryotes, such as harvesting and converting energy for use in biological processes. Consistent with their evolutionary origins, plastids and mitochondria proliferate by the binary fission of pre-existing organelles. Here, I review the structures and functions of the supramolecular machineries driving plastid and mitochondrial division, which were discovered and first studied in the primitive red alga Cyanidioschyzon merolae. In the past decade, intact division machineries have been isolated from plastids and mitochondria and examined to investigate their underlying structure and molecular mechanisms. A series of studies has elucidated how these division machineries assemble and transform during the fission of these organelles, and which of the component proteins generate the motive force for their contraction. Plastid- and mitochondrial-division machineries have important similarities in their structures and mechanisms despite sharing no component proteins, implying that these division machineries evolved in parallel. The establishment of these division machineries might have enabled the host eukaryotic ancestor to permanently retain these endosymbiotic organelles by regulating their binary fission and the equal distribution of resources to daughter cells. These findings provide key insights into the establishment of endosymbiotic organelles and have opened new avenues of research into their evolution and mechanisms of proliferation.

Lamelloplasts and minichloroplasts in Begoniaceae: iridescence and photosynthetic functioning.

Iridoplasts (modified plastids in adaxial epidermal cells) reported from Begonia were originally hypothesized to cause iridescence, which was broadly accepted for decades. However, several species of Begonia with iridoplasts are not iridescent causing confusion. Here chloroplast ultrastructure was observed in 40 taxa of Begoniaceae to explore the phenomenon of iridescence. However, 22 Begonias and Hillebrandia were found to have iridoplasts, but only nine display visually iridescent blue to blue-green leaves. Unexpectedly, a new type of plastid, a 'minichloroplast,' was found in the abaxial epidermal cells of all taxa, but was present in adaxial epidermal cells only if iridoplasts were absent. Comparative ultrastructural study of iridoplasts and a shading experiment of selected taxa show that a taxon with iridoplasts does not inevitably have visual iridescence, but iridescence is greatly affected by the spacing between thylakoid lamellae (stoma spacing). Thus, we propose instead the name 'lamelloplast' for plastids filled entirely with regular lamellae to avoid prejudging their function. To evaluate photosynthetic performance, chlorophyll fluorescence (F v /F m ) was measured separately from the chloroplasts in the adaxial epidermis and lower leaf tissues by using leaf dermal peels. Lamelloplasts and minichloroplasts have much lower photosynthetic efficiency than mesophyll chloroplasts. Nevertheless, photosynthetic proteins (psbA protein of PSII, RuBisCo and ATPase) were detected in both plastids as well as mesophyll chloroplasts in an immunogold labeling. Spectrometry revealed additional blue to blue-green peaks in visually iridescent leaves. Micro-spectrometry detected a blue peak from single blue spots in adaxial epidermal cells confirming that the color is derived from lamelloplasts. Presence of lamelloplasts or minichloroplasts is species specific and exclusive. High prevalence of lamelloplasts in Begoniaceae, including the basal clade Hillebrandia, highlights a unique evolutionary development. These new findings clarify the association between iridescence and lamelloplasts, and with implications for new directions in the study of plastid morphogenesis.

TSC1 enables plastid development under dark conditions, contributing to rice adaptation to transplantation shock.

Since its domestication from wild rice thousands of years ago, rice has been cultivated largely through transplantation. During transplantation from the nursery to the paddy field, rice seedlings experience transplantation shock which affects their physiology and production. However, the mechanisms underlying transplantation shock and rice adaptation to this shock are largely unknown. Here, we isolated a transplant-sensitive chloroplast-deficient (tsc1) rice mutant that produces albino leaves after transplantation. Blocking light from reaching the juvenile leaves and leaf primordia caused chloroplast deficiencies in transplanted tsc1 seedlings. TSC1 encodes a noncanonical adenosine triphosphate-binding cassette (ABC) transporter homologous to AtNAP14 and is of cyanobacterial origin. We demonstrate that TSC1 controls plastid development in rice under dark conditions, and functions independently of light signaling. However, light rescued the tsc1 mutant phenotype in a spectrum-independent manner. TSC1 was upregulated following transplantation, and modulated the iron and copper levels, thereby regulating prolamellar body formation during the early P4 stage of leaf development. Therefore, TSC1 is indispensable for plastid development in the absence of light, and contributes to adaptation to transplantation shock. Our study provides insight into the regulation of plastid development and establishes a framework for improving recovery from transplantation shock in rice.

Development of the photosynthetic apparatus of Cunninghamia lanceolata in light and darkness.

Here, we compared the development of dark- and light-grown Chinese fir (Cunninghamia lanceolata) cotyledons, which synthesize chlorophyll in the dark, representing a different phenomenon from angiosperm model plants. We determined that the grana lamellar membranes were well developed in both chloroplasts and etiochloroplasts. The accumulation of thylakoid membrane protein complexes was similar between chloroplasts and etiochloroplasts. Measurement of chlorophyll fluorescence parameters indicated that photosystem II (PSII) had low photosynthetic activities, whereas the photosystem I (PSI)-driven cyclic electron flow (CEF) rate exceeded the rate of PSII-mediated photon harvesting in etiochloroplasts. Analysis of the protein contents in etiochloroplasts indicated that the light-harvesting complex II remained mostly in its monomeric conformation. The ferredoxin NADP+ oxidoreductase and NADH dehydrogenase-like complexes were relatively abundantly expressed in etiochloroplasts for Chinese fir. Our transcriptome analysis contributes a global expression database for Chinese fir cotyledons, providing background information on the regulatory mechanisms of different genes involved in the development of dark- and light-grown cotyledons. In conclusion, we provide a novel description of the early developmental status of the light-dependent and light-independent photosynthetic apparatuses in gymnosperms.