Early Development of the Seed Coat of Soybean (Glycine max)

Although the development of the soybean ovule has been fairlywell studied, knowledge of the sequence of events in the seedcoat during the first 3 weeks after flowering is incomplete.The goal of the present study was to document, using light microscopy,the early development of the soybean seed coat with respectto changes in structure and histochemistry. At anthesis, theseed coat consists of an outer layer of cuboidal epidermal cellssurrounding several layers of undifferentiated parenchyma (whichtogether constitute the outer integument), and an inner layerof cuboidal endothelial cells (the inner integument). At 3 dpost anthesis (dpa), the inner integument has expanded to includethree to five layers of relatively large cells with thick, heavily-stainingcell walls immediately adjacent to the endothelium. By 18 dpa,the outer integument has developed into a complex of tissuescomprised of an inner layer of thick-walled parenchyma, an outerlayer of thin-walled parenchyma containing vascular tissue whichhas grown down from the lateral vascular bundles in the hilumregion, a hypodermis of hourglass cells, and palisade layer(epidermis). The thick-walled parenchyma of the inner integumenthas become completely stretched and compressed, leaving a single,deeply staining wall layer directly above the endothelium. At21 dpa, the outermost cells of the endosperm have begun to compressthe endothelium. At 45 dpa (physiological maturity) the seedcoat retains only the palisade layer, hourglass cells, and afew layers of thin-walled parenchyma. The innermost layer ofthe endosperm, the aleurone layer, adheres to the inside ofthe seed coat. This knowledge will be invaluable in future studiesof manipulation of gene expression in the seed coat to modifyseed or seed coat characteristics. Copyright 1999 Annals ofBotany Company Soybean, Glycine max, seed coat, development, aleurone.     

Seed coat development, anatomy and scanning electron microscopy of Harpagophytum procumbens (Devil's Claw), Pedaliaceae

Seed coat development of Harpagophytum procumbens (Devil's Claw) and the possible role of the mature seed coat in seed dormancy were studied by light microscopy (LM), transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM). Very young ovules of H. procumbens have a single thick integument consisting of densely packed thin-walled parenchyma cells that are uniform in shape and size. During later developmental stages the parenchyma cells differentiate into 4 different zones. Zone 1 is the multi-layered inner epidermis of the single integument that eventually develops into a tough impenetrable covering that tightly encloses the embryo. The inner epidermis is delineated on the inside by a few layers of collapsed remnant endosperm cell wall layers and on the outside by remnant cell wall layers of zone 2, also called the middle layer. Together with the inner epidermis these remnant cell wall layers from collapsed cells may contribute towards seed coat impermeability. Zone 2 underneath the inner epidermis consists of large thin-walled parenchyma cells. Zone 3 is the sub-epidermal layers underneath the outer epidermis referred to as a hypodermis and zone 4 is the single outer seed coat epidermal layer. Both zones 3 and 4 develop unusual secondary wall thickenings. The primary cell walls of the outer epidermis and hypodermis disintegrated during the final stages of seed maturation, leaving only a scaffold of these secondary cell wall thickenings. In the mature seed coat the outer fibrillar seed coat consists of the outer epidermis and hypodermis and separates easily to reveal the dense, smooth inner epidermis of the seed coat. Outer epidermal and hypodermal wall thickenings develop over primary pit fields and arise from the deposition of secondary cell wall material in the form of alternative electron dense and electron lucent layers. ESEM studies showed that the outer epidermal and hypodermal seed coat layers are exceptionally hygroscopic. At 100% relative humidity within the ESEM chamber, drops of water readily condense on the seed surface and react in various ways with the seed coat components, resulting in the swelling and expansion of the wall thickenings. The flexible fibrous outer seed coat epidermis and hypodermis may enhance soil seed contact and retention of water, while the inner seed coat epidermis maintains structural and perhaps chemical seed dormancy due to the possible presence of inhibitors.     

长豇豆种皮和种脐的发育

长豇豆的胚珠具内外两层珠被,内珠被在种子发育早期退化消失,种皮仅由外珠被发育而成。外珠被的外表皮细胞径向伸长,外壁和经向壁增厚,形成约占成熟种皮厚度一半的栅栏层;亚表皮细胞发育为骨状石细胞层。第三层细胞类似于亚表皮层但细胞壁增厚不明显,其内方的多层薄壁细胞形成海绵组织。种脐具两层栅栏细胞,外栅栏层及其以外部分由珠柄组织发育而成管胞群。本文还对脐缝和管胞群的作用以及豆科种子的吸水机制进行了讨论。     

In situ expression of two storage protein genes in relation to histo-differentiation at mid-embryogenesis in Medicago truncatula and Pisum sativum seeds

The seed consists of several layers of specialized cell-types that divide and differentiate following a highly regulated programme in time and space. A cytological approach was undertaken in order to study the histo-differentiation at mid-embryogenesis in Medicago truncatula as a model legume, and in Pisum sativum using serial sections of embedded immature seed. Little published information is available about seed development in Medicago species. The observations from this study revealed a number of distinctive features of Medicago seed development and differentiation. Transfer cells, involved in nutrient transfer to the embryo, were clearly identified in the thin-walled parenchyma of the innermost integument. Histological Schiff-naphthol enabled carbohydrate accumulation to be followed in the different seed compartments, and revealed the storage protein bodies. Non-radioactive mRNA in situ hybridization, was carried out using mRNA probes from two highly expressed genes encoding the major vicilin and legumin A storage protein types. The timing of mRNA expression was related to that of the corresponding proteins already identified.     

Expression patterns and subcellular localization of a 52 kDa sucrose-binding protein homologue of Vicia faba (VfSBPL) suggest different functions during development

A cDNA coding for a 54 kDa signal sequence containing protein has been isolated from a faba bean cotyledonary library and characterized. The deduced protein is designated Vicia faba SBP-like protein (VfSBPL) since it shares 58% homology to a 62 kDa soybean (Glycine max) protein (GmSBP) which has been described as a sucrose-binding and sucrose-transporting protein (SBP). VfSBPL as well as GmSBP are outgroup members of the large vicilin storage protein family. We were unable to measure any sucrose transport activity in mutant yeast cells expressing VfSBPL. During seed maturation in late (stage VII) cotyledons mRNA was localized by in situ hybridization in the storage parenchyma cells. At the subcellular level, immunolocalization studies proved VfSBPL accumulation in storage protein vacuoles. However, mRNA localization in stage VI cotyledons during the pre-storage/storage transition phase was untypical for a storage protein in that, in addition to storage parenchyma cell labelling, strong labelling was found over seed coat vascular strands and the embryo epidermal transfer cell layer reminiscent of sucrose transporter localization. The VfSBPL gene is composed of 6 exons and 5 introns with introns located at the same sites as in a Vicia faba 50 kDa vicilin storage protein gene. The time pattern of expression as revealed by northern blotting and the GUS accumulation pattern caused by a VfSBPL-promoter/GUS construct in transgenic tobacco seeds was similar to a seed protein gene with increasing expression during seed maturation. Our data suggest different functions of VfSBPL during seed development.     

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.     

抱茎独行菜MUM4基因的克隆及分析

抱茎独行菜(Lepidium perfoliatum L.)为十字花科具典型粘液繁殖体植物,为探究该植物中种皮粘液质基因（MUCILAGE-MODIFIED4,MUM4,该基因在拟南芥中编码NDP-L-鼠李糖合成酶)的功能,通过生物信息学分析设计引物克隆得到抱茎独行菜MUM4基因,命名为LpMUM4。同源比对分析结果表明,LpMUM4与拟南芥AtMUM4基因具有很高的一致性。qRT-PCR结果表明,该基因在抱茎独行菜各组织中均有表达,在角果和根中的表达量最高,且其表达量随角果的发育表现出渐强的趋势。免疫组织化学定位分析表明,LpMUM4基因于角果发育的早期阶段在内珠被和外珠被都有表达,而在外珠被的表皮和亚表皮中表达量更高,至角果发育的最后阶段,其表达集中于表皮和亚表皮层,这可能与抱茎独行菜的外珠被发育成种皮及粘液质的生成有关。将LpMUM4基因转化拟南芥,该基因的过表达对位于粘液质合成途径中的上游基因AtTTG1具有显著的抑制作用。表型比对观察显示,转基因拟南芥与其野生型植株形态无显著差异,这可能是因为抱茎独行菜种皮的发育和粘液质的形成是一个多基因调控的复杂过程,某一基因的过表达或许不会引起明显的表型变化。     

Histological Study of Seed Coat Development in Arabidopsis thaliana

Arabidopsis seed coat development using light and transmission electron microscopy revealed major morphological changes associated with the transition of the integuments into the mature seed coat. By the use of a metachromatic staining procedure, cytological events such as the production of phenolic compounds and acidic polysaccharides were followed. Immediately after fertilization, the cells of the inner epidermis of the inner integument became vacuolated and subsequently accumulated pigment within them. This pigment started to disappear from the cytoplasm at the torpedo stage of the embryo, as it became green. During the torpedo stage, mucilage began to accumulate in the cells of the external epidermis of the outer integument. Furthermore, starch grains accumulated against the central part of the inner periclinal wall of these cells, resulting in the formation of small pyramidal domes that persisted until seed maturity. At the maturation stage, when the embryo became dormant and colourless, a new pigment accumulation was observed in an amorphous layer derived from remnants of crushed integument layers. This second pigment layer was responsible for the brown seed colour. These results show that seed coat formation may proceed in a coordinated way with the developmental phases of embryogenesis. Received 25 May 1999/ Accepted in revised form 10 February 2000     

兰花蕉种子的解剖学和组织化学研究

兰花蕉种子球形或近球形,具表皮毛,种脊不明显。种子包括假种皮、种皮、外胚乳、内胚乳和胚五部分。假种皮具３～４条粗毛状裂片,包围种子或不定向伸展;裂片最外方为１层表皮细胞和１～３层厚壁细胞,内方为薄壁细胞;表皮细胞和厚壁细胞的壁增厚并木质化;成熟时裂片下部１／２段中空。种皮由外珠被发育而来,但内珠被在种子发育后期才萎缩。种皮分化为外种皮,中种皮与内种皮;外种皮由１层表皮细胞构成,其细胞壁增厚并木质化;中种皮外方为２～３层厚壁细胞,内方由１２～１５层薄壁细胞构成;内种皮由１层径向延长的石细胞构成,其细胞壁网状增厚,胞腔不明显。外胚乳极不显眼,大部分只由１层切向延长的长方形细胞构成,局部为２～１７层细胞;外胚乳细胞主要含许多脂类物质及少量蛋白质颗粒,不含淀粉。内胚乳占据种子很大的体积,由通常径向延长的长方形、长条形或方形薄壁细胞构成;细胞内充满淀粉粒和通常一颗亦有２至多颗菱形或方形蛋白质晶体,脂类物质极少。胚圆柱形,胚根和胚芽不明显。种子珠孔区不分化出珠孔领和孔盖,但具柄,柄的远轴端边缘大部分着生假种皮,着生假种皮一侧柄略膨大。合点区内种皮出现极宽的缺口,缺口间为整体呈弧状长条形的合点区厚壁细胞群。较粗的种脊维管?     

Expression of TaCYP78A3, a gene encoding cytochrome P450 CYP78A3 protein in wheat (Triticum aestivum L.), affects seed size

Several studies have described quantitative trait loci (QTL) for seed size in wheat, but the relevant genes and molecular mechanisms remain largely unknown. Here we report the functional characterization of the wheat TaCYP78A3 gene and its effect on seed size. TaCYP78A3 encoded wheat cytochrome P450 CYP78A3, and was specifically expressed in wheat reproductive organs. TaCYP78A3 activity was positively correlated with the final seed size. Its silencing caused a reduction of cell number in the seed coat, resulting in an 11% decrease in wheat seed size, whereas TaCYP78A3 over‐expression induced production of more cells in the seed coat, leading to an 11–48% increase in Arabidopsis seed size. In addition, the cell number in the final seed coat was determined by the TaCYP78A3 expression level, which affected the extent of integument cell proliferation in the developing ovule and seed. Unfortunately, TaCYP78A3 over‐expression in Arabidopsis caused a reduced seed set due to an ovule developmental defect. Moreover, TaCYP78A3 over‐expression affected embryo development by promoting embryo integument cell proliferation during seed development, which also ultimately affected the final seed size in Arabidopsis. In summary, our results indicated that TaCYP78A3 plays critical roles in influencing seed size by affecting the extent of integument cell proliferation. The present study provides direct evidence that TaCYP78A3 affects seed size in wheat, and contributes to an understanding of the cellular basis of the gene influencing seed development.     

The seed coat-specific expression of a subtilisin-like gene, SCS1, from soybean

A seed coat-specific gene, SCS1 (Seed Coat Subtilisin 1), from soybean, Glycine max [L.] Merill, has been identified and studied. The gene belongs to a small family of genes with sequence similarity to the subtilisins, which are serine proteases. Northern blot analysis showed that SCS1 RNA accumulates to maximal levels in seed coats at 12 days post anthesis, preceding the final stages of seed coat differentiation. The SCS1 RNA was not found in other tissues including embryos, seed pods, flowers, stems, roots or leaves. In-situ hybridization studies confirmed the temporal pattern of expression observed by Northern blot analysis and further revealed a restricted pattern of RNA accumulation in thick-walled parenchyma cells of the seed coats. These cells are important in the apoplastic translocation of nutrients en route to the embryo from the vascular tissues. The tissue-specific subtilisin-like gene may be required for regulating the differentiation of the thick-walled parenchyma cells. Received: 10 January 2000 / Accepted: 22 February 2000     

Pathway of Photosynthate Transfer in the Developing Seed of Vicia faba L. Transfer in Relation to Seed Anatomy

The seed coat vascular system of the developing seed of Viciafaba consists of a chalazal and two lateral veins. The veinsare embedded in parenchymatous tissue which lies beneath thehypodermis and is divided into chlorenchyma, ground parenchymaand thin-walled parenchyma. The thin-walled parenchyma cellsand, in old seed coats, the vascular parenchyma of the veinsundergo additional secondary wall development to form transfercells. Thus, transfer cells line the entire inner surface ofthe seed coat. Initial distribution of ¹⁴C-photosynthates andsodium fluorescein within the seed coat was in the vascularsystem. Subsequent transfer towards the embryo was either radiallythrough vascular parenchyma and thin-walled parenchyma to thin-walledparenchyma/transfer cells, or by lateral spread within the groundand thin-walled parenchyma/transfer cells of the non-vascularregion of the seed coat prior to radial transfer. One-thirdof the ¹⁴C-photosynthate delivered to the enclosed embryo wasestimated to be transferred via the non-vascular region of theseed coat. The cotyledons consist of a single-layered epidermisenclosing storage parenchyma in which a differentiating reticulatevascular system is embedded. Epidermal cells juxtaposed to theseed coat develop wall ingrowths characteristic of transfercells. Initial distribution of ¹⁴C-photosynthate within thecotyledons reflected the unequal delivery to the seed apoplastfrom the vascular and non-vascular regions of the seed coat.Subsequent even distribution of photosynthate within the cotyledonspossibly occurred by transfer within their vascular system. Key words: Cellular pathway, photosynthate transfer, seed anatomy, transfer cell     

Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator

A maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identified a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modifications. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows approximately 72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.     

Systematic and phylogenetic significance of the seed coat in the shrubby African Iridaceae, Nivenia, Klattia and Witsenia

MANNINGJ. C. & GOLDBLATT, P., 1991. Systematic and phylogenetic significance of the seed coat in the shrubby African Iridaceae, Nivenia, Klattia and Witsenia. The seeds of Nivenia, Klattia and Witsenia, a natural alliance within Nivenioideae, are among their most distinctive features, and seed number and shape constitute at least two synapomorphies for this monophyletic lineage. In this paper we survey the structure of the seed surface of five species of Nivenia and one each of Klattia and Witsenia by scanning electron microsopy (SEM), and study the development of the seed coat in a representative species of each of the three genera. Outgroups for comparison were selected from genera inside and outside the subfamily. The six genera of Nivenioideae are united in having a 3 (– 2) seriate outer integument but the shrubby genera are unique in the family in their tangentially flattened ovules which mature into depressed, flat, scutiform seeds. They display a range of variation in seed coat characters but are specialized in the subfamily and family in having a transparent outer integument. Other genera have pigmented deposits in the outer epidermis of the outer integument. Nivenia and Witsenia are further specialized in having a partially exfoliating outer integument. Klattia is unique among the shrubby genera in having deposits in the second layer of the tegmen, in retaining an intact outer epidermis of the tegmen, and in the fusiform shape of the epidermal cells and striate surface of the seed. Our results are incorporated in a cladogram that expresses our current understanding of the phylogenetic relationships of Nivenioideae.     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Deposition and localization of lipid polyester in developing seeds of Brassica napus and Arabidopsis thaliana

Mature seeds of Arabidopsis thaliana and Brassica napus contain complex mixtures of aliphatic monomers derived from non-extractable lipid polyesters. Most of the monomers are deposited in the seed coat, and their compositions suggest the presence of both cutin and suberin layers. The location of these polyesters within the seed coat, and their contributions to permeability of the seed coat and other functional properties are unknown. Polyester deposition was followed over Brassica seed development and distinct temporal patterns of monomer accumulation were observed. Octadecadiene-1,18-dioate, the major leaf cutin monomer, was transiently deposited. In contrast, the saturated dicarboxylates maintained a constant level during seed desiccation, whereas the fatty alcohols and saturated omega-hydroxy fatty acids continually increased. Dissection and analysis of Brassica seed coats showed that suberization is not specific to the chalaza. Analysis of the Arabidopsis ap2-7 mutant suggested that suberin monomers are preferentially associated with the outer integument. Several Arabidopsis knockout mutant lines for genes involved in polyester biosynthesis (att1, fatB and gpat5) were examined for seed monomer load and composition. The variance in polyester monomers of these mutants is correlated with dye penetration assays. Furthermore, stable transgenic plants expressing promoter::YFP fusions showed ATT1 promoter activity in the inner integument, whereas GPAT5 promoter is active in the outer integument. Together, the Arabidopsis data indicated that there is a suberized layer associated with the outer integument and a cutin-like polyester layer associated with the inner seed coat.     

Temporary storage compounds and sucrose-starch metabolism in seed coats during pea seed development (Pisum sativum)

Quantitative data for growth, carbohydrate, protein and free amino acid nitrogen content of pea ( Pisum sativum L. cv. Finale) seed coat were obtained during the main stage of seed development. These data allowed us to define the role of the seed coat storage compounds. High amounts of arginine were measured in the seed coat and this amino acid is hypothesized to be synthesized de novo in the seed coat cells. Starch appeared to be stored in a specific parenchyma layer of the seed coat. Starch storage was shown to occur from phloem-unloaded sucrose and high activities of some enzymes of sucrose-starch metabolism (sucrose synthase, EC 2.4.1.13 and ADP glucose pyrophosphorylase, EC 2.7.7.27) were measured. The contribution of seed storage compounds is discussed in terms of buffering embryo nutrition. The sink strength of the young pea seed may be located within the seed coat.     

Cell-specific expression of the carrot EP2 lipid transfer protein gene.

A cDNA corresponding to a 10-kD protein, designated extracellular protein 2 (EP2), that is secreted by embryogenic cell cultures of carrot was obtained by expression screening. The derived protein sequence and antisera against heterologous plant lipid transfer proteins identified the EP2 protein as a lipid transfer protein. Protein gel blot analysis showed that the EP2 protein is present in cell walls and conditioned medium of cell cultures. RNA gel blot analysis revealed that the EP2 gene is expressed in embryogenic cell cultures, the shoot apex of seedlings, developing flowers, and maturing seeds. In situ hybridization showed expression of the EP2 gene in protoderm cells of somatic and zygotic embryos and transient expression in epidermis cells of leaf primordia and all flower organs. In the shoot apical meristem, expression is found in the tunica and lateral zone. In maturing seeds, the EP2 gene is expressed in the outer epidermis of the integument, the seed coat, and the pericarp epidermis, as well as transiently in between both mericarps. Based on the extracellular location of the EP2 protein and the expression pattern of the encoding gene, we propose a role for plant lipid transfer proteins in the transport of cutin monomers through the extracellular matrix to sites of cutin synthesis.     

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.