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In most dicotyledonous plants, leaf epidermal pavement cells develop jigsaw puzzle-like shapes during cell expansion. The rapid growth and complicated cell shape of pavement cells is suggested to be achieved by targeted exocytosis that is coordinated with cytoskeletal rearrangement to provide plasma membrane and/or cell wall materials for lobe development during their morphogenesis. Therefore, visualization of membrane trafficking in leaf pavement cells should contribute an understanding of the mechanism of plant cell morphogenesis. To reveal membrane trafficking in pavement cells, we observed monomeric red fluorescent protein-tagged rat sialyl transferases, which are markers of trans-Golgi cisternal membranes, in the leaf epidermis of Arabidopsis thaliana. Quantitative fluorescence imaging techniques and immunoelectron microscopic observations revealed that accumulation of the red fluorescent protein occurred mostly in the curved regions of pavement cell borders and guard cell ends during leaf expansion. Transmission electron microscopy observations revealed that apoplastic vesicular membrane structures called paramural bodies were more frequent beneath the curved cell wall regions of interdigitated pavement cells and guard cell ends in young leaf epidermis. In addition, pharmacological studies showed that perturbations in membrane trafficking resulted in simple cell shapes. These results suggested possible heterogeneity of the curved regions of plasma membranes, implying a relationship with pavement cell morphogenesis. [ABSTRACT FROM AUTHOR]

Cyanophora is an important glaucophyte genus of unicellular biflagellates that may have retained ancestral features of photosynthetic eukaryotes. The nuclear genome of Cyanophora was recently sequenced, but taxonomic studies of more than two strains are lacking for this genus. Furthermore, no study has used molecular methods to taxonomically delineate Cyanophora species. Here, we delimited the species of Cyanophora using light and electron microscopy, combined with molecular data from several globally distributed strains, including one newly established. Using a light microscope, we identified two distinct morphological groups: one with ovoid to ellipsoidal vegetative cells and another with dorsoventrally flattened or broad, bean-shaped vegetative cells containing duplicated plastids. Our light and scanning electron microscopy clearly distinguished three species with ovoid to ellipsoidal cells ( C. paradoxa Korshikov, C. cuspidata Tos. Takah. & Nozaki sp. nov., and C. kugrensii Tos. Takah. & Nozaki sp. nov.) and two species with broad, bean-shaped cells ( C. biloba Kugrens, B.L.Clay, C.J.Mey. & R.E.Lee and C. sudae Tos. Takah. & Nozaki sp. nov.) based on differences in cell shape and surface ornamentations of the vegetative cells under the field-emission scanning electron microscope. Molecular phylogenetic analyses of P700 chl a apoprotein A2 ( psaB) genes and internal transcribed spacer ( ITS) regions of nuclear ribosomal DNA ( rDNA), as well as a comparison of secondary structures of nuclear rDNA ITS-2 and genetic distances of psaB genes, supported the delineation of five morphological species of Cyanophora. [ABSTRACT FROM AUTHOR]

Rapid growth of plant cells by cell division and expansion requires an endomembrane trafficking system. The endomembrane compartments, such as the Golgi stacks, endosome and vesicles, are important in the synthesis and trafficking of cell wall materials during cell elongation. However, changes in the morphology, distribution and number of these compartments during the different stages of cell proliferation and differentiation have not yet been clarified. In this study, we examined these changes at the ultrastructural level in tobacco Bright yellow 2 (BY-2) cells during the log and stationary phases of growth. We analyzed images of the BY-2 cells prepared by the high-pressure freezing/freeze substitution technique with the aid of an auto-acquisition transmission electron microscope system. We quantified the distribution of secretory and endosomal compartments in longitudinal sections of whole cells by using wide-range gigapixel-class images obtained by merging thousands of transmission electron micrographs. During the log phase, all Golgi stacks were composed of several thick cisternae. Approximately 20 vesicle clusters (VCs), including the trans-Golgi network and secretory vesicle cluster, were observed throughout the cell. In the stationary-phase cells, Golgi stacks were thin with small cisternae, and only a few VCs were observed. Nearly the same number of multivesicular body and small high-density vesicles were observed in both the stationary and log phases. Results from electron microscopy and live fluorescence imaging indicate that the morphology and distribution of secretory-related compartments dramatically change when cells transition from log to stationary phases of growth. [ABSTRACT FROM PUBLISHER]

Over-expression of sigE, a gene encoding an RNA polymerase sigma factor in the unicellular cyanobacterium Synechocystis sp. PCC 6803, is known to activate sugar catabolism and bioplastic production. In this study, we investigated the effects of sigE over-expression on cell morphology, photosynthesis and hydrogen production in this cyanobacterium. Transmission electron and scanning probe microscopic analyses revealed that sigE over-expression increased the cell size, possibly as a result of aberrant cell division. Over-expression of sigE reduced respiration and photosynthesis activities via changes in gene expression and chlorophyll fluorescence. Hydrogen production under micro-oxic conditions is enhanced in sigE over-expressing cells. Despite these pleiotropic phenotypes, the sigE over-expressing strain showed normal cell viability under both nitrogen-replete and nitrogen-depleted conditions. These results provide insights into the inter-relationship among metabolism, cell morphology, photosynthesis and hydrogen production in this unicellular cyanobacterium. [ABSTRACT FROM AUTHOR]

Bacterial endosymbionts belonging to the family Rickettsiaceae were recently identified in the unicellular green alga Carteria cerasiformis, providing the first molecular evidence of rickettsial endosymbionts within photosynthetic eukaryotes. However, previous morphological studies using transmission electron microscopy (TEM) with conventional chemical fixation did not demonstrate whether the endosymbionts of C. cerasiformis have the diagnostic characteristics of the family Rickettsiaceae. In this study, we observed the rickettsial endosymbionts ' MIDORIKO' within C. cerasiformis cells by TEM with high-pressure freezing and freeze-substitution fixation. The rickettsial endosymbionts resided directly in the C. cerasiformis cytoplasm without engulfing or encompassing membranes or vacuoles. The endosymbionts had a Gram-negative cell envelope composed of outer and inner bilayer membranes. The thicknesses of the outer and inner leaflets of the bacterial cell wall were almost identical. These morphological characteristics are consistent with those of the genus Rickettsia, but the cell wall structure differed from that of the genus Orientia within the family Rickettsiaceae. [ABSTRACT FROM AUTHOR]

The cotyledon of legume seeds is a storage organ that provides nutrients for seed germination and seedling growth. The spatial and temporal control of the degradation processes within cotyledons has not been elucidated. Calcium oxalate (CaOx) crystals, a common calcium deposit in plants, have often been reported to be present in legume seeds. In this study, micro-computed tomography (micro-CT) was employed at the SPring-8 facility to examine the three-dimensional distribution of crystals inside cotyledons during seed maturation and germination of Lotus miyakojimae (previously Lotus japonicus accession Miyakojima MG-20). Using this technique, we could detect the outline of the embryo, void spaces in seeds and the cotyledon venation pattern. We found several sites that strongly inhibited X-ray transmission within the cotyledons. Light and polarizing microscopy confirmed that these areas corresponded to CaOx crystals. Three-dimensional observations of dry seeds indicated that the CaOx crystals in the L. miyakojimae cotyledons were distributed along lateral veins; however, their distribution was limited to the abaxial side of the procambium. The CaOx crystals appeared at stage II (seed-filling stage) of seed development, and their number increased in dry seeds. The number of crystals in cotyledons was high during germination, suggesting that CaOx crystals are not degraded for their calcium supply. Evidence for the conservation of CaOx crystals in cotyledons during the L. miyakojimae germination process was also supported by the biochemical measurement of oxalic acid levels. [ABSTRACT FROM PUBLISHER]

The galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) are the predominant lipids in thylakoid membranes and indispensable for photosynthesis. Among the three isoforms that catalyze MGDG synthesis in Arabidopsis thaliana, MGD1 is responsible for most galactolipid synthesis in chloroplasts, whereas MGD2 and MGD3 are required for DGDG accumulation during phosphate (Pi) starvation. A null mutant of Arabidopsis MGD1 (mgd1-2), which lacks both galactolipids and shows a severe defect in chloroplast biogenesis under nutrient-sufficient conditions, accumulated large amounts of DGDG, with a strong induction of MGD2/3 expression, during Pi starvation. In plastids of Pi-starved mgd1-2 leaves, biogenesis of thylakoid-like internal membranes, occasionally associated with invagination of the inner envelope, was observed, together with chlorophyll accumulation. Moreover, the mutant accumulated photosynthetic membrane proteins upon Pi starvation, indicating a compensation for MGD1 deficiency by Pi stress-induced galactolipid biosynthesis. However, photosynthetic activity in the mutant was still abolished, and light-harvesting/photosystem core complexes were improperly formed, suggesting a requirement for MGDG for proper assembly of these complexes. During Pi starvation, distribution of plastid nucleoids changed concomitantly with internal membrane biogenesis in the mgd1-2 mutant. Moreover, the reduced expression of nuclear- and plastid-encoded photosynthetic genes observed in the mgd1-2 mutant under Pi-sufficient conditions was restored after Pi starvation. In contrast, Pi starvation had no such positive effects in mutants lacking chlorophyll biosynthesis. These observations demonstrate that galactolipid biosynthesis and subsequent membrane biogenesis inside the plastid strongly influence nucleoid distribution and the expression of both plastidand nuclear-encoded photosynthetic genes, independently of photosynthesis. [ABSTRACT FROM AUTHOR]

Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens , Echinosophora koreensis , Thermopsis chinensis , and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of l -lysine and l -ornithine. L.   angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis   thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La- L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QA-producing plants. [ABSTRACT FROM AUTHOR]

Tight coordination between plastid differentiation and plant development is best evidenced by the synchronized development of photosynthetic tissues and the biogenesis of chloroplasts. Here, we show that Arabidopsis thaliana roots demonstrate accelerated chlorophyll accumulation and chloroplast development when they are detached from shoots. However, this phenomenon is repressed by auxin treatment. Mutant analyses suggest that auxin transported from the shoot represses root greening via the function of INDOLE-3-ACETIC ACID14, AUXIN RESPONSE FACTOR7 (ARF7), and ARF19. Cytokinin signaling, on the contrary, is required for chlorophyll biosynthesis in roots. The regulation by auxin/cytokinin is dependent on the transcription factor LONG HYPOCOTYL5 (HY5), which is required for the expression of key chlorophyll biosynthesis genes in roots. The expression of yet another root greening transcription factor, GOLDEN2-LIKE2 (GLK2), was found to be regulated in opposing directions by auxin and cytokinin. Furthermore, both the hormone signaling and the GLK transcription factors modified the accumulation of HY5 in roots. Overexpression of GLK s in the hy5 mutant provided evidence that GLKs require HY5 to maximize their activities in root greening. We conclude that the combination of HY5 and GLKs, functioning downstream of light and auxin/cytokinin signaling pathways, is responsible for coordinated expression of the key genes in chloroplast biogenesis. [ABSTRACT FROM AUTHOR]

In angiosperms, a zygote generally divides into an asymmetric two-celled embryo consisting of an apical and a basal cell. This unequal division of the zygote is a putative first step for formation of the apical–basal axis of plants and is a fundamental feature of early embryogenesis and morphogenesis in angiosperms. Because fertilization and subsequent embryogenesis occur in embryo sacs, which are deeply embedded in ovular tissue, in vitro fertilization of isolated gametes is a powerful system to dissect mechanisms of fertilization and post-fertilization events. Rice is an emerging molecular and experimental model plant, however, profile of the first zygotic division within embryo sac and thus origin of apical–basal embryo polarity has not been closely investigated. Therefore, in the present study, the division pattern of rice zygote in planta was first determined accurately by observations employing serial sections of the egg apparatus, zygotes and two-celled embryos in the embryo sac. The rice zygote divides asymmetrically into a two-celled embryo consisting of a statistically significantly smaller apical cell with dense cytoplasm and a larger vacuolated basal cell. Moreover, detailed observations of division profiles of zygotes prepared by in vitro fertilization indicate that the zygote also divides into an asymmetric two-celled embryo as in planta. Such observations suggest that in vitro-produced rice zygotes and two-celled embryos may be useful as experimental models for further investigations into the mechanism and control of asymmetric division of plant zygotes. [ABSTRACT FROM AUTHOR]

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (Amyl-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed Amyl-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of Amyl-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with Amyl-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gin-369 is necessary for plastid targeting of Amyl-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the Amyl-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal-dependent manner. [ABSTRACT FROM AUTHOR]

Sex-dependent thermogenesis during reproductive organ development in the inflorescence is a characteristic feature of some of the protogynous arum species. One such plant, skunk cabbage (Symplocarpus renifolius), can produce massive heat during the female stage but not during the subsequent male stage in which the stamen completes development, the anthers dehisce, and pollen is released. Unlike other thermogenic species, skunk cabbage belongs to the bisexual flower group. Although recent studies have identified the spadix as the thermogenic organ, it remains unclear how individual tissues or intracellular structures are involved in thermogenesis. In this study, reproductive organ development and organelle biogenesis were examined during the transition from the female to the male stage. During the female stage, the stamens exhibit extensive structural changes including changes in organelle structure and density. They accumulate high levels of mitochondrial proteins, including possible thermogenic factors, alternative oxidase, and uncoupling protein. By contrast, the petals and pistils do not undergo extensive changes during the female stage. However, they contain a larger number of mitochondria than during the male stage in which they develop large cytoplasmic vacuoles. Comparison between female and male spadices suggests that mitochondrial number rather than their level of activity correlates with thermogenesis. Their spadices, even in the male, contain a larger amount of mitochondria that had greater oxygen consumption, compared with non-thermogenic plants. Taken together, our data suggest that the extensive maturation process in stamens produces massive heat through increased metabolic activities. The possible mechanisms by which petal and pistil metabolism may affect thermogenesis are also discussed. [ABSTRACT FROM PUBLISHER]

Secretory proteins and extracellular glycans are transported to the extracellular space during cell growth. These materials are carried in secretory vesicles generated at the trans-Golgi network (TGN). Analysis of the mammalian post- Golgi secretory pathway demonstrated the movement of separated secretory vesicles in the cell. Using secretory carrier membrane protein 2 (SCAMP2) as a marker for secretory vesicles and tobacco (Nicotiana tabacum) BY-2 cell as a model cell, we characterized the transport machinery in plant cells. A combination of analyses, including electron microscopy of quick-frozen cells and four-dimensional analysis of cells expressing fluorescent-tagged SCAMP2, enabled the identification of a clustered structure of secretory vesicles generated from TGN that moves in the cell and eventually fuses with plasma membrane. This structure was termed the secretory vesicle cluster (SVC). The SVC was also found in Arabidopsis thaliana and rice (Oryza sativa) cells and moved to the cell plate in dividing tobacco cells. Thus, the SVC is a motile structure involved in mass transport from the Golgi to the plasma membrane and cell plate in plant cells. [ABSTRACT FROM AUTHOR]

Xylem vessel elements are hollow cellular units that assemble end-to-end to form a continuous vessel throughout the plant body; the xylem vessel is strengthened by the xylem elements' reinforced secondary cell walls (SCWs). This work aims to unravel the contribution of unknown actors in xylem vessel differentiation using the model in vitro cell culture system of Zinnia elegans differentiating cell cultures and the model in vivo system of Arabidopsis thaliana plants. Tracheary Element Differentiation-Related6 (TED6) and TED7 were selected based on an RNA interference (RNAi) screen in the Zinnia system. RNAi reduction of TED6 and 7 delayed tracheary element (TE) differentiation and co-overexpression of TED6 and increased TE differentiation in cultured Zinnia cells. Arabidopsis TED6 and 7 were expressed preferentially in differentiating vessel elements in seedlings. Aberrant SCW formation of root vessel elements was induced by transient RNAi of At TED7 alone and enhanced by inhibition of both TED6 and 7. Protein-protein interactions were demonstrated between TED6 subunit of the SCW-related cellulose synthase complex. Our strategy has succeeded in finding two novel components in SCW formation and has opened the door for in-depth analysis of their molecular functions. [ABSTRACT FROM AUTHOR]

Plants synthesize a sulfur-containing lipid, sulfoquinovosyldiacylglycerol, which is one of three nonphosphorus glycerolipids that provide the bulk of the structural lipids in photosynthetic membranes. Here, the identification of a novel gene, UDP-glucose pyrophosphorylase3 (UGP3), required for sulfolipid biosynthesis is described. Transcriptome coexpression analysis demonstrated highly correlated expression of UGP3 with known genes for sulfolipid biosynthesis in Arabidopsis thaliana. Liquid chromatography-mass spectrometry analysis of leaf lipids in two Arabidopsis ugp3 mutants revealed that no sulfolipid was accumulated in these mutants, indicating the participation of UGP3 in sulfolipid biosynthesis. From the deduced amino acid sequence, UGP3 was presumed to be a UDP-glucose pyrophosphorylase (UGPase) involved in the generation of UDP-glucose, serving as the precursor of the polar head of sulfolipid. Recombinant UGP3 was able to catalyze the formation of UDP-glucose from glucose-l-phosphate and UTP. A transient assay using fluorescence fusion proteins and UGPase activity in isolated chloroplasts indicated chloroplastic localization of UGP3. The transcription level of UGP3 was increased by phosphate starvation. A comparative genomics study on UGP3 homologs across different plant species suggested the structural and functional conservation of the proteins and, thus, a committing role for UGP3 in sulfolipid synthesis. [ABSTRACT FROM AUTHOR]

Nicotine is a major alkaloid accumulatmg in the vacuole of tobacco (Nicotiana tabacum), but the transporters involved in the vacuolar sequestration are not known. We here report that tobacco genes (NtMATEI and NtMATE2) encoding transporters of the multidrug and toxic compound extrusion (MATE) family are coordinately regulated with structural genes for nicotine biosynthesis in the root, with respect to spatial expression patterns, regulation by NIC regulatory loci, and induction by methyl jasmonate. Subcellular fractionation, immunogold electron microscopy, and expression of a green fluorescent protein fusion protein all suggested that these transporters are localized to the vacuolar membrane. Reduced expression of the transporters rendered tobacco plants more sensitive to the application of nicotine. In contrast, overexpression of NtMATE1 in cultured tobacco cells induced strong acidification of the cytoplasm after jasmonate elicitation or after the addition of nicotine under nonelicited conditions. Expression of NtMATE1 in yeast (Saccharomyces cerevisiae) cells compromised the accumulation of exogenously supplied nicotine into the yeast cells. The results imply that these MATE-type proteins transport tobacco alkaloids from the cytosol into the vacuole in exchange for protons in alkaloid-synthesizing root cells. [ABSTRACT FROM AUTHOR]

SUMMARY Land plants have evolved a hydrophobic cuticle on the surface of aerial organs as an adaptation to ensure survival in terrestrial environments. Cuticle is mainly composed of lipids, namely cutin and intracuticular wax, with epicuticular wax deposited on plant surface. The composition and permeability of cuticle have a large influence on its ability to protect plants against drought stress. However, the regulatory mechanisms underlying cuticular wax biosynthesis in response to drought stress have not been fully elucidated. Here, we identified three AP2/ERF transcription factors (DREB26/ERF12, ERF13 and ERF14) involved in the regulation of water permeability of the plant surface. Transmission electron microscopy revealed thicker cuticle on the leaves of DREB26‐overexpressing (DREB26OX) plants, and thinner cuticle on the leaves of transgenic plants expressing SRDX repression domain‐fused DREB26 (DREB26SR). Genes involved in cuticular wax formation were upregulated in DREB26OX and downregulated in DREB26SR. The levels of very‐long chain (VLC) alkanes, which are a major wax component, increased in DREB26OX leaves and decreased in DREB26SR leaves. Under dehydration stress, water loss was reduced in DREB26OX and increased in DREB26SR. The erf12/13/14 triple mutant showed delayed growth, decreased leaf water content, and reduced drought‐inducible VLC alkane accumulation. Taken together, our results indicate that the DREB26/ERF12 and its closed family members, ERF13 and ERF14, play an important role in cuticular wax biosynthesis in response to drought stress. The complex transcriptional cascade involved in the regulation of cuticular wax biosynthesis under drought stress conditions is discussed. [ABSTRACT FROM AUTHOR]