The orchid seed coat: a developmental and functional perspective.

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.

Germacrene A Synthases for Sesquiterpene Lactone Biosynthesis Are Expressed in Vascular Parenchyma Cells Neighboring Laticifers in Lettuce.

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 ß-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.

The promoter sequences of lettuce cis-prenyltransferase and its binding protein specify gene expression in laticifers.

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 ß-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 ß-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.

Identification and characterization of a female gametophyte defect in sdk1-7 +/- abi3-6 +/- heterozygotes of Arabidopsis thaliana.

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.

The overexpression of rice ACYL-CoA-BINDING PROTEIN2 increases grain size and bran oil content in transgenic rice.

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 ß-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.

Elevated carbon dioxide decreases the adverse effects of higher temperature and drought stress by mitigating oxidative stress and improving water status in Arabidopsis thaliana.

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 (ECO2), 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, CO2, 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 CO2 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 ECO2 had larger effects on plants than higher temperatures. ECO2 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 ECO2 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.

Paracrine/autocrine control of spermatogenesis by gonadotropin-inhibitory hormone.

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.

Immunolocalization and Changes of Hydroxyproline-Rich Glycoproteins During Symbiotic Germination of Dendrobium officinale.

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.

Arabidopsis ACYL-COA-BINDING PROTEIN1 interacts with STEROL C4-METHYL OXIDASE1-2 to modulate gene expression of homeodomain-leucine zipper IV transcription factors.

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 ß-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.

A perspective on orchid seed and protocorm development.

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.

Acyl-CoA-Binding Protein ACBP1 Modulates Sterol Synthesis during Embryogenesis.

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.

Dynamic distribution and the role of abscisic acid during seed development of a lady's slipper orchid, Cypripedium formosanum.

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.

Transcriptome atlas of the Arabidopsis funiculus--a study of maternal seed subregions.

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.

The Arabidopsis cytosolic Acyl-CoA-binding proteins play combinatory roles in pollen development.

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 ß-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.

Preservation of high phenylalanine ammonia lyase activities in roots of Japanese Striped corn: a potential oral therapeutic to treat phenylketonuria.

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.

Orchid protocorm-like bodies are somatic embryos.

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.

The evolution of floral nectaries in Disa (Orchidaceae: Disinae): recapitulation or diversifying innovation?

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.

Comprehensive developmental profiles of gene activity in regions and subregions of the Arabidopsis seed.

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.

Ethylene involvement in silique and seed development of canola, Brassica napus L.

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.

The study of in vitro development in plants: general approaches and photography.

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.