A seed coat outer integument-specific promoter for Brassica napus

In search for seed coat-specific promoters for canola (Brassica napus), transgenic plants carrying a 2,121 bp fragment of Arabidopsis thaliana At4g12960 promoter (AtGILTpro) fused to the uidA reporter gene (GUS) were generated. Out of 7 independent events in transgenic canola plants raised, 2 exhibited GUS activity exclusively in the outer integument of the seed coat. GUS activity in other tissues was also observed in the remaining five transformants. Therefore, the AtGILT promoter can be used as a canola seed coat outer integument-specific promoter after the generation and selection of desired transformants from several transgenic lines.     

Two seed coat-specific promoters are functionally conserved between <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> and <Emphasis Type="Italic">Brassica napus</Emphasis>

Canola is one of the most important cash crops in Canada, and a national project named “Designing Oilseeds for Tomorrow’s Market” was undertaken to improve seed meal quality of this strategically important crop. As a part of this project, our group is focusing on identifying seed coat-specific promoters for canola (Brassica napus). These promoters will be used to genetically modify canola seed coat to reduce or eliminate anti-nutritional components from the meal. The Arabidopsis thaliana BAN promoter (AtBANpro) and δVPE promoter (AtδVPEpro) were isolated and fused to GUS reporter gene to generate transgenic canola plants. These plants were analyzed by GUS staining and microtome sectioning which showed that both promoters are seed coat-specific in canola: AtBANpro in inner seed coat layer and AtδVPEpro in outer seed coat layer. Therefore, the two Arabidopsis promoters can be used to modify genes in seed coat of canola for further improving its seed qualities.     

<Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> outer ovule integument morphogenesis: Ectopic expression of <Emphasis Type="Italic">KNAT1</Emphasis>reveals a compensation mechanism

Background  

The Arabidopsis outer ovule integument is a simple two-cell layered structure that grows around the developing embryo and develops into the outer layer of the seed coat. As one of the functions of the seed coat is the protection of the plant embryo, the outer ovule integument is an example for a plant organ whose morphogenesis has to be precisely regulated.     

Promoter Activity of Phenylalanine Ammonia-Lyase Gene of Pharbitis nil in Arabidopsis

Promoter activity of phenylalanine ammonia-lyase (PAL) gene of Pharbitis nil was examined by introducing a PAL:GUS construct into Arabidopsis. GUS staining was observed in vascular bundles of hypocotyl and cotyledons, endodermal cells of the primary root, hydathodes, stigma and pollens of mature flower, abscission zones of petals and sepals and inner layer of seed coat. Light induced GUS expression in cotyledons and the upper part of hypocotyl in which anthocyanin was accumulated. Wounding also induced GUS expression. Endogenous PAL activity increased earlier than the GUS activity directed by the PAL promoter.     

Isolation and functional characterization of two novel seed-specific promoters from sunflower (Helianthus annuus L.)

The promoter region of two sunflower (Helianthus annuus L. HA89 genotype) seed specifically expressed genes, coding for an oleate desaturase (HaFAD2-1) and a lipid transfer protein (HaAP10), were cloned and in silico characterized. The isolated fragments are 867 and 964 bp long, respectively, and contain several seed-specific motifs, such as AACA motif, ACGT element, E-Boxes, SEF binding sites and GCN4 motif. Functional analysis of these promoters in transgenic Arabidopsis plants was investigated after fusing them with the β-glucuronidase (GUS) reporter gene. None of the promoters triggered GUS activity in any vegetative tissue, with the exception of early seedling cotyledons. HaFAD2-1 and HaAP10 promoters were tested along seed development from globular stage to mature seeds. GUS staining was restricted to embryonic tissue and quantitative fluorometric assays showed high activity values at the later stages of development. In this work we demonstrate that HaFAD2-1 promoter is as strong as 35S promoter even though it is a tissue-specific promoter and its activity derived just from the embryo, thus confirming that it can be considered a strong highly specific seed promoter useful for biotechnology applications.     

Identification and preliminary analysis of a new PCP promoter from Brassica rapa ssp. chinensis

The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation method, Arabidopsis transgenic Kan^R plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls, upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any expression in sepals and pistils.     

Global analysis of canola genes targeted by SHORT HYPOCOTYL UNDER BLUE 1 during endosperm and embryo development

Seed development in dicots includes early endosperm proliferation followed by growth of the embryo to replace the endosperm. Endosperm proliferation in dicots not only provides nutrient supplies for subsequent embryo development but also enforces a space limitation, influencing final seed size. Overexpression of Arabidopsis SHORT HYPOCOTYL UNDER BLUE1::uidA (SHB1:uidA) in canola produces large seeds. We performed global analysis of the canola genes that were expressed and influenced by SHB1 during early endosperm proliferation at 8 days after pollination (DAP) and late embryo development at 13 DAP. Overexpression of SHB1 altered the expression of 973 genes at 8 DAP and 1035 genes at 13 DAP. We also surveyed the global SHB1 association sites, and merging of these sites with the RNA sequencing data identified a set of canola genes targeted by SHB1. The 8‐DAP list includes positive and negative genes that influence endosperm proliferation and are homologous to Arabidopsis MINI3, IKU2, SHB1, AGL62, FIE and AP2. We revealed a major role for SHB1 in canola endosperm development based on the dynamics of SHB1‐altered gene expression, the magnitude of SHB1 chromatin immunoprecipitation enrichment and the over‐representation of eight regulatory genes for endosperm development. Our studies focus on an important agronomic trait in a major crop for global agriculture. The datasets on stage‐specific and SHB1‐induced gene expression and genes targeted by SHB1 also provide a useful resource in the field of endosperm development and seed size engineering. Our practices in an allotetraploid species will impact similar studies in other crop species.     

Seed coat development in Leucospermum cordifolium (Knight) Fourcade (Proteaceae) and a clarification of the seed covering structures in Proteaceae

MANNING, J. C. & BRITS, G. J., 1993. Seed coat development in Leucospermum cordifolium (Knight) Fourcade (Proteaceae) and a clarification of the seed covering structures in Proteaceae . The development of the seed coat and pericarp is studied in Leucospermum cordifolium from ovule to mature seed. The ovule and seed are characterized by a tegmic pachychalaza. The pericarp is adnate to the integuments from anthesis and remains unthickened to maturity. The outer integument forms the seed coat and the seed is endotestal: the outer epidermis becomes tanniniferous and the inner epidermis develops into a crystalliferous palisade. The inner integument degenerates at an early stage. Examination of the literature reveals that the crystal palisade layer of the outer integument has been erroneously assumed to constitute an endocarp. This finding indicates that a re-interpretation of all published information on the seed coat in indehiscent Proteaceae is necessary before any speculations on the phylogenetic significance of the seed coat can be entertained.     

Ephemeral and non-ephemeral structures during seed development in two Cytisus species (Papilionoideae,Leguminosae)

Abstract

Seed formation involves not only the embryo and endosperm development, but also the formation of a series of either ephemeral or non-ephemeral structures. In this article, we study several of those structures in Cytisus multiflorus and Cytisus striatus. The endosperm development is first nuclear and later cellular, except for the chalazal area, whose development is always nuclear. It generates, in the early developmental stages, a sac-like haustorium. As the seed develops, two structures seem to be closely related to nutrient mobilization to the embryo sac: on the one hand, a group of cells and a channel, located in the chalazal area and closely related between them and to the endosperm haustorium, which could be interpreted as a hypostase and on the other hand, an endothelium, derived from the inner integument, which later degenerates leaving no trace in the mature seed. All of these structures would be associated with the directionality of assimilates from ovule tissues to embryo sac. In mature seed and surrounding the embryo appears a unicellular layer of cells rich in proteins (aleurone layer), which is the origin of the outermost layer of the cellular endosperm. The seed coat is made up only of the outer integument.     

Expression of the Commelina yellow mottle virus promoter in transgenic oat

The Commelina yellow mottle virus (CoYMV) infects the monocot weed Commelina diffusa. The objective of this study was to investigate the transgene expression conferred by the CoYMV promoter in a monocot species. Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with the CoYMV promoter fused to the coding region of E. coli β-glucuronidase (uidA, GUS). Developmental and tissue-specific expression of the CoYMV-GUS construct was investigated in regenerated plants and their progeny. Histochemical GUS staining was primarily localized in the vascular tissues of shoots, leaves, floral bracts and in roots. While ovaries stained intensely, no staining was detected in anthers or the endosperm in mature seed. The scutellum of mature and germinating seed exhibited GUS activity. Received: 16 April 1997 / Revision received: 23 July 1997 / Accepted: 2 August 1997     

Involvement of AtLAC15 in lignin synthesis in seeds and in root elongation of Arabidopsis

Laccase, EC 1.10.3.2 or p-diphenol:dioxygen oxidoreductase, has been proposed to be involved in lignin synthesis in plants based on its in vitro enzymatic activity and a close correlation with the lignification process in plants. Despite many years of research, genetic evidence for the role of laccase in lignin synthesis is still missing. By screening mutants available for the annotated laccase gene family in Arabidopsis, we identified two mutants for a single laccase gene, AtLAC15 (At5g48100) with a pale brown or yellow seed coat which resembled the transparent testa (tt) mutant phenotype. A chemical component analysis revealed that the mutant seeds had nearly a 30% decrease in extractable lignin content and a 59% increase in soluble proanthocyanidin or condensed tannin compared with wild-type seeds. In an in vitro enzyme assay, the developing mutant seeds showed a significant reduction in polymerization activity of coniferyl alcohol in the absence of H₂O₂. Among the dimers formed in the in vitro assay using developing wild-type seeds, 23% of the linkages were β-O-4 which resembles the major linkages formed in native lignin. The evidence strongly supports that AtLAC15 is involved in lignin synthesis in plants. To our knowledge, this is the first genetic evidence for the role of laccase in lignin synthesis. Changes in seed coat permeability, seed germination and root elongation were also observed in the mutant.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

THE DEVELOPMENT OF THE SEED OF ABUTILON THEOPHRASTI. II. SEED COAT

Winter , Dorothy M. (Iowa State U., Ames.) The development of the seed of Abutilon theophrasti. II. Seat coat. Amer. Jour. Bot. 47(3) : 157—162. Illus. 1960.–The integuments of Abutilon theophrasti Medic. undergo a rapid increase in size, predominantly by anticlinal cell divisions during the first 3 days after fertilization. Within 7 days, the outer epidermis of the inner integument becomes thick walled. At maturity this compact, lignified, and cutinized palisade layer accounts for more than half the thickness of the seed coat. During early growth, the palisade cells form a continuous layer in the micropylar region. In the chalazal region the palisade layer is discontinuous in a slit-shaped region, 60 × 740 microns. The shape of this discontinuity constitutes a major difference between dormant-seeded Abutilon and non-dormant Gossypium seeds. Exterior to the palisade layer is the outer integument which consists of a small-celled layer and a large-celled layer sparsely covered with unicellular, lignified hairs. Interior to the palisade is the thick mesophyll of the inner integument which is largely digested during seed growth and leaves only 2 pigmented cell layers in most regions at maturity. The inner epidermis is small-celled, pigmented and cutinized and adheres tightly to the endosperm. Seed coat impermeability increases with seed maturity. Even immature seeds will germinate, if scarified, indicating a lack of embryo dormancy.     

STUDIES ON THE SEED OF BANANA. I. ANATOMY OF THE SEED AND EMBRYO OF MUSA BALBISIANA

Mc Gahan , Merritt W. (United Fruit Co., Norwood, Mass.) Studies on the seed of banana. I. Anatomy of the seed and embryo of Musa balbisiana. Amer. Jour. Bot. 48(3): 230–238. Illus. 1961.—The seed coat of Musa balbisiana Colla consists of a relatively thick outer integument and a 2–cell-layered inner integument. The entire seed coat is sclerified, but routine tests for lignin are negative. Within the outer integument there is a zone of unusual sclereids tentatively termed “multiluminate.” Between the inner integument and the remnants of the nucellus is a cuticle 10–12 μ thick. The micropylar plug and collar are typical of the genus. The chalazal mass is an annular region of gelatinous cells. The mature embryo is comprised of a massive cotyledon, an epicotyl with 1 leaf primordium, a primary root primordium, and several adventitious root primordia. Procambium is well developed, but no mature vascular elements are present in the embryo.     

Fruit harvesting time and corresponding morphological changes of seed integuments influence in vitro seed germination of <Emphasis Type="Italic">Dendrobium nobile</Emphasis> Lindl.

In vitro asymbiotic seed germination of Dendrobium nobile varied significantly with fruit harvesting time and growth medium used for culturing seeds. Seeds harvested 129 days after pollination (DAP) possessing globular shaped embryos and a discontinuous cuticle layer showed a substantially greater germination on P668 medium. Alternatively, immature seeds harvested 96 and 116 DAP displayed a significantly lower germination response on various growth media. Most of the ovules at 96 DAP are in archesporial and megaspore mother cell stages, whereas the majority of ovules are mature and fertilized at 116 DAP. Mature seeds harvested 158 DAP also germinated at a higher frequency at Stage 5 (emergence of the first leaf) after 8 weeks of culture on different growth media indicating the absence of testa imposed dormancy in this endangered epiphytic orchid.     

Characterization of the mannan synthase promoter from guar (Cyamopsis tetragonoloba)

Guar seed gum, consisting primarily of a high molecular weight galactomannan, is the most cost effective natural thickener, having broad applications in the food, cosmetics, paper, pharmaceutical and petroleum industries. The properties of the polymer can potentially be enhanced by genetic modification. Development of suitable endosperm-specific promoters for use in guar is desirable for metabolic engineering of the seed gum. A ~1.6 kb guar mannan synthase (MS) promoter region has been isolated. The MS promoter sequence was fused with the GUS reporter gene and overexpressed in the heterologous species alfalfa (Medicago sativa). The potential strength and specificity of the MS promoter was compared with those of the constitutive 35S promoter and the seed specific β-phaseolin promoter. Quantitative GUS assays revealed that the MS promoter directs GUS expression specifically in endosperm in transgenic alfalfa. Thus, the guar MS promoter could prove generally useful for directing endosperm-specific expression of transgenes in legume species.     

Embryogenesis and seed development in Sinomanglietia glauca (Magnoliaceae)

The development of the floral bud, especially the ovule and seed coat, of Sinomanglietia glauca was observed. Floral buds were covered by eight to nine hypsophyll pieces. The hypsophyll nearest the tepal was closed completely and characterized by two arrays of densely stained cells with dense cytoplasm, which split longitudinally at flowering. The perianth consisted of 16 tepals arranged in three whorls. The gynoecium was composed of numerous apocarpous carpels; the ovule was anatropous with two integuments. Embryogenesis was of the Polygonum type, and the endosperm was nuclear. The inner integument degenerated during seed development. The seed of S. glauca had an endotestal seed coat comprised of a sclerotic layer derived from the inner adaxial epidermis of the outer integument and a sarcotesta derived mainly from the middle cells between the inner and outer epidermis of the outer integument. The embryo developed normally, so embryogenesis is not the cause of difficult regeneration.     

Analysis of kafirin promoter activity in transgenic tobacco seeds

Sequences corresponding to 855 bp of 5 promoter region and the transit peptide from GK.1, a genomic clone encoding a 22 kDa -kafirin seed protein from sorghum, were translationally fused to a cloned -glucuronidase (GUS) coding sequence from uidA and transferred to tobacco via Agrobacterium tumefaciens-mediated transformation. No GUS expression was detectable at any stage of growth in stems or leaves of these plants. However, GUS expression was detected in both embryo and endosperm tissues of resulting tobacco seeds 10–15 days after flowering. Dissected tissues indicate endosperm expression was localized within the bulk endosperm and not within the parenchyma cell layer underlying the integument. These studies also demonstrate that within dissected tobacco embryos, expression from the kafirin promoter was restricted to the mesocotyl region.     

Upstream regulatory regions from the maize Sh1 promoter confer tissue-specific expression of the ,-glucuronidase gene in tomato

A promoter fusion (Sh35) combining upstream regulatory regions from the maize Sh1 promoter with a truncated 35S promoter, Δ9035 (–90 to +8) has been compared with the original Sh1 promoter for its capacity to promote expression of the β-glucuronidase (GUS) gene in stably transformed tomato plants. For both promoters, very faint GUS expression was detected in the vegetative tissues, and no expression was detected in the fruit pericarp tissues. However, in the seed, Sh1 promoted low GUS expression but Sh35 directed 25-fold higher GUS expression. For both constructs, the profile of GUS expression was similar to that of endogenous sucrose synthase activity, but maximal GUS activity was reached 15 days after the peak of sucrose synthase activity. Received: 20 October 1998 / Revision received: 1 December 1998 / Accepted: 14 December 1998