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Orchid seeds are 'dust-like.' The seed coat is usually thin, with only one to a few cell layers. It originates from the integuments formed during ovule development. In orchids, the outer integument is primarily responsible for forming a mature seed coat. The inner integument usually fails to develop after fertilization, becomes compressed, and collapses over the expanding embryo. Hence, the seed coat is formed from the funiculus, chalaza, and outer integumentary cells. The outermost layer of the seed coat, the testa, is lignified, usually at the radial and inner tangential walls. The subepidermal thin-walled layer(s), the tegmen, subsequently cold, resulting in seeds having only a single layer of seed coat cells. In some species, cells of the inner integument remain alive with the ability to synthesize and accumulate lipidic and or phenolic compounds in their walls covering the embryo. This cover is called the 'carapace,' a protective shield contributing to the embryo's added protection. A developmental and functional perspective of the integuments and seed coat during seed development and germination is presented in this review., (© 2023. Institute of Plant and Microbial Biology.)

Sesquiterpene lactone (STL) and natural rubber (NR) are characteristic isoprenoids in lettuce ( Lactuca sativa ). Both STL and NR co-accumulate in laticifers, pipe-like structures located along the vasculature. NR-biosynthetic genes are exclusively expressed in laticifers, but cell-type specific expression of STL-biosynthetic genes has not been studied. Here, we examined the expression pattern of germacrene A synthase ( LsGAS ), which catalyzes the first step in STL biosynthesis in lettuce. Quantitative PCR and Illumina read mapping revealed that the transcripts of two GAS isoforms ( LsGAS1 / LsGAS2 ) are expressed two orders of magnitude (~100-200) higher in stems than laticifers. This result implies that the cellular site for LsGAS1 / 2 expression is not in laticifers. To gain more insights, promoters of LsGAS1 / 2 were cloned and fused to β-glucuronidase ( GUS ), followed by transformations of lettuce with these promoter- GUS constructs. In in situ GUS assays, the GUS expression driven by the LsGAS1/2 promoters was tightly associated with vascular bundles. High-resolution microsections showed that GUS signals are not present in laticifers but are detected in the vascular parenchyma cells neighboring the laticifers. These results suggest that expression of LsGAS1/2 occurs in the parenchyma cells neighboring laticifers, while the resulting STL metabolites accumulate in laticifers. It can be inferred that active metabolite-trafficking occurs from the parenchyma cells to laticifers in lettuce.

Main Conclusion: Promoters of lettuce cis-prenyltransferase 3 (LsCPT3) and CPT-binding protein 2 (LsCBP2) specify gene expression in laticifers, as supported by in situ β-glucuronidase stains and microsection analysis. Lettuce (Lactuca sativa) has articulated laticifers alongside vascular bundles. In the cytoplasm of laticifers, natural rubber (cis-1,4-polyisoprene) is synthesized by cis-prenyltransferase (LsCPT3) and CPT-binding protein (LsCBP2), both of which form an enzyme complex. Here we determined the gene structures of LsCPT3 and LsCBP2 and characterized their promoter activities using β-glucuronidase (GUS) reporter assays in stable transgenic lines of lettuce. LsCPT3 has a single 7.4-kb intron while LsCBP2 has seven introns including a 940-bp intron in the 5'-untranslated region (UTR). Serially truncated LsCPT3 promoters (2.3 kb, 1.6 kb, and 1.1 kb) and the LsCBP2 promoter with (1.7 kb) or without (0.8 kb) the 940-bp introns were fused to GUS to examine their promoter activities. In situ GUS stains of the transgenic plants revealed that the 1.1-kb LsCPT3 and 0.8-kb LsCBP2 promoter without the 5'-UTR intron are sufficient to express GUS exclusively in laticifers. Fluorometric assays showed that the LsCBP2 promoter was several-fold stronger than the CaMV35S promoter and was ~ 400 times stronger than the LsCPT3 promoter in latex. Histochemical analyses confirmed that both promoters express GUS exclusively in laticifers, recognized by characteristic fused multicellular structures. We concluded that both the LsCPT3 and LsCBP2 promoters specify gene expression in laticifers, and the LsCBP2 promoter displays stronger expression than the CaMV35S promoter in laticifers. For the LsCPT3 promoter, it appears that unknown cis-elements outside of the currently examined LsCPT3 promoter are required to enhance LsCPT3 expression.

Successful reproduction in angiosperms is dependent on the highly synchronous development of their male and female gametophytes and the ensuing fusion of the gametes from these reproductive tissue types. When crossing a T-DNA insertion line sdk1-7-/- (Salk_024564), one of the S-domain receptor kinases involved in ABA responses with a fast neutron deletion line abi3-6-/- , the F1 heterozygotes ( sdk1-7+/-abi3-6 +/-) displayed 50% ovule abortion suggesting a likely gametophytic defects. We identified and characterized an early stage female gametophyte developmental defect in the heterozygous mutant ovules. Recombination frequency analysis of the F2 progenies from selfed heterozygotes revealed a possible pseudo-linkage of sdk1-7 and abi3-6 suggesting a reciprocal translocation event in the heterozygote. Our study emphasizes the importance of robust analysis to distinguish gametophytic defect phenotypes caused by genetic interactions and that resulting from possible chromosomal translocation events.

As Oryza sativa (rice) seeds represent food for over three billion people worldwide, the identification of genes that enhance grain size and composition is much desired. Past reports have indicated that Arabidopsis thaliana acyl-CoA-binding proteins (ACBPs) are important in seed development but did not affect seed size. Herein, rice OsACBP2 was demonstrated not only to play a role in seed development and germination, but also to influence grain size. OsACBP2 mRNA accumulated in embryos and endosperm of germinating seeds in qRT-PCR analysis, while β-glucuronidase (GUS) assays on OsACBP2pro::GUS rice transformants showed GUS expression in embryos, as well as the scutellum and aleurone layer of germinating seeds. Deletion analysis of the OsACBP2 5'-flanking region revealed five copies of the seed cis-element, Skn-I-like motif (-1486/-1482, -956/-952, -939/-935, -826/-822, and -766/-762), and the removal of any adversely affected expression in seeds, thereby providing a molecular basis for OsACBP2 expression in seeds. When OsACBP2 function was investigated using osacbp2 mutants and transgenic rice overexpressing OsACBP2 (OsACBP2-OE), osacbp2 was retarded in germination, while OsACBP2-OEs performed better than the wild-type and vector-transformed controls, in germination, seedling growth, grain size and grain weight. Transmission electron microscopy of OsACBP2-OE mature seeds revealed an accumulation of oil bodies in the scutellum cells, while confocal laser scanning microscopy indicated oil accumulation in OsACBP2-OE aleurone tissues. Correspondingly, OsACBP2-OE seeds showed gain in triacylglycerols and long-chain fatty acids over the vector-transformed control. As dietary rice bran contains beneficial bioactive components, OsACBP2 appears to be a promising candidate for enriching seed nutritional value., (© 2019 The Authors The Plant Journal © 2019 John Wiley & Sons Ltd.)

Main Conclusion: This study revealed that elevated carbon dioxide increases Arabidopsis tolerance to higher temperature and drought stress by mitigating oxidative stress and improving water status of plants. Few studies have considered multiple aspects of plant responses to key components of global climate change, including higher temperature, elevated carbon dioxide (ECO 2 ), and drought. Hence, their individual and combinatorial effects on plants need to be investigated in the context of understanding climate change impact on plant growth and development. We investigated the interactive effects of temperature, CO 2 , watering regime, and genotype on Arabidopsis thaliana (WT and ABA-insensitive mutant, abi1-1). Plants were grown in controlled-environment growth chambers under two temperature regimes (22/18 °C and 28/24 °C, 16 h light/8 h dark), two CO 2 concentrations (400 and 700 μmol mol -1 ), and two watering regimes (well-watered and water-stressed) for 18 days. Plant growth, anatomical, physiological, molecular, and hormonal responses were determined. Our study provided valuable information about plant responses to the interactive effects of multiple environmental factors. We showed that drought and ECO 2 had larger effects on plants than higher temperatures. ECO 2 alleviated the detrimental effects of temperature and drought by mitigating oxidative stress and plant water status, and this positive effect was consistent across multiple response levels. The WT plants performed better than the abi1-1 plants; the former had higher rosette diameter, total dry mass, leaf and soil water potential, leaf moisture, proline, ethylene, trans-zeatin, isopentyladenine, and cis-zeatin riboside than the latter. The water-stressed plants of both genotypes accumulated more abscisic acid (ABA) than the well-watered plants; however, higher temperatures decreased the ability of WT plants to produce ABA in response to drought. We conclude that drought strongly, while higher temperature to a lesser extent, affects Arabidopsis seedlings, and ECO 2 reduces the adverse effects of these stressors more efficiently in the WT plants than in the abi1-1 plants. Findings from this study can be extrapolated to other plant species that share similar characteristics and/or family with Arabidopsis.

Control of testicular development is multifactorial and involves a number of hypothalamic, hypophyseal and peripheral hormones. Here, we investigated direct action of zebrafish gonadotropin-inhibitory hormone (zGnih) which is expressed in the testis, on spermatogenesis in zebrafish, in vitro. Treatment with zGnih at the lower doses (10 and 100 nM) inhibited gonadotropin-induced spermatids/spermatozoa (SPD/SPZ) production. However, at the highest dose (1000 nM), zGnih increased basal number of SPD/SPZ and showed paradoxical effect. The effects of zGnih on testosterone and SPD/SPZ production was blocked in the presence of androgen receptor antagonist, flutamide (FLU). A number of transcripts were also measured to better understand zGnih mechanisms of action on zebrafish spermatogenesis. Our results provide strong support for the hypothesis that locally produced zGnih is a component of the complex multifactorial system that regulates testicular development and function in adult zebrafish, in part, by changes in testicular steroidogenesis and regulation of gonadotropin-induced response., (Copyright © 2019. Published by Elsevier B.V.)

Hydroxyproline-rich glycoproteins (HRGPs) are abundant cell wall components involved in mycorrhizal symbiosis, but little is known about their function in orchid mycorrhizal association. To gain further insight into the role of HRGPs in orchid symbiosis, the location and function of HRGPs were investigated during symbiotic germination of Dendrobium officinale . The presence of JIM11 epitope in developing protocorms was determined using immunodot blots and immunohistochemical staining procedures. Real-time PCR was also employed to verify the expression patterns of genes coding for extensin-like genes selected from the transcriptomic database. The importance of HRGPs in symbiotic germination was further investigated using 3,4-dehydro-L-proline (3,4-DHP), an inhibitor of HRGP biosynthesis. In symbiotic cultures, immunodot blots of JIM11 signals were moderate in mature seeds, and the signals became stronger in swollen embryos. After germination, signal intensities decreased in developing protocorms. In contrast, in asymbiotic cultures, JIM11 signals were much lower as compared with those stages in symbiotic cultures. Immunofluorescence staining enabled the visualization of JIM11 epitope in mature embryo and protocorm cells. Positive signals were initially localized in the larger cells near the basal (suspensor) end of uninfected embryos, marking the future colonization site of fungal hyphae. After 1 week of inoculation, the basal end of embryos had been colonized, and a strong signal was detected mostly at the mid- and basal regions of the enlarging protocorm. As protocorm development progressed, the signal was concentrated in the colonized cells at the basal end. In colonized cells, signals were present in the walls and intracellularly associated with hyphae and the pelotons. The precise localization of JIM11 epitope is further examined by immunogold labeling. In the colonized cells, gold particles were found mainly in the cell wall and the interfacial matrix near the fungal cell wall. Four extensin-like genes were verified to be highly up-regulated in symbiotically germinated protocorms as compared to asymbiotically germinated ones. The 3,4-DHP treatment inhibited the accumulation of HRGPs and symbiotic seed germination. In these protocorms, fungal hyphae could be found throughout the protocorms. Our results indicate that HRGPs play an important role in symbiotic germination. They can serve as markers for fungal colonization, establishing a symbiotic compartment and constraining fungal colonization inside the basal cells of protocorms.

Fatty acids (FAs) and sterols constitute building blocks of eukaryotic membranes and lipid signals. Co-regulation of FA and sterol synthesis is mediated by sterol regulatory element-binding proteins in animals but remains elusive in plants. We reported recently that Arabidopsis ACYL-COA-BINDING PROTEIN1 (ACBP1) modulates sterol synthesis via protein-protein interaction with STEROL C4-METHYL OXIDASE1-1 (SMO1-1). Herein, ACBP1 was demonstrated to co-express and interact with SMO1-2 by yeast two-hybrid, co-localization, pull-down, co-immunoprecipitation and β-glucuronidase assays. SMO1-2 silenced in acbp1 was used in phenotyping, GC-MS and expression profiling. ACBP1 co-expressed with SMO1-2 in embryo sacs, pollen and trichomes, corroborating with cooperative tissue-specific functions unseen with SMO1-1. SMO1-2 silencing in acbp1 impaired seed development, male and female gamete transmission, and pollen function. Genes encoding homeodomain-leucine zipper IV transcription factors (HDG5, HDG10, HDG11 and GLABRA2), which potentially bind phospholipids/sterols, were transcribed aberrantly. GLABRA2 targets (MYB23, MUM4 and PLDα1) were misregulated, causing glabra2-resembling trichome, seed coat mucilage and oil-accumulating phenotypes. Together with altered sterol and FA compositions upon ACBP1 mutation and/or SMO1-2 silencing, ACBP1-SMO1 interaction appears to mediate homeostatic co-regulation of FAs and sterols, which serve as lipid modulators for gene expression of homeodomain-leucine zipper IV transcription factors., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

This perspective draws attention to the functional organization of orchid seed and protocorm during the course of development. The orchid embryos have a well-organized developmental plan generating a blue-print of a protocorm as they mature. The different phases of embryo development in orchids, i.e. histodifferentiation, storage product synthesis and accumulation, and maturation are essentially similar to other flowering plants. The protocorm is considered as a unique structure designed to establish symbiotic association with mycorrhizal fungi and with the primary goal to form a shoot apical meristem. This perspective brings forth arguments that the processes of embryo and protocorm development are highly programmed events, enhancing survival of orchid seeds and plantlets in their natural habitats. Furthermore, the ability of protocorm cells to divide, makes them ideal explants for micropropagation and transformation studies. Through seed germination and micropropagation using protocorms as explants, orchid conservation efforts are greatly enhanced.

Fatty acids (FAs) and sterols are primary metabolites that exert interrelated functions as structural and signaling lipids. Despite their common syntheses from acetyl-coenzyme A, homeostatic cross talk remains enigmatic. Six Arabidopsis ( Arabidopsis thaliana ) acyl-coenzyme A-binding proteins (ACBPs) are involved in FA metabolism. ACBP1 interacts with PHOSPHOLIPASE Dα1 and regulates phospholipid composition. Here, its specific role in the negative modulation of sterol synthesis during embryogenesis is reported. ACBP1, likely in a liganded state, interacts with STEROL C4-METHYL OXIDASE1-1 (SMO1-1), a rate-limiting enzyme in the sterol pathway. Proembryo abortion in the double mutant indicated that the ACBP1-SMO1-1 interaction is synthetic lethal, corroborating with their strong promoter activities in developing ovules. Gas chromatography-mass spectrometry revealed quantitative and compositional changes in FAs and sterols upon overexpression or mutation of ACBP1 and/or SMO1-1 Aberrant levels of these metabolites may account for the downstream defect in lipid signaling. GLABRA2 ( GL2 ), encoding a phospholipid/sterol-binding homeodomain transcription factor, was up-regulated in developing seeds of acbp1 , smo1-1 , and ACBP1 +/- smo1-1 in comparison with the wild type. Consistent with the corresponding transcriptional alteration of GL2 targets, high-oil, low-mucilage phenotypes of gl2 were phenocopied in ACBP1 +/- smo1-1 Thus, ACBP1 appears to modulate the metabolism of two important lipid classes (FAs and sterols) influencing cellular signaling., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Background and Aims: Although abscisic acid (ABA) is commonly recognized as a primary cause of seed dormancy, there is a lack of information on the role of ABA during orchid seed development. In order to address this issue, the localization and quantification of ABA were determined in developing seeds of Cypripedium formosanum., Methods: The endogenous ABA profile of seeds was measured by enzyme-linked immunosorbent assay (ELISA). Temporal and spatial distributions of ABA in developing seeds were visualized by immunohistochemical staining with monoclonal ABA antibodies. Fluoridone was applied to test the causal relationship between ABA content and seed germinability., Key Results: ABA content was low at the proembryo stage, then increased rapidly from 120 to 150 days after pollination (DAP), accompanied by a progressive decrease in water content and seed germination. Immunofluorescence signals indicated an increase in fluorescence over time from the proembryo stage to seed maturation. From immunogold labelling, gold particles could be seen within the cytoplasm of embryo-proper cells during the early stages of seed development. As seeds approached maturity, increased localization of gold particles was observed in the periplasmic space, the plasmalemma between embryo-proper cells, the surface wall of the embryo proper, and the inner walls of inner seed-coat cells. At maturity, gold particles were found mainly in the apoplast, such as the surface wall of the embryo proper, and the shrivelled inner and outer seed coats. Injection of fluoridone into capsules resulted in enhanced germination of mature seeds., Conclusions: The results indicate that ABA is the key inhibitor of germination in C. formosanum. The distinct accumulation pattern of ABA suggests that it is synthesized in the cytosol of embryo cells during the early stages of seed development, and then exported to the apoplastic region of the cells for subsequent regulatory processes as seeds approach maturity., (© The Author 2015. Published by Oxford University Press on behalf of the Annals of Botany Company.All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Follicular dendritic cell sarcoma is a rare neoplasm of immune accessory cells. It occurs primarily in lymph nodes. Occurrences in the mediastinum are rarely reported. Diagnosis and management of follicular dendritic cell sarcoma remain unclear, and it is an under-recognized clinical entity. Only a few cases of paraneoplastic pemphigus as the first presentation of follicular dendritic cell sarcoma have been reported. We report an unusual case of follicular dendritic cell sarcoma of the anterior mediastinum, presenting as paraneoplastic pemphigus in a 62-year-old man. Typical histological features confirmed the diagnosis of follicular dendritic cell sarcoma, and surgical resection was successfully performed., (© The Author(s) 2014.)

The funiculus anchors the structurally complex seed to the maternal plant, and is the only direct route of transport for nutrients and maternal signals to the seed. While our understanding of seed development is becoming clearer, current understanding of the genetics and cellular mechanisms that contribute to funiculus development is limited. Using laser microdissection combined with global RNA-profiling experiments we compared the genetic profiles of all maternal and zygotic regions and subregions during seed development. We found that the funiculus is a dynamic region of the seed that is enriched for mRNAs associated with hormone metabolism, molecular transport, and metabolic activities corresponding to biological processes that have yet to be described in this maternal seed structure. We complemented our genetic data with a complete histological analysis of the funiculus from the earliest stages of development through to seed maturation at the light and electron microscopy levels. The anatomy revealed signs of photosynthesis, the endomembrane system, cellular respiration, and transport within the funiculus, all of which supported data from the transcriptional analysis. Finally, we studied the transcriptional programming of the funiculus compared to other seed subregions throughout seed development. Using newly designed in silico algorithms, we identified a number of transcriptional networks hypothesized to be responsible for biological processes like auxin response and glucosinolate biosynthesis found specifically within the funiculus. Taken together, patterns of gene activity and histological observations reveal putative functions of the understudied funiculus region and identify predictive transcriptional circuits underlying these biological processes in space and time., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

In Arabidopsis, six acyl-CoA-binding proteins (ACBPs) have been identified and they have been demonstrated to function in plant stress responses and development. Three of these AtACBPs (AtACBP4-AtACBP6) are cytosolic proteins and all are expressed in floral organs as well as in other tissues. The roles of cytosolic AtACBPs in floral development were addressed in this study. To this end, a T-DNA insertional knockout mutant of acbp5 was characterized before use in crosses with the already available acbp4 and acbp6 T-DNA knockout mutants to examine their independent and combinatory functions in floral development. The single-gene knockout mutations did not cause any significant phenotypic changes, while phenotypic deficiencies affecting siliques and pollen were observed in the double mutants (acbp4acbp6 and acbp5acbp6) and the acbp4acbp5acbp6 triple mutant. Vacuole accumulation in the acbp4acbp6, acbp5acbp6 and acbp4acbp5acbp6 pollen was the most severe abnormality occurring in the double and triple mutants. Furthermore, scanning electron microscopy and transmission electron microscopy revealed exine and oil body defects in the acbp4acbp5acbp6 mutant, which also displayed reduced ability in in vitro pollen germination. Transgenic Arabidopsis expressing β-glucuronidase (GUS) driven from the various AtACBP promoters indicated that AtACBP6pro::GUS expression overlapped with AtACBP4pro::GUS expression in pollen grains and with AtACBP5pro::GUS expression in the microspores and tapetal cells. Taken together, these results suggest that the three cytosolic AtACBPs play combinatory roles in acyl-lipid metabolism during pollen development., (© The Author 2014. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Phenylketonuria (PKU) is an inherited metabolic disorder caused by deficient phenylalanine hydroxylase (PAH) activity, the enzyme responsible for the disposal of excess amounts of the essential amino acid phenylalanine (Phe). Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) has potential to serve as an enzyme substitution therapy for this human genetic disease. Using 7-day-old Japanese Striped corn seedlings (Japonica Striped maize, Zea mays L. cv. japonica) that contain high activities of PAL, we investigated a number of methods to preserve the roots as an intact food and for long-term storage. The cryoprotectant effects of maple syrup and other edible sugars (mono- and oligosaccharides) were evaluated. Following thawing, the preserved roots were then examined to determine whether the rigid plant cell walls could protect the PAL enzyme from proteolysis during simulated (in vitro) digestion comprised of gastric and intestinal phases. While several treatments led to retention of PAL activity during freezing, upon thawing and in vitro digestion, root tissues that had been previously frozen in the presence of maple syrup exhibited the highest residual PAL activities (∼50% of the initial enzyme activity), in marked contrast to all of the treatments using other edible sugars. The structural integrity of the root cells, and the stability of the functional PAL tetramer were also preserved with the maple syrup protocol. These results have significance for the formulation of oral enzyme/protein therapeutics. When plant tissues are adequately preserved, the rigid cell walls constitute a protective barrier even under harsh (e.g. gastrointestinal-like) conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Background and Aims: The Orchidaceae have a history of recurring convergent evolution in floral function as nectar production has evolved repeatedly from an ancestral nectarless state. However, orchids exhibit considerable diversity in nectary type, position and morphology, indicating that this convergence arose from alternative adaptive solutions. Using the genus Disa, this study asks whether repeated evolution of floral nectaries involved recapitulation of the same nectary type or diversifying innovation. Epidermis morphology of closely related nectar-producing and nectarless species is also compared in order to identify histological changes that accompanied the gain or loss of nectar production., Methods: The micromorphology of nectaries and positionally equivalent tissues in nectarless species was examined with light and scanning electron microscopy. This information was subjected to phylogenetic analyses to reconstruct nectary evolution and compare characteristics of nectar-producing and nectarless species., Key Results: Two nectary types evolved in Disa. Nectar exudation by modified stomata in floral spurs evolved twice, whereas exudation by a secretory epidermis evolved six times in different perianth segments. The spur epidermis of nectarless species exhibited considerable micromorphological variation, including strongly textured surfaces and non-secreting stomata in some species. Epidermis morphology of nectar-producing species did not differ consistently from that of rewardless species at the magnifications used in this study, suggesting that transitions from rewardlessness to nectar production are not necessarily accompanied by visible morphological changes but only require sub-cellular modification., Conclusions: Independent nectary evolution in Disa involved both repeated recapitulation of secretory epidermis, which is present in the sister genus Brownleea, and innovation of stomatal nectaries. These contrasting nectary types and positional diversity within types imply weak genetic, developmental or physiological constraints in ancestral, nectarless Disa. Such functional convergence generated by morphologically diverse solutions probably also underlies the extensive diversity of nectary types and positions in the Orchidaceae.

Premise of the Study: Protocorm-like bodies (PLBs) of orchids are important in orchid micropropagation and outwardly resemble somatic embryos in form and development. To determine whether PLBs are truly embryogenetic, we compared PLBs with somatic embryos and zygotic embryos to determine whether they had similar surface molecules and whether hydroxyproline-rich glycoprotein (HRGP) inhibitors similarly alter their growth., Methods: Embryogenic calluses (ECs), zygotic embryos, and protocorms were collected for histological and histochemical studies with light microscopy. The presence of JIM11 and JIM20 epitopes was determined using immunodot blots and immunolocalization procedures. The importance of wall proteins in the formation of PLBs was investigated using 3,4-dehydro-l-proline (3,4-DHP), an inhibitor of HRGP biosynthesis., Key Results: At the early stages of PLB formation, the cytoplasm of the globular cell clusters and meristemoids took on a vacuolated appearance. Starch granules and protein bodies appeared, albeit transitory in nature. Positive localizations of JIM11 and JIM20 were noted in the embryogenic culture and developing PLBs similar to zygotic embryos. The inclusion of an inhibitor to HRGPs inhibited PLB formation., Conclusions: This study demonstrates that during the early stages of PLB formation, the cells show cytological characteristics and cell wall markers similar to zygotic embryo development, justifying the statement that PLBs are indeed somatic embryos of orchids. Thus, these results suggest that PLBs could be used as an experimental embryological system for physiological or molecular characterization.

Monte Carlo (MC) simulation is currently the most accurate dose calculation algorithm in radiotherapy planning but requires relatively long processing time. Faster model-based algorithms such as the anisotropic analytical algorithm (AAA) by the Eclipse treatment planning system and multigrid superposition (MGS) by the XiO treatment planning system are 2 commonly used algorithms. This study compared AAA and MGS against MC, as the gold standard, on brain, nasopharynx, lung, and prostate cancer patients. Computed tomography of 6 patients of each cancer type was used. The same hypothetical treatment plan using the same machine and treatment prescription was computed for each case by each planning system using their respective dose calculation algorithm. The doses at reference points including (1) soft tissues only, (2) bones only, (3) air cavities only, (4) soft tissue-bone boundary (Soft/Bone), (5) soft tissue-air boundary (Soft/Air), and (6) bone-air boundary (Bone/Air), were measured and compared using the mean absolute percentage error (MAPE), which was a function of the percentage dose deviations from MC. Besides, the computation time of each treatment plan was recorded and compared. The MAPEs of MGS were significantly lower than AAA in all types of cancers (p<0.001). With regards to body density combinations, the MAPE of AAA ranged from 1.8% (soft tissue) to 4.9% (Bone/Air), whereas that of MGS from 1.6% (air cavities) to 2.9% (Soft/Bone). The MAPEs of MGS (2.6%±2.1) were significantly lower than that of AAA (3.7%±2.5) in all tissue density combinations (p<0.001). The mean computation time of AAA for all treatment plans was significantly lower than that of the MGS (p<0.001). Both AAA and MGS algorithms demonstrated dose deviations of less than 4.0% in most clinical cases and their performance was better in homogeneous tissues than at tissue boundaries. In general, MGS demonstrated relatively smaller dose deviations than AAA but required longer computation time., (Copyright © 2013 American Association of Medical Dosimetrists. Published by Elsevier Inc. All rights reserved.)

Seeds are complex structures that consist of the embryo, endosperm, and seed-coat regions that are of different ontogenetic origins, and each region can be further divided into morphologically distinct subregions. Despite the importance of seeds for food, fiber, and fuel globally, little is known of the cellular processes that characterize each subregion or how these processes are integrated to permit the coordinated development of the seed. We profiled gene activity genome-wide in every organ, tissue, and cell type of Arabidopsis seeds from fertilization through maturity. The resulting mRNA datasets offer the most comprehensive description of gene activity in seeds with high spatial and temporal resolution,providing unique insights into the function of understudied seed regions. Global comparisons of mRNA populations reveal unexpected overlaps in the functional identities of seed subregions. Analyses of coexpressed gene sets suggest that processes that regulate seed size and filling are coordinated across several subregions. Predictions of gene regulatory networks based on the association of transcription factors with enriched DNA sequence motifs upstream of coexpressed genes identify regulators of seed development. These studies emphasize the utility of these data sets as an essential resource for the study of seed biology.

A wide range of plant hormones, including gibberellins (GAs) and auxins are known to be involved in regulating seed and fruit growth and development. Changes in ethylene biosynthesis are also associated with seed and fruit development, but ethylene's role in these processes is poorly understood, as is its possible interaction with the other plant hormones. A major complication of investigating ethylene-induced regulation of developmental processes is ethylene's biphasic mode of action. To investigate ethylene's actions and interactions we used a 1-amino-cyclopropane-1-carboxylic acid (ACC) deaminase transgenic canola line. This line evolves significantly less ethylene from its siliques and seeds, relative to plants from a wild type (WT) background. Plants of the transgenic line also had smaller siliques which were associated with reductions in both seed size and seed number. Application of ethephon, a compound that produces ethylene, to plants of the transgenic line restored the WT phenotype for both siliques and seeds. Application of the same dose of ethephon to WT plants diminished both silique and seed development, showing ethylene's biphasic effect and effectively producing the ACC deaminase transgenic phenotype. There were significant decreases in endogenous concentrations of GA(1) and GA(4) and also of indole-3-acetic acid (IAA), between WT seeds and seedless siliques and seeds and siliques from the transgenic line plants. These differences were emphasized during early stages (10-20 days after pollination) of seed and silique development. The above results strongly suggest that ethylene interacts with other endogenous plant hormones in regulating silique and seed development and growth in WT lines of canola., (Copyright © 2012 Elsevier Masson SAS. All rights reserved.)

Careful examination of how explants react to experimental conditions is the first step of a successful research program. Photographing the specimen is an extremely important method that can be used to document changes of an explant throughout an experiment. This article serves to draw attention to the utility of macroscopic and microscopic examinations in the study of in vitro development, and details some useful methods in the study of the cultured explants.

Arabidopsis thaliana is an excellent experimental system to study various aspects of plant biology. An efficient regeneration protocol of the plant is important to produce in vitro fertile plants, as well as a fundamental step to study the cellular, physiological, and molecular mechanisms of plant development. Here, we describe an efficient and fast regeneration system from root explants of A. thaliana ecotypes Columbia-0 and Landsberg erecta. Culture conditions and media compositions were optimized for the development of higher number of plantlets. We demonstrate that the combination of the plant regulators abscisic acid and cytokinin in the shoot induction medium increases the formation of shoots as well as reduces the proliferation of abnormal tissues.

In safflower, the anther wall at maturity consists of a single epidermis, an endothecium, a middle layer and the tapetum. The tapetum consists mainly of a single layer of cells. However, this single-layer appearance is punctuated by loci having 'two-celled' groupings due to additional periclinal divisions in some tapetal cells. Meiotic division in microsporocytes gives rise to tetrads of microspores. The primexine is formed around the protoplasts of microspores while they are still enveloped within the callose wall. Just prior to microgametogenesis, the microspores enlarge through the process of vacuolation, and the exine wall pattern becomes established. Microgametogenesis results in the formation of 3-celled pollen grains. The two elongated sperm cells appear to be connected. The exine wall is highly sculptured with a distinct tectum, columellae, a foot layer, an endexine and a thin intine. Similar to other members of the Asteraceae family, the tapetum is of the invasive type. The most novel finding of this study is that in addition to the presence of invasive tapetal cells, a small population of 'non-invasive' tapetal cells is also present. The tapetal cells next to the anther locules in direct contact with the microspores become invasive and start to grow into the space between developing microspores. These tapetal cells synthesize tryphine and eventually degenerate at the time of gametogenesis releasing their content into the anther locules. A smaller population of non-invasive tapetal cells is formed as a result of periclinal divisions at the time of tapetum differentiation. These cells are not exposed to the anther locules until the degeneration of the invasive tapetal cells. The non-invasive tapetal cells have a different cell fate as they synthesize pollenkitt. This material is responsible for allowing some pollen grains to adhere to each other and to the anther wall after anther dehiscence. This observation explains the out-crossing ability of Carthamus species and varieties in nature.

Spontaneous haemopneumothorax (SHP) can be life threatening and is an important cause for unexplained signs of significant hypovolaemia. There is still some debate relating to patient selection and timing of surgery, particularly in those who become stable following chest tube insertion without further blood loss. Review of the literature over the past decade in the management of SHP are presented and discussed. Surgery should be considered early in the management of SHP to reduce morbidity associated with continued haemorrhage and inadequate drainage. Lower postoperative complications and shorter hospital stay following video assisted thoracic surgery compared with thoracotomy have led to its increased acceptance as an alternative approach for SHP patients who are haemodynamically stable.

To further understand the events occurring during embryogenesis, it is imperative to have an experimental system. While somatic embryo is the system of choice due to the prolific number and success of protocols available, it is necessary to compare in vitro results with those in vivo. This process is often difficult due to the inaccessibility of the developing embryo and the complications of manipulating the embryo in vivo. The development of protocols that allow for manipulation and comparison of both somatic and zygotic embryos is key to elucidating the differences between the two types of embryos and determining if results observed using one system can be applied to the other. This chapter details a simple protocol for the culture of zygotic embryos of canola that allows for the processing of large numbers of embryos with little physical damage. Furthermore, this protocol allows for the experimental manipulation of zygotic embryos in vitro and comparisons to be made with a well-established microspore-derived embryo system.

Zygotic embryo culture has proven itself an invaluable method in plant science for both pure and applied research. The composition of medium used to sustain embryos is a key to successful culture. Optimal composition of the medium changes during embryonic development; generally, the younger the embryo, the more complex is its nutritional requirements. Feeder cell and "double medium" culture methods have been developed to improve the survival of zygotes and proembryos in vitro. In this chapter, we discuss the nutritional requirements of cultured embryos and the importance of the osmotic environment for nurturing young embryos. Specific methodological adaptations used in the culture of Capsella are outlined to demonstrate how standard protocols can be manipulated to suit one's needs.

The suspensor plays an active role during the early embryo development of flowering plants. In orchids, the suspensor cells are highly vacuolated without structural specializations, and the possible mechanism(s) that enable the suspensor to serve as the nutrient uptake site is virtually unknown. Here, we used the fluorescent tracer CFDA to characterize the pathway for symplastic transport in the suspensor cells of developing embryos and to provide direct visual evidence that the orchid suspensor has unique physiological properties. The embryo proper uptakes the fluorescent dye through the suspensor. CF could first be detected throughout the suspensor cell and then subsequently in the embryo proper. A plasmolysis experiment clearly indicates that suspensor cells have a more negative osmotic potential than the adjoining testa cells. It is proposed that the preferential entry of CFDA into the suspensor cell of the Nun orchid is aided by the more negative osmotic potential of the suspensor than neighboring cells, providing a driving force for the uptake of water from the apoplast into the symplast.

Minimal invasive video-assisted thoracic surgery can be a safe alternative technique in the assessment, diagnosis and surgical resection of posterior mediastinal tumours. Video-assisted thoracic surgery may be particularly suited for the management of posterior mediastinal tumours as most are benign. Surgical technique continues to evolve from the classic 3-port access in order to tackle more complex tumours positioned at the apical and inferior recesses of the posterior mediastinum. The preoperative identification of dumbbell tumours is important to facilitate arrangements for a single-stage combined resection for both the intra-thoracic and intraspinal tumour. Results from Video-assisted thoracic surgery posterior mediastinal tumour resection are comparable with conventional surgical techniques in terms of symptomatic improvement, recurrence and survival. Video-assisted thoracic surgery approach has been shown to result in less post-operative pain, improved cosmesis, shorter hospital stay, and more rapid recovery and return to normal activities. In over a decade, video-assisted thoracic surgery has gradually matured and is now a promising therapeutic alternative to open approach. In certain selected patients, video-assisted thoracic surgery may be considered the standard of care for conditions of the posterior mediastinum. Recent developments in robotic surgery for the management of mediastinal tumours are promising, however, long-term results are pending., (Copyright © 2010 Royal College of Surgeons of Edinburgh (Scottish charity number SC005317) and Royal College of Surgeons in Ireland. Published by Elsevier Ltd. All rights reserved.)

White spruce is an important commercial species for reforestation. The success in its propagation through somatic embryogenesis is well documented; however the physiological processes involved are poorly understood and remain unoptimized. The variable quality embryos generated in vitro from the same genotype suggest control at the protein and metabolite level. In order to probe metabolic changes, we have conducted a "metabolic footprinting" study, whereby culture media from growing cells was quantitatively analyzed to determine which metabolites were consumed and excreted. Such experiments are advantageous in that there is no need to quench cellular metabolism or extract intracellular metabolites through time-consuming protocols. In this paper we demonstrate the application of the footprinting assay to somatic embryo cells of white spruce (Picea glauca) using 1D (1)H NMR spectroscopy. We have surveyed embryogenesis metabolism in two types of media, maintenance (MN) and maturation (MT). MN medium does not result in shoot apical meristem (SAM) formation, while MT medium induces the necessary changes leading to fully developed somatic embryos. The two types of media were easily distinguished using metabolomics analysis, namely multivariate pattern recognition statistics (orthogonal partial least squares discriminatory analysis). From this analysis, we have identified numerous compounds involved with branched chain amino acid pathways such as valine and isoleucine. These results are explained on the basis of known metabolic pathways implicated in plant and animal developmental processes, and ultimately implicate altered CoA biosynthesis.

Mago Nashi (Mago) is involved in several processes related to mRNA physiology in animal cells, including mRNA export from the nucleus, cytoplasmic mRNA localization, non-sense mediated mRNA decay, and translation. These cellular roles are visible as defects in development when Mago gene expression is modified in mutant model animal systems. Mago gene orthologs exist in plants, however, their functional roles in growth and development have not been well studied. Using an RNA interference (RNAi) approach, we produced transgenic Arabidopsis plants that had reduced levels of AtMago mRNA. RNAi-AtMago plants were delayed in their overall development, produced a greater number of leaves, and possessed short and occasionally fasciated stems. The leaves were small in size and demonstrated enhanced curling along their length. Shoot meristems of RNAi-AtMago plants lacked the cellular organization of wildtype meristems. Shoot meristematic cells were extensively vacuolated and large intercellular spaces were evident. RNAi-AtMago plants produced short lateral roots that lacked normal cell profiles and demonstrated premature root hair differentiation. The arrangement of microspore tetrads in RNAi-AtMago plants was aberrant, and microspores were extensively vacuolated. Pollen production and pollen germination rates were also reduced. RNAi-AtMago plants occasionally produced aborted seeds, or demonstrated delayed seed development that resulted in non-viable seed. The range of developmental defects visible in RNAi-AtMago plants and the ubiquitous expression of AtMago indicates that Mago has essential functions in most, if not all plant cell types., (Copyright © 2009 Elsevier Ireland Ltd. All rights reserved.)

Arabidopsis contains only one functional dihydroflavonol 4-reductase (DFR) gene, but several DFR-like genes encoding proteins with the conserved NAD(P)H binding domain. At4g35420, named DRL1 (Dihydroflavonol 4-reductase-like1), is a closely related homolog of the rice anther-specific gene OsDFR2 reported previously. Two T-DNA mutants (drl1-1 and drl1-2) were found to have impaired pollen formation and seed production. Histological analysis revealed defective microspore development after tetrad release in both mutants. Microspore walls were found to rupture, releasing the protoplasts which eventually degenerated. The DRL1 promoter is anther-specific in closed flower buds. Promoter-GUS analysis in transgenic Arabidopsis revealed expression in tapetum, tetrads, and developing microspores, but not in mature anthers. Enhanced yellow fluorescent protein (EYFP)-localization analysis demonstrated that DRL1 is a soluble cytosolic protein that may also be localized in the nucleus. Restoration of male fertility and seed formation was only achieved by a native promoter-DRL1 construct, but not by a 35S-DRL1 construct, demonstrating the importance of spatial and temporal specificities of DRL1 expression. DRL1 may be involved in a novel metabolic pathway essential for pollen wall development. DRL1 homologs were identified as anther- and floral-specific expressed sequence tags from different species, suggesting that DRL1 may have a conserved functional role in male fertility in flowering plants.

Laticifers are specialized cells that occur in over 20 plant families in several unrelated angiosperm orders. Although laticifers are likely to be of polyphyletic origin, their occurrence is considered a morphological indicator of relatedness among species. The classification of laticifers is based on developmental patterns and overall morphology. The cytoplasmic latex exuded in response to damage often includes specialized metabolites, such as cardenolides, alkaloids and natural rubber. Laticifers provide an effective location to store defense metabolites, although not all latex-bearing plants accumulate bioactive natural products. Ecophysiological studies have shown that latex and its associated metabolites are vital for the defense of plants against insects. The anatomy, development and physiology of laticifers are discussed with a focus on evolutionary and ecological perspectives.

Pyrimidine nucleotide metabolism was studied in tobacco callus cultured for 21days under shoot-forming (SF) and non-shoot-forming (NSF) conditions by following the metabolic fate of orotic acid, a precursor of the de novo pathway, and uridine and uracil, intermediates of the salvage and degradation pathways respectively. Nucleic acid synthesis was also investigated by measuring the incorporation of labeled thymidine into different cellular components. Our results indicate that with respect to nucleotide metabolism, the organogenic process in tobacco can be divided in two "metabolic phases": a de novo phase followed by a salvage phase. The initial stages of meristemoid formation during tobacco organogenesis (up to day 8) are characterized by a heavy utilization of orotic acid into nucleotides and nucleic acids. Utilization of this intermediate for the de novo synthesis of nucleotides, which is limited in NSF tissue, is mainly due to the activity of orotate phosphoribosyltransferase (OPRT), which increases in tissue cultured under SF conditions. After day 8, nucleotide synthesis during shoot growth seems to be mainly due to the salvage activity of both uridine and uracil. Both intermediates are preferentially utilized in SF tissue for the formation of nucleotides and nucleic acids through the activities of their respective salvage enzymes: uridine kinase (URK), and uracil phosphoribosyltransferase (UPRT). Metabolic studies on thymidine indicate that in SF tissue maximal nucleic acid synthesis occurs at day 4, in support of the initiation of meristemoid formation. Overall these results suggest that the organogenic process in tobacco is underlined by precise fluctuations in pyrimidine metabolism which delineate structural events culminating in shoot formation.

Indole-3-butyric acid at 25 microM with methyl jasmonate (MJ) at 100 microM in Panax ginseng synergistically stimulated both root growth and ginsenoside accumulation compared with 100 microM MJ alone. Productivity of ginsenoside was 10 mg l(-1) d(-1) compared to 7.3 mg l(-1) d(-1) with MJ elicitation alone.

Changes in the pattern of pyrimidine nucleotide metabolism were investigated in Pinus radiata cotyledons cultured under shoot-forming (SF; +N(6)-benzyladenine) and non-shoot-forming (NSF, -N(6)-benzyladenine) conditions, as well as in cotyledons unresponsive (OLD) to N(6)-benzyladenine. This was carried out by following the metabolic fate of externally supplied (14)C-labeled orotic acid, intermediate of the de novo pathway, and (14)C-labeled uridine and uracil, substrates of the salvage pathway. Nucleic acid synthesis was also investigated by following the metabolic fate of (14)C-labeled thymidine during shoot bud formation and development. The de novo synthesis of pyrimidine nucleotides was operative under both SF and NSF conditions, and the activity of orotate phosphoribosyltransferase (OPRT), a key enzyme of the de novo pathway, was higher in SF tissue. Utilization of both uridine and uracil for nucleotide and nucleic acid synthesis clearly indicated that the salvage pathway of pyrimidine metabolism is also operative during shoot organogenesis. In general, uridine was a better substrate for the synthesis of salvage products than uracil, possibly due to the higher activity of uridine kinase (UK), compared to uracil phosphoribosyltransferase (UPRT). Incorporation of uridine into the nucleic acid fraction of OLD cotyledons was lower than that observed for their responsive (day 0) counterparts. Similarly, uracil utilization for nucleic acid synthesis was lower in NSF cotyledons, compared to that observed for SF tissue after 10 days in culture. This difference was ascribed to higher UPRT activity measured in the latter. Thus, there was an apparent difference in the utilization of nucleotides derived from uracil and uridine for nucleotide synthesis. The increased ability to produce pyrimidine nucleotides via the salvage pathway during shoot bud formation may be required in support of nucleic acid synthesis occurring during the process. Studies on thymidine metabolism confirmed this notion.

In previous studies we have reported that applications of ascorbic acid (ACS) enhance the conversion frequency of white spruce somatic embryos by "rescuing" structurally disorganized meristems and inducing cell proliferation in the apical poles [C. Stasolla, E.C. Yeung, Ascorbic acid improves the conversion of white spruce somatic embryos, In Vitro Cell. Dev. Biol. Plant 35 (1999) 316-319]. In order to determine if the role played by this metabolite during embryo conversion is mediated by cellular peroxidases, the activity of guaiacol-, ferulic acid-, and ascorbic acid-dependent peroxidases were measured in the apical poles of germinating embryos with altered ASC levels. Changes in the endogenous ASC pool were achieved by treating the embryos with exogenously supplied ASC, L-galactono-gamma-lactone (GL) the last precursor of the de novo biosynthesis of ASC, and lycorine (L), an inhibitor of the last reaction leading to the synthesis of ASC. Our studies demonstrate the existence of a negative correlation between cellular ASC levels and activities of both guaiacol and ferulic acid peroxidases in root and shoot apices. A depletion of cellular ASC enhanced the rate of both guaiacol and ferulic acid oxidation at the apical poles of the embryos and resulted in meristem abortion. In contrast, the activity of guaiacol and ferulic acid peroxidases decreased below control levels if the endogenous ASC content of the embryos was experimentally increased. Fluctuations of total peroxidase activity following alterations in ASC pool were also confirmed by histochemical staining and in vitro studies. Overall our results suggest that a threshold of ASC level must be maintained in the apical poles of germinating embryos in order to inhibit peroxidase activities from cross-linking cell wall components and preventing post-embryonic growth.

Somatic embryos of Arabidopsis thaliana can be produced from explants of developing zygotic embryos. Cultivation of explants on maturation medium results in development of three main classes of regenerative structures: adventitious shoots, fused shoots, and complete somatic embryos. The ontogeny and anatomy of these structures was examined using serial plastic sections. Furthermore, two molecular markers were assayed to monitor transcriptional auxin responses and formation of a root meristem in this process: the LENNY allele of PIN4, a transposon insertion creating a fusion to the reporter gene GUS; and DR5::GUS, a synthetic reporter of auxin-induced transcription. In zygotic embryogenesis, PIN4 expression is confined to the center of the root meristem and begins to be detectable by the globular stage of embryogenesis, while DR5::GUS expression marks an "auxin perception maximum" in the more distal regions of the root. Adventitious and fused shoots develop no anatomically recognizable root meristem and do not express either of the two markers at their basal pole. Instead, the vasculature of their axis is directly connected to the vasculature of the explant. By contrast, complete somatic embryos were only loosely attached to the explant, had an anatomically defined root meristem and showed expression of both markers at their root pole. Our results suggest that the establishment of a root meristem in somatic embryos required appropriate auxin levels during the course of their development.

Background and Aims: Owing to large-scale collecting, the lady's slipper orchid, Paphiopedilum delenatii, is under threat of extinction. Asymbiotic germination provides a useful way to re-establish plants in the wild and for commercial propagation. A detailed study of embryo development would provide information on subsequent germination events and aid in the propagation of the species., Methods: Developing capsules were collected for histochemical and ultrastructural studies by using both light and transmission electron microscopy., Key Results: The suspensor of this species consists of three vacuolated cells. During the early globular stage of embryo development, structural differentiation occurs, revealing an abundance of smooth endoplasmic reticulum cisternae and wall ingrowths within the suspensor cells. These features are not present in cells of the embryo proper. Furthermore, the results of Nile red staining demonstrate that a cuticular layer is present only in the embryo proper, but absent from the suspensor. Cuticular material is also present in the inner walls of the seed coat, and persists through seed maturation., Conclusions: The morphological features of the transfer cell and the absence of cuticular material in the suspensor cell wall corroborate the hypothesis that the suspensor is the major nutrient uptake site for the developing embryo in the lady's slipper orchid. The absence of an endosperm and presence of cuticular material in the inner walls of the seed coat enclosing the embryo proper further support the notion that nutrient uptake by the embryo is confined to the micropylar end of the seed through the suspensor.

Previously, we demonstrated that, in some cell lines, exogenous applications of ascorbic acid (ASC) enhance the conversion frequency of white spruce (Picea glauca (Moench) Voss) somatic embryos, by stimulating mitotic activity in the apical meristems. To examine this event in more detail, we investigated the effects of ASC on de novo, salvage and degradation pathways of pyrimidine metabolism by following the metabolic fate of (14)C-labeled orotic acid, thymidine, uridine and uracil in shoot and root poles of germinating embryos, after altering the cellular ASC content of the embryos. Alterations in endogenous ASC content did not affect the utilization of either orotic acid or uracil, but affected the metabolism of thymidine and uridine. Specifically, a lowering of endogenous ASC content by applications of lycorine (L), an inhibitor of the last enzyme of the ASC de novo biosynthetic pathway, resulted in a lower embryo conversion frequency, as well as a reduced percentage of thymidine and uridine incorporated into nucleotides and nucleic acids. The reduction in thymidine and uridine anabolism was mainly ascribed to the decreased activities of thymidine kinase (TRK) and uridine kinase (URK), the respective salvage enzymes of thymidine and uridine, measured in L-treated embryos. These effects were solely a result of a decrease in endogenous ASC content because applications of ascorbic acid plus lycorine (ASC + L) increased embryo conversion frequency, thymidine and uridine salvage activities, and TRK and URK activities to near control values. Inclusion of exogenous ASC in the germination medium did not affect the percentage of embryos able to convert to viable plantlets, although it increased thymidine and uridine utilization for nucleic acid synthesis in the shoot and root poles of the embryos. Taken together, these findings confirm that cellular ASC plays a key role in the reactivation of the apical meristems of germinating white spruce somatic embryos.

Nucleotide metabolism was investigated during white spruce organogenesis by following the metabolic fate of (14)C-labeled adenine, adenosine and inosine, as purine precursors, and orotic acid, uridine, and uracil, as pyrimidine intermediates. Key enzymes of purine and pyrimidine metabolism were also assayed during the organogenic process. White spruce epicotyl explants cultured on shoot-forming (SF) medium had a better ability to utilize adenine and adenosine for nucleotide and nucleic acid synthesis, compared to tissue cultured on non-shoot forming (NSF) medium. High levels of salvage products were observed in SF tissue after 10 days in culture, when shoot formation was initiated along the epicotyl axis of the explants. Such a differential utilization of purine precursors was mainly due to the higher specific activity of the two adenine and adenosine salvage enzymes, adenine phosphoribosyltransferase (APRT) and adenosine kinase (AK), measured in SF tissue. Similar catabolism of inosine was observed in both SF and NSF conditions during the 30 days of culture. For pyrimidines, the higher activities of the de novo, salvage, and degradation pathways observed in SF tissue, compared to NSF tissue throughout the course of the experiment, clearly denote a faster turnover of pyrimidine nucleotides in the former. Taken together, these results suggest that a better utilization of purine bases and nucleosides for nucleotide and nucleic acid synthesis, as well as a more rapid turnover of pyrimidine nucleotides, represent a physiological switch, which occurs during the initiation and continuation of the organogenic process in white spruce., (Copyright © 2006 Elsevier Ireland Ltd. All rights reserved.)

We investigated the role of the oilbody proteins in developing and germinating Arabidopsis thaliana seeds. Seed oilbodies are simple organelles comprising a matrix of triacylglycerol surrounded by a phospholipid monolayer embedded and covered with unique proteins called oleosins. Indirect observations have suggested that oleosins maintain oilbodies as small single units preventing their coalescence during seed desiccation. To understand the role of oleosins during seed development or germination, we created lines of Arabidopsis in which a major oleosin is ablated or severely attenuated. This was achieved using RNA interference techniques and through the use of a T-DNA insertional event, which appears to interrupt the major (18 kD) seed oleosin gene of Arabidopsis and results in ablation of expression. Oleosin suppression resulted in an aberrant phenotype of embryo cells that contain unusually large oilbodies that are not normally observed in seeds. Changes in the size of oilbodies caused disruption of storage organelles, altering accumulation of lipids and proteins and causing delay in germination. The aberrant phenotypes were reversed by reintroducing a recombinant oleosin. Based on this direct evidence, we have shown that oleosins are important proteins in seed tissue for controlling oilbody structure and lipid accumulation.

Proteinase inhibitor II proteins (PIN2) are serine proteinase inhibitors found in the Solanaceae. Here, we assign functions in seed development to two Solanum americanum genes, SaPIN2a and SaPIN2b, encoding proteinase inhibitor II. Their mRNAs and proteins have been previously localized to the reproductive tissues, including the inner cell layers of ovules in senescent flowers at the beginning of fruit development, suggestive of their endogenous roles in reproductive development. We have employed RNA interference (RNAi)-induced post-transcriptional gene silencing (PTGS) to further investigate the role of SaPIN2a and SaPIN2b during seed development. A SaPIN2a-derived construct that shared 83% nucleotide homology to SaPIN2b was used in PTGS to silence both genes. Northern blot analyses confirmed that the PIN2-RNAi transgenic plants contain small interfering RNAs (siRNAs) and exhibit reduced levels of SaPIN2a and SaPIN2b mRNAs at various stages of floral development. A reduction in seed set due to seed abortion was observed in PIN2-RNAi transgenic lines. Cytological and molecular analyses of these lines showed the lack of SaPIN2a and SaPIN2b mRNAs and proteins at the inner cell layers of the ovules in senescent flowers. Aborted seeds in transgenic fruits had an abnormal endothelium. The anomalous expansion of the endothelium prevented proper development of the endosperm and embryo, leading to seed abortion. Our observations indicate that SaPIN2a and SaPIN2b are essential for seed development and suggest that the endothelium may protect the embryo sac, allowing proper formation of the endosperm and embryo, as a result of its ability to produce proteinase inhibitors.

Softwoods or gymnosperms, which make up 60% of the forested areas of the world, are economically important as a source of lumber, pulp, and paper. Reforestation is a major activity worldwide and the potential benefits of using clonal planting stock have long been recognized. Tissue culture clonal methods or micropropagation is a newer approach that can be achieved by enhancing axillary bud breaking, production of adventitious buds (organogenesis), and somatic embryogenesis. Plantlet production via organogenesis requires at least four stages: (1) bud induction on the explant, (2) shoot development and multiplication, (3) rooting of developed shoots, and (4) hardening of plantlets. Similarly, the production of plantlets via somatic embryogenesis, which has the potential to produce a larger number of plantlets, and in a shorter period of time, also requires several stages. These include (1) induction, maintenance, and proliferation of embryogenic tissue; (2) maturation (both morphological and physiological) of somatic embryos; and (3) germination and conversion of the somatic embryos. In this chapter, plantlet production via organogenesis from seedling and adolescent/mature explants and somatic embryogenesis from immature and mature seeds of white spruce (Picea glauca) are outlined.

In white spruce, an improvement of somatic embryo number and quality can be achieved through experimental manipulations of the endogenous levels of reduced (GSH) and oxidized (GSSG) glutathione. An optimal protocol for embryo production included an initial application of GSH in the maturation medium, followed by replacement with GSSG during the remaining maturation period. Under these conditions, the overall embryo population more than doubled, and the percentage of fully developed embryos increased from 22% to almost 70%. These embryos showed improved post-embryonic growth and conversion frequency. Structural studies revealed remarkable differences between embryo types, especially in storage product deposition pattern and organization of the shoot apical meristem (SAM). Compared with their control counterparts, glutathione-treated embryos accumulated a larger amount of starch during the early stages of development, and more protein and lipid bodies during the second half of development. Differences were also noted in the organization of SAMs. Shoot meristems of control embryos were poorly organized and were characterized by the presence of intercellular spaces, which caused separation of the subapical cells. Glutathione-treated embryos had well-organized meristems composed of tightly packed cells which lack large vacuoles. The improved organization of the shoot apical meristems in treated embryos was ascribed to a lower production of ethylene. Differences in meristem structure between control and treated embryos were also related to the localization pattern of HBK1, a shoot apical meristem 'molecular marker' gene with preferential expression to the meristematic cells of the shoot pole. Expression of this gene, which was localized to the apical cells in control embryos, was extended to the subapical cells of treated embryos. Overall, it appears that meristem integrity and embryo quality are under the direct control of the glutathione redox state.

Water stress is an important factor which regulates organized development of both zygotic and somatic embryos. Somatic embryos of white spruce were cultured in the presence of polyethylene glycol (PEG), a non-plasmolyzing agent which increases embryo quality and number, and mannitol, a plasmolyzing agent. The effects of these two compounds on both ascorbate and glutathione metabolism were investigated at different stages of embryo development. Compared to control and mannitol-treated embryos, embryos treated with PEG accumulated higher levels of endogenous ascorbate (ASC) in its reduced form, especially during the first half of the maturation period. This increase, also observed in immature seeds, was mainly the result of two different processes: activation of the de novo ASC machinery, and recycling of ASC from ascorbate free radicals (AFR) which was modulated by the activity of ascorbate free radical reductase (AFRR, EC. 1.6.5.4). The activity of this enzyme increased during the early phases of development in both PEG-treated somatic embryos and seeds. Compared to control somatic embryos, mannitol and PEG were shown to change the levels of reduced (GSH) and oxidized glutathione (GSSG). In particular, a constant decline in the GSH/GSSG ratio was observed in the presence of PEG. This pattern was also observed in maturing white spruce seeds. Overall, these data indicate that applications of non-plasmolyzing agents in the culture medium of spruce somatic embryos result in seed-like fluctuations of the ascorbate-glutathione metabolism, which may have a positive effect on embryo yield.

In vitro culture of hypocotyl explants from Kandelia candel, a common mangrove species, on hormone-free Murashige and Skoog (MS) medium resulted in shoot formation. Since the hypocotyls showed good potential for in vitro shoot multiplication, the process of bud primordium formation was analyzed from a histological viewpoint. A wound periderm first appeared at the top, exposed cut surface of the explants. The wound-induced meristem continued to divide giving rise to suberized cells oriented towards the cut surface. After formation of the suberized cell layers, the meristem and its inner derivatives differentiated into multilayered, uniformly packed parenchyma cells. Bud primordia differentiated from the dense cytoplasmic cells of the wound-induced meristem just beneath the suberized layer near the severed vascular bundles. Each explant produced several visible shoot buds. Furthermore, histological sections revealed that additional bud primordia were present within the explant just underneath the suberized cells and that these bud primordia appeared to be arrested in their development. The fact that additional bud primordia were present within the explant suggests that further manipulation of the explant is helpful to maximize the potential of this system.

Changes in pyrimidine metabolism were investigated during programmed cell death (PCD) of tobacco BY-2 cells, induced by a simultaneous increase in the endogenous levels of nitric oxide (NO) and hydrogen peroxide. The de novo synthesis of pyrimidine nucleotides was estimated by following the metabolic fate of the (14)C-labelled orotic acid, whereas the rates of salvage and degradation pathways were studied by measuring the respective incorporation of (14)C-labelled uridine and uracil under different treatments. Nucleic acid metabolism was also examined using labelled thymidine as a marker. The results show that specific alterations in the balance of pyrimidine nucleotide synthesis, which include a decreased rate of salvage activity of uracil and uridine and increased salvage activity of thymidine, represent a metabolic switch that establishes proper cellular conditions for the induction of PCD. In particular, a reduction in the utilization of uracil for salvage products occurs very early during PCD, before the appearance of typical cytological features of the death programme, thus representing an early metabolic marker for PCD. These changes are strictly associated with PCD, since they do not occur if NO or hydrogen peroxide are increased individually, or if actinomycin, which inhibits the death programme, is added into the medium in the presence of NO and hydrogen peroxide. The possible roles of these fluctuations in pyrimidine metabolism on the cellular nucleotide pool are discussed in relation to the induction of cell death.

Inclusions of reduced glutathione (GSH) in the maturation medium increased the conversion frequency of white spruce somatic embryos without the need of a partial drying treatment (PDT). This beneficial effect was the result of major alterations in morphology and gene expression during the maturation period. Compared with control embryos, GSH-treated embryos showed a differential accumulation of storage products, i.e. preferential deposition of starch, the reduced formation of protein bodies, and increased vacuolation of cells. These morphological changes correlated with extensive alterations of gene expression occurring throughout the maturation period. The transcript profiles of stage-specific embryos matured with or without GSH were analysed using a DNA microarray containing 2 178 cDNAs from loblolly pine (Pinus taeda). The efficiency of heterologous hybridization between spruce and pine species on microarrays has previously been documented. The results indicate that several genes involved in a variety of signal regulatory pathways were differentially expressed in developing GSH- treated embryos. The transcript levels of many genes involved in carbohydrate metabolism and protein synthesis were altered by the presence of GSH and denoted differences in physiology between treatments. Extensive changes in the expression of genes participating in hormone synthesis, nucleotide metabolism, and meristem formation were also observed and related to the post-embryonic performance of the embryos.