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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.

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.)

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.)

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.)

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.

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.

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.

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.

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.

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.

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.

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.

The role of gibberellins (GAs) during early embryo development was examined using microspore-derived embryos (MDEs) of Brassica napus. At the globular stage of development, 10 d after initial culture (DAC) when endogenous GA(1) levels are increasing rapidly, a triazole, uniconazole, was used at 1, 33 and 100 microM to inhibit GA biosynthesis. Within this dose range there was no apparent effect of the inhibitor on embryo growth through to the early torpedo stage. However, by 25 DAC uniconazole-treated MDEs showed significantly reduced (50%) axis elongation. Addition of GA(1) at 33 microM on 14 DAC to embryos pretreated with 1 microM uniconazole on 10 DAC prevented this reduction in axis length, giving axis elongation equivalent to untreated MDEs. Application of GA(1) alone, however, did not significantly increase axis elongation. The reduced axis growth seen with uniconazole treatment was due to reduced cell elongation, but not cell number, and the co-applied GA(1) thus prevented the uniconazole-induced reduction in cell length. The elongating axis of MDEs may thus be a useful tool for examining the role of GAs in cell elongation.

Fertilization in flowering plants involves the fusion of one sperm with the egg cell and a second sperm with the central cell. In the Nun orchid, Phaius tankervilliae (Aiton) Bl., the pollen tube deposits two sperms in the "cytoplasmic mass" of the degenerating synergid. Initially both sperms stay close together. Soon afterwards, the two sperms undergo migration. The leading sperm migrates towards the central cell, while the other sperm moves laterally towards the egg cell. The degenerated synergid cytoplasmic content fills in the available space between the central cell and the egg cell, forming the actin coronas. Due to the high actin content, the bright fluorescence initially prevents the visualization of cellular details. With the subsequent reduction in fluorescence, actin staining reveals that the two sperms are pear-shaped with pointed tails. As the sperms approach their respective target cells, cellular extensions form near the point of sperm entry in both the egg cell and the central cell. These structures appear to aid in the cell fusion process. The morphological and structural features observed provide evidence that the process of double fertilization requires the active participation of not only the two sperms but also the egg cell and the central cell.

It has previously been shown that exogenous applications of ascorbic acid (AA) increase the conversion frequency of somatic embryos of white spruce (Picea glauca (Moench) Voss). To determine whether ascorbic acid alters purine metabolism during the early phases of embryo germination, the relative rates of purine salvage and degradation were investigated by following the metabolic fates of exogenously applied [8-14C]adenine, [8-14C]adenosine, and [8-14C]inosine, and the activities of several key enzymes. We demonstrated that both the salvage and the degradation pathways operate during germination. Specifically, adenine and adenosine were mainly salvaged to nucleotides and nucleic acids, whereas an appreciable amount of inosine was degraded to CO2 and ureides. Comparisons of purine metabolism between control and AA-treated embryos showed that exogenous applications of ascorbic acid enhanced the ability of the embryos to take up adenine and adenosine throughout the germination period. Furthermore, the higher enzymatic activities of adenosine kinase and adenine phosphoribosyltransferase were responsible for the larger proportion of adenine and adenosine being salvaged in AA-treated embryos compared with control embryos. Thus, there was a positive correlation between the ability to anabolize purine precursors and successful embryo conversion.

Ethylene production during seed development in Brassica napus occurs first at 20 d after pollination (DAP), while a second greater peak occurs at 35 DAP. Because of the inaccessible location of the embryo within the maternal tissue, microspore-derived embryos (MDEs) of B. napus were used as a model for studying the role of ethylene during embryo development. The MDEs also produced a peak in ethylene evolution at 20 DAC (i.e. the early cotyledonary stage), dropping to minimal levels by 25-30 DAC. At 20 DAC the excised cotyledon evolved 85% of the ethylene found in the whole MDE. To determine the role of ethylene, MDEs were treated with aminoethoxyvinylglycine (AVG, an inhibitor of ethylene biosynthesis), CoCl(2) (an inhibitor of 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase), and silver thiosulphate (STS, an inhibitor of ethylene action). An inhibition in ethylene production or action at 20 DAC resulted in diminished lateral cotyledon expansion, due to a reduction in the lateral expansion of cells within the cotyledon. Recovery to 'control-type' levels of cotyledon cell expansion was achieved by application of ACC (the metabolic precursor of ethylene) to AVG-treated MDEs. Thus, ethylene production at 20 DAP likely controls cotyledon expansion during embryo development.

We have previously described a homeotic leafy cotyledon (lec) mutant of Arabidopsis that exhibits striking defects in embryonic maturation and produces viviparous embryos with cotyledons that are partially transformed into leaves. In this study, we present further details on the developmental anatomy of mutant embryos, characterize their response to abscisic acid (ABA) in culture, describe other mutants with related phenotypes, and summarize studies with double mutants. Our results indicate that immature embryos precociously enter a germination pathway after the torpedo stage of development and then acquire characteristics normally restricted to vegetative parts of the plant. In contrast to other viviparous mutants of maize (vp1) and Arabidopsis (abi3) that produce ABA-insensitive embryos, immature lec embryos are sensitive to ABA in culture. ABA is therefore necessary but not sufficient for embryonic maturation in Arabidopsis. Three other mutants that produce trichomes on cotyledons following precocious germination in culture are described. One mutant is allelic to lec1, another is a fusca mutant (fus3), and the third defines a new locus (lec2). Mutant embryos differ in morphology, desiccation tolerance, pattern of anthocyanin accumulation, presence of storage materials, size and frequency of trichomes on cotyledons, and timing of precocious germination in culture. The leafy cotyledon phenotype has therefore allowed the identification of an important network of regulatory genes with overlapping functions during embryonic maturation in Arabidopsis.

The bulk modulus of elasticity (E) for Pinus contorta (lodgepole pine) x Pinus banksiana (jack pine) hybrids was compared between a site (AI) close to a sour gas processing plant and a control site (AV). The mean bulk modulus of elasticity for branches from AI was 47.5 MPa vs 18.5 MPa for the control site (AV). Site AI had been exposed to S-gas emissions and large amounts of elemental S deposition and had an acidic soil (pH 4.0 at 10 cm depth). During 1981 the needles at AI had more aluminum and iron compared to those at AV (900 ppm vs 390 ppm AI in the 3-year-old needles). Mean leader growth was measured over a 3-year period and was observed to be greater at AI than AV (46+/-7 cm vs 29+/-9 cm for 1988). Histochemically, the needles at AI had higher phenol and lignin content than AV. These results suggest that the S-gas fumigation, S-dust deposition, plus increased concentrations of soluble aluminum and iron had altered the cell wall elastic properties resulting in altered water relations. The implications of this on leaf diffusive resistance and photosynthesis are discussed.

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [(13)C(6)]-IAA or [(2)H(6)]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.

Structural changes during development of the caudicle of Epidendrum ibaguense (Orchidaceae) were followed using light and electron microscopy. The caudicle begins as a mass of small meristematic cells in the microsporangium. The central cells of the mass enlarge and enter a division cycle. Completion of this cycle produces linear tetrads of cells which remain attached to each other throughout the life of the caudicle. These tetrads then accumulate a thick secondary wall on their outer surfaces. These thick-walled cells form the main structural element of the caudicle at maturity. The cells on the periphery of the mass differentiate into a gradation of cell types characterized by their very high content of smooth ER. The majority of these thin-walled cells are located at the junction points between adjacent caudicles and between caudicle and pollinium. The cells in these positions produce large quantities of a lipid polymer. This product is synthesized in association with large tubular ER complexes present in discrete areas in the cytoplasm. Plastids of these cells also produce a lipidic substance. Some thin-walled cells are located among the thick-walled structural cells; however, the lipid polymer is not synthesized in these cells. At maturity, all thin-walled cells undergo breakdown and autolysis resulting in gradual obliteration of cytoplasmic detail and organelles. In the lipid containing thin-walled cells, lysis of the cells releases the lipidic masses which then form the elastic joining compound between adjacent caudicles and between caudicles and pollinia., (© 1983 Botanical Society of America.)

Abscisic acid (ABA) levels in 3-mm apical root segments of slowly droughted sunflower plants (Helianthus annuus L. cv Russian Giant) were analyzed as the methyl ester by selected ion monitoring gas chromatography-mass spectrometry using characteristic ions. An internal standard, hexadeuterated ABA (d6ABA) was used for quantitative analysis. Sunflower seedlings, grown in aeroponic chambers, were slowly droughted over a 7-day period. Drought stress increased ABA levels in the root tips at 24, 72, and 168 hour sample times. Control plants had 57 to 106 nanograms per gram ABA dry weight in the root tips (leaf water potential, -0.35 to -0.42 megapascals). The greatest increase in ABA, about 20-fold, was found after 72 hours of drought (leaf water potential, -1.34 to -1.47 megapascals). Levels of ABA also increased (about 7- to 54-fold) in 3-mm apical root segments which were excised and then allowed to dessicate for 1 hour at room temperature.

THERE ARE TWO TYPES OF MITOCHONDRIA PRESENT IN IMBIBED PEANUT COTYLEDONS: a light type (density 1.182 grams per cubic centimeter) and a heavy type (density 1.205 grams per cubic centimeter). The membrane fractions from these two types can be distinguished using sucrose density gradient analysis, and differences in membrane density between the light and heavy types are reflected in differences in their protein N and phospholipid P composition. With increasing time after imbibition, there is a substantial increase in the amount and activity of the light type of mitochondria due to their de novo synthesis. The membrane density of the light mitochondrial fraction declines over 5 days after the start of imbibition as the phospholipid P to protein N ratio increases. The heavy mitochondrial fraction declines during the first 3 days after the start of imbibition, and then it remains at a low, but constant, level thereafter. Even during the decline, however, there is synthesis of proteins comparable to that into light mitochondria. The mitochondrial biogenesis that has been observed in peanut cotyledons is of the light type, the function and physiological importance of the minor heavy type is not known.

Xylem transfer cells in the rhizome of Hieracium floribundum are described for the first time and several methods used in visualizing these cells are discussed. The most marked wall ingrowths occur in transfer cells associated with the xylem of foliar traces.