CELL WALL CHEMISTRY AND ULTRASTRUCTURE OF CHLOROCOCCUM OLEOFACIENS (CHLOROPHYCEAE)1,2

Cell walls of Chlorococcum oleofadens Trainor & Bold were examined ultrastructurally and chemically. The wall of zoospores has a uniform 30 nm width and a regular lamellar pattern. Zoospores and young vegetative cell walk exhibit periodicities, consisting of 20 nm ridges on the outer layer. Vegetative cell walls have a variable thickness of Up to 800 nm and are composed of multiple layers of electron dense material. Further, vegetative walk contain a microfibrillar material composed predominantly of glucose and presumed to be cellulose. Except for this cellulose, vegetative cell wall chemistry is very similar to that of Chlamydomemas being composed of glycoprotein rich in hydroxyproline. The hydroxyproline in Chlorococcum walls is linked glycosidically to a mixture of hetrooligosaccharides composed of arabinose and galactose, and in one instance, an unknown 6-deoxyhexose. Altogether, the glycoprotein complex accounts for at least 52% of the wall. The amino acid composition of the walls is stikingly similar to those of widely different plant species. Indirect evidence indicates zoospore cell walls are also chemically similar to those of Chlamydomonas, and like them, are cellulose free. Thus a major chemical difference between zoospore and vegetative cell walk of Chlorococcum is the presence of cellulose in the latter. The contribution of this microfibrillar cellulose to the physical properties of the vegetative wall is discussed.     

THE STRUCTURE OF THE PRIMARY EPIDERMAL CELL WALL OF AVENA COLEOPTILES

The primary walls of epidermal cells in Avena coleoptiles ranging in length from 2 to 40 mm. have been studied in the electron and polarizing microscopes and by the low-angle scattering of x-rays. The outer walls of these cells are composed of multiple layers of cellulose microfibrils oriented longitudinally; initially the number of layers is between 10 and 15 but this increases to about 25 in older tissue. Where epidermal cells touch, these multiple layers fuse gradually into a primary wall of the normal type between cells. In these radial walls, the microfibrils are oriented transversely. Possible mechanisms for the growth of the multilayered outer wall during cell elongation are discussed.     

DEMONSTRATION OF A FIBRILLAR COMPONENT IN THE CELL WALL OF THE YEAST SACCHAROMYCES CEREVISIAE AND ITS CHEMICAL NATURE

The ultrastructure of isolated cell walls of Saccharomyces cerevisiae from the log and stationary phases of growth was studied after treatment with the following enzymes: purified endo-β-(1 → 3)-glucanase and endo-β-(1 → 6)-glucanase produced by Bacillus circulans; purified exo-β-glucanase and endo-β-(1 → 3)-glucanase produced by Schizosaccharomyces versatilis; commercial Pronase. While exo-β-glucanase from S. versatilis had no electron microscopically detectable effect on the walls, Pronase removed part of the external amorphous wall material disclosing an amorphous wall layer in which fibrils were indistinctly visible. Amorphous wall material was completely removed by the effect of either endo-β-(1 → 3)- or endo-β-(1 → 6)-glucanase of B. circulans or by a mixture of the two enzymes. As a result of these treatments a continuous fibrillar component appeared, composed of densely interwoven microfibrils resisting further action by both of the B. circulans enzymes. The fibrillar wall component was also demonstrated in untreated cell walls by electron microscopy after negative staining. Because of the complete disappearance of the fibrils following treatment with the S. versatilis endo-β-(1 → 3)-glucanase it can be concluded that this fibrillar component is composed of β-(1 → 3)-linked glucan. Bud scars were the only wall structures resistant to the effect of the latter enzyme.     

THE CELL WALL OF COSMARIUM BOTRYTIS12

The cell wall of Cosmarium botrytis was studied through the use of the freeze-etch technique. The cell wall consists of many thin layers. Fracturing along one layer reveals the positioning of the wall sculpturing, wall pores, and wall microfibrils. The individual microfibrils are grouped together in bands of parallel oriented fibrils. The different bands of parallel microfibrils were apparently arranged at random angles with regard to each other. Small particles may also be present in the cell walls. The cell wall pore unit of Cosmarium botrytis was studied through the use of scanning, freeze-etching, and thin sectioning techniques. The pore sheaths, on the outside of the cell wall, form a collar around the mouth of each pore. The pore sheath is composed of needle-like fibrils radiating outward from the pore. A pore channel traverses the cell wall and leads to a complex pore bulb region between the cell wall and the plasmalemma. The pore bulb contains many small fibrils which radiate toward the plasmalemma from a number of net-like fibril layers which in turn merge into a very electron dense region near the base of the pore.     

THE COMPOSITION AND PHYLOGENETIC SIGNIFICANCE OF THE MOUGEOTIA (CHAROPHYCEAE) CELL WALL1

The two-layered, fibrillar cell wall of Mougeotia C. Agardh sp. consisted of 63.6% non-cellulosic carbohydrates and 13.4% cellulose. The orientation of cellulose microfibrils in the native cell wall agrees with the multinet growth hypothesis, which has been employed to explain the shift in microfibril orientation from transverse (inner wall) toward axial (outer wall). Monosaccharide analysis of isolated cell walls revealed the presence of ten sugars with glucose, xylose and galactose most abundant. Methylation analysis of the acid-modified, 1 N NaOH insoluble residue fraction showed that it was composed almost exclusively of 4-linked glucose, confirming the presence of cellulose. The major hemicellulosic carbohydrate was semi-purified by DEAE Sephacel (Cl^?) anion-exchange chromatography of the hot 1 N NaOH soluble fraction. This hemicellulose was a xylan consisting of a 4-xylosyl backbone and 2,4-xylosyl branch points. The major hot water soluble neutral polysaccharide was identified as a 3-linked galactan. Mougeotia cell wall composition is similar to that of (Charophyceae) and has homologies with vascular plant cell walls. Our observations support transtructural evidence which suggests that members of the Charophyceae represent the phylogenetic line that gave rise to vascular plants. Therefore, the primary cell walls of vascular plants many have evolved directly from structures typical of the filamentous green algal cell walls found in the Charophyceae.     

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES,CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.     

Synthesis and Transport of Hydroxyproline-rich Components in Suspension Cultures of Sycamore-Maple Cells

Plant cell walls contain a glycoprotein rich in hydroxyproline. To determine how Acer pseudoplatanus L. cells transport this glycoprotein to the wall, the pulse-chase technique was used to follow changes in specific radio-activity of hydroxyproline and proline in isolated, mitochondrial, Golgi, microsomal, soluble protein, and wall fractions. The turnover rates or changes in specific radioactivity of cytoplasmic hydroxyproline in these cell fractions indicated that the bulk of this hydroxyproline was transferred not by the Golgi apparatus but by a smooth membranous component.     

THE CELL WALL OF PYROCYSTIS SPP. (DINOCOCCALES)1,2

The cell of Pyrocystis spp. is covered by an outer layer of material resistant to strong acids and bases. Internal to this layer much of the cell wall is composed of cellulose fibrils. The presence of cellulose fibrils was established by staining raw and ultra-violet–peroxide-cleaned cell walls and by combining X-ray diffraction spectroscopy with electron microscope observation. Carbon replicas of freeze-etched preparations and thin sections of P. lunula walls show outer layers, inside them ca. 24 layers of crossed parallel cellulose fibrils (4–5 nm thick, ca. 12 nm wide), then a region of smaller (ca. 6–12 nm diameter) fibrils in a disperse texture, and then the plasma membrane. Cellulose fibrils in the parallel texture are constructed of 3–5 elementary fibrils ca. 3 nm in diameter. Walls of P. fusiformis and P. pseudonctiluca also have cellulose fibrils in a crossed parallel texture similar to those of P. lunula. The Gymnodinium-type swarmer from lunate P. lunula appears to have a cell wall ultrastructure typical of other “naked” dinoflagellates.     

THE MICROFIBRILLAR STRUCTURE OF THE CELL WALLS OF THE FILAMENTOUS FUNGUS, Allomyces

Cell walls of the fungus, Allomyces, were isolated by chemical procedures, using either potassium permanganate oxidation or glacial acetic acid-hydrogen peroxide treatment followed by dilute mineral acid. The structure of the treated walls was investigated by means of electron microscopy and electron diffraction analysis which showed that rhizoidal walls were especially suitable for observation. Chitin microfibrils exist in the extreme tips of rhizoidal walls, and tend to lie in a preferred longitudinal orientation. Older rhizoidal wall segments show a crossed fibrillar structure under a thin layer of short randomly arranged microfibrils. In the possession of systems of crossed fibrils these walls are like the cell walls of certain green algae. Walls of branch rhizoidal filaments were observed in the early stages of development, in which case the observed microfibrillar orientations are such that it is possible to envisage their origin from pre-existing fibrils that have passively reoriented. With respect to the continued growth of the filaments, however, it is difficult to explain the observed microfibrillar arrangements in terms of the "multi-net" theory. Hyphal walls usually show two layers, the outer consisting of microfibrils arranged randomly, and the inner consisting of well oriented microfibrils running parallel with the longitudinal axis of the hypha. The oriented inner layer appears to be similar in structure to the secondary wall of the Phycomyces sporangiophore.     

FUNCTIONS OF CALCIUM IN THE CELL WALL OF RICE LEAVES

Rice plants were grown in nutrient solutions containing 0.5,20, and 500 ppm of calcium as CaO. Microscopic observation revealedthat the mesophyll structure in the leaves was disordered atthe lowest Ca level and became healthy with increasing supplyof calcium in the culture solution. The chemical constitutionof cell wall and the composition of sugar in each chemical constituentof wall differed little among the leaves grown with differentlevels of calcium. The calcium contents in the walls of calciumdeficient leaves were extremely low as compared with those ofnormal leaves. The amount of calcium extractable in the ligninfraction of Ca 0.5 leaves was only about one-sixth of that ofCa 500 leaves. Furthermore, calcium in the lignin fraction wasleached out by treating the wall previously with 2% acetic acidsolution. These results suggest a close association of calciumwith ligneous substances in combined forms of physiologicalimportance in the cell walls. (Received April 11, 1967; )     

A MODIFIED CELL WALL MUTANT OF THE RED MICROALGA RHODELLA RETICULATA RESISTANT TO THE HERBICIDE 2,6-DICHLOROBENZONITRILE1

The rigid component of the cell walls of red macroalgae, cellulose, is lacking in the red microalgae. Instead, the cells are encapsulated within an amorphous polysaccharide. These complex sul fated polysaccharides are composed of at least 10 different sugars, but their structure is not known, When the herbicide 2,6-dichlorobenzonitrile (DCB), a compound that specifically inhibits cellulose biosynthesis, was applied to cultures of the red microalga Rhodella reticulata upon inoculation, growth was inhibited. When added during the stationary phase of growth (after cell division had ceased), DCB did not affect cell number but it did inhibit polysaccharide production. A spontaneous mutant resistant to DCB was selected; it had physiological characteristics similar to those of the wild-type parent. The composition of the cell wall polysaccharide of the mutant was totally modified, being composed almost entirely (98% of its dry matter, as compared to 2.9% in the wild type) of methyl galactose, but retaining the same sulfate content. The molecular mass of the mutant polysaccharide was, however, similar to that of the wild-type parent (～6 × 10⁶ daltons), although its viscosity was significantly lower.     

COMPOSITION AND SYNTHESIS OF THE PECTIN AND PROTEIN COMPONENTS OF THE CELL WALL OF CLOSTERIUM ACEROSUM (CHLOROPHYTA)

Closterium acerosum Ehrenberg (Chlorophyta) possesses a trilayered cell wall consisting of an outer tri-laminate stratum, a fibrous middle layer, and a thick inner fibrous layer. The outermost layer has a series of external parallel ridges and valleys. At the bases of the valleys are the wall pores, the site of mucilage release. Pure fractions of cell walls were isolated and inclusive pectin and wall protein fractions were extracted and characterized. Two pectin-like fractions were isolated: a CDTA-extracted polymer consisting of 60.1% galacturonic acid and a Na₂CO₃-extracted fraction consisting of 39.9% galacturonic acid. Two major protein fractions, one with a molecular mass of 23.5 kDa and one with a molecular mass of 28.5 kDa, were isolated by preparative gel electrophoresis. The former was glycine-rich, whereas the latter contained both significant amounts of glycine and hydroxyproline. Antibodies were raised to both the pectin fractions and the 23.5-kDa wall protein fraction. Immunocytochemical labeling of whole cells and wall fragments using antibodies raised against CDTA and Na₂CO₃ extracts showed that these pectin-like components were found throughout the wall strata and were more concentrated at the polar tips, the site of new wall synthesis in growing semicells. Immunogold labeling showed that their production was focused on the trans- Golgi network of the Golgi apparatus. Immunolabeling with an antibody raised against the 23.5-kDa glycine-rich wall protein showed close association of the protein with the wall pores. Similarly, immunogold labeling revealed that the protein was processed throughout the entire Golgi body even when large mucilage-containing vesicles were being processed. The roles of the secretory apparatus and putative spitzenkorper-like regions of the cell are discussed.     

被子植物生殖细胞的细胞壁

本文综述了国内外有关被子植物生殖细胞壁的资料,概述了它的形成、发育、性质和功能;在这些方面,生殖细胞壁的特征因植物种类而异。     

GLYCOPROTEIN MOIETY IN THE CELL WALL OF THE RED MICROALGA PORPHYRIDIUM SP. (RHODOPHYTA) AS THE BIORECOGNITION SITE FOR THE CRYPTHECODINIUM COHNII-LIKE DINOFLAGELLATE

The Crypthecodinium cohnii -like heterotrophic dinoflagellate preys on the cells of the red microalga Porphyridium sp. UTEX 637, and not on other microalgae. The dinoflagellate contains enzymes that degrade the cell wall complex of this species of alga and not that of other red microalgae. The cells of the red microalgae are encapsulated within a cell wall complex composed of about 10 sugars, sulfate, and proteins. We previously hypothesized that the dinoflagellate recognizes the cell wall of this alga. In this study, we have shown that the biorecognition site is the 66-kDa glycoprotein in the algal cell wall complex. The methodology used in this study was based on changing the algal cell wall composition and examining the prey and chemosensory response of the dinoflagellate. The dinoflagellate was not attracted to the cell wall of other red microalgae, which are similar to that of Porphyridium sp., or to sugars composing its cell wall. However, the dinoflagellate preyed on and was attracted to Porphyridium sp. mutants (DCB resistant) having modified cell wall polysaccharide composition, probably because the 66-kDa cell wall glycoprotein was not changed. The dinoflagellate did not respond chemotactically to enzymatically degraded cell wall complex. Treatment of the cell wall complex with antiserum to the 66-kDa glycoprotein or with the lectin concanavalin A (con A), which binds specifically to α-d-mannosyl and α-d-glucosyl residues, did not affect the chemotactic attraction. However, prey by the dinoflagellate was prevented when the algal cells were blocked with antiserum specific to the 66-kDa glycoprotein or with con A. These latter results provide direct proof that the 66-kDa cell wall glycoprotein isthe recognition site and prey-prevention results from the blocking of this site on the cell wall.     

Host-Pathogen Interactions : XIX. THE ENDOGENOUS ELICITOR, A FRAGMENT OF A PLANT CELL WALL POLYSACCHARIDE THAT ELICITS PHYTOALEXIN ACCUMULATION IN SOYBEANS

An elicitor of phytoalexin accumulation (endogenous elicitor) is solubilized from purified cell walls of soybean (Glycine max [L.] Merr., cv. Wayne) by extracting the walls with hot water or by subjecting the walls to partial acid hydrolysis. The endogenous elicitor obtained from soybean cell walls binds to an anion exchange resin. The elicitor-active material released from the resin contains oligosaccharides rich in galacturonic acid; small amounts of rhamnose and xylose are also present. The preponderance of galacturonic acid in the elicitor-active fragments suggests that the elicitor is, in fact, a fragment of a pectic polysaccharide. This possibility is supported by the observation that treatment of the wall fragments with a highly purified endopolygalacturonase destroys their ability to elicit phytoalexin accumulation. This observation, together with other evidence presented in this paper, suggests that galacturonic acid is an essential constituent of the elicitor-active wall fragments. Endogenous elicitors were also solubilized by partial hydrolysis from cell walls of suspension-cultured tobacco, sycamore, and wheat cells.     

TAXONOMIC REVISION OF THE CONJUGATOPHYCEAN FAMILY PENIACEAE ON THE BASIS OF CELL WALL ULTRASTRUCTURE1

An ultrastructural investigation of the cell wall of Penium silvae-nigrae Raban. and P. spinospermum Josh. showed that these species possess true pores with a pore apparatus and overlapping semi-cell walls. It follows that these two taxa belong not to the Peniaceae, but to the Desmidiaceae sensu stricto; they are referred to the genus Actinotaenium Teil. on account of the shape of their cells and chloroplasts. Two other species previously included in Penium Bréb. are referred to Actinotaenium. Although their cell wall structure could not be studied, they are distinguished from “typical” representatives of Penium by the following photomicroscopically observable complex of features: (pseudo-) girdle bands none, cell wall pores in longitudinal rows, zygospores not globose but of irregular shape. The following new combinations ensued: Actinotaenium borgeanum (Skuja), A. phymatosporum (Nordst.), A. silvae-nigrae (Raban.), A. silvaenigrae var. parallelum (Krieger) and A. spinospermum (Josh.). In addition the diagnosis of the genus Penium was emended and P. margaritaceum (Ehr.) ex Bréb. was selected as the lectotype species. The family Gonatozy-gaceae is merged into the Peniaceae on the basis of cell wall structure.     

CELL‐WALL POLYMER MAPPING IN THE COENOCYTIC MACROALGA CODIUM VERMILARA (BRYOPSIDALES,CHLOROPHYTA)1

Cell walls in the coenocytic green seaweed Codium vermilara (Olivi) Chiaje (Bryopsidales, Chlorophyta) are composed of ～32% (w/w) β‐(1→4)‐d‐mannans, ～12% sulfated polysaccharides (SPs), and small amounts of hydroxyproline‐rich glycoprotein‐like (HRGP‐L) compounds of the arabinogalactan proteins (AGPs) and arabinosides (extensins). Similar quantities of mannans and SPs were reported previously in the related seaweed C. fragile (Suringar) Hariot. Overall, both seaweed cell walls comprise ～40%–44% of their dry weights. Within the SP group, a variety of polysaccharide structures from pyruvylated arabinogalactan sulfate and pyruvylated galactan sulfate to pyranosic arabinan sulfate are present in Codium cell walls. In this paper, the in situ distribution of the main cell‐wall polymers in the green seaweed C. vermilara was studied, comparing their arrangements with those observed in cell walls from C. fragile. The utricle cell wall in C. vermilara showed by TEM a sandwich structure of two fibrillar‐like layers of similar width delimiting a middle amorphous‐like zone. By immuno‐ and chemical imaging, the in situ distribution of β‐(1→4)‐d‐mannans and HRGP‐like epitopes was shown to consist of two distinct cell‐wall layers, whereas SPs are distributed in the middle area of the wall. The overall cell‐wall polymer arrangement of the SPs, HRGP‐like epitopes, and mannans in the utricles of C. vermilara is different from the ubiquitous green algae C. fragile, in spite of both being phylogenetically very close. In addition, a preliminary cell‐wall model of the utricle moiety is proposed for both seaweeds, C. fragile and C. vermilara.     

A ZYGOTE-SPECIFIC PROTEIN WITH HYDROXYPROLINE-RICH GLYCOPROTEIN DOMAINS AND LECTIN-LIKE DOMAINS INVOLVED IN THE ASSEMBLY OF THE CELL WALL OF CHLAMYDOMONAS REINHARDTII (CHLOROPHYTA)

The cell wall of Chlamydomonas reinhardtii zygotes, which forms rapidly after the fusion of wall-free gametes, provides a tractable system for studying the properties and assembly of hydroxyproline-rich glycoproteins, the major proteinaceous components of green algal and plant cell walls. We report the cloning of the zsp2 gene and the analysis of its ZSP-2 product, a 58.9 kDa polypeptide that is synthesized exclusively by zygotes. The protein contains two (SP)_x repeats, establishing it as a member of the cell wall hydroxyproline-rich glycoproteins family. It also contains a 4-fold iteration of an amino acid sequence centered around cysteine residues, a configuration found in both plant and animal lectins. Furthermore, we report four observations on pellicle composition and production. First, cell-free preparations of the pellicle matrix are rich in hydroxyproline, arabinose, and galactose and contain bundles of very long fibrils. Second, glutathione blocks pellicle formation and results in the accumulation of long fibrils in the growth medium. Third, antibody to ZSP-2 also blocks pellicle formation. Fourth, ZSP-2 immunolocalizes to the boundary between the outer layers of the wall proper and the pellicle matrix. These observations are consistent with the possibility that the Cys-rich (glutathione-sensitive) lectin-like domains of ZSP-2 may bind to sugar residues on the long fibrils and anchor them to the cell wall, thereby initiating and maintaining pellicle formation.     

LIGNOFIBRILS ON THE EXTERNAL CELL WALL SURFACE OF CULTURED PLANT CELLS

Small strands and bundles of strands extend from the outside surface of suspension-cultured cells of Daucus, Ipomoea, and Phaseolus into the medium. This fibrous cell coat is present in all samples from various growth stages but appears to increase in quantity in the order Ipomoea < Phaseolus < Daucus. The bundles are often many microns in length and display great variation in frequency, size, and form. Identification of the composition of the strands and bundles as lignin is consistent with the following observations: alkaline nitrobenzene oxidation of the strands to compounds which resemble monomers of wood lignin; resistance of the strands to pronase, trypsin, pectinase, and lipase; strong irreversible adsorption of heavy metals; deposition of silver granules by treatment with silver nitrate-hexamine reagent; extraction of the bundles with aqueous dioxane (Björkman procedure); presence in quantity of a structured form of Klason lignin; and existence of material giving a positive test with the Wiesner reagent. Large individual strands (lignofibrils) from Phaseolus show the form of a flat ribbon with very thin branches at irregular intervals. This form does not vary with preparatory techniques, although its electron opacity does. Intercellular spaces display considerable structure and sometimes contain sheets of fibrillar material merging with both the middle lamella between the cells and the surface bundles facing the medium. These sheets are probably another form of association of the lignofibrils. It is suggested that natural fibrous lignin may be a much commoner component of plant tissue than suspected hitherto.     

CELL WALL CONFORMATION IN DRY SEEDS IN RELATION TO THE PRESERVATION OF STRUCTURAL INTEGRITY DURING DESICCATION

Internal tissues of mature air-dry seeds, prepared anhydrously for observation with the scanning electron microscope, exhibit cell wall structure which is different from that observed in aqueously fixed (hydrated) seed tissues. In a wide range of dry seeds observed (six members of the Cucurbitaceae, two species of Yucca, Hibiscus esculentus, Phaseolus vulgaris, and Helianthus annuus) cell walls exhibit a unique collapsed structure. The manner of cell wall collapse is characteristic for a given species and ranges from a highly regular folding pattern in the Cucurbitaceae to random wrinkling of the walls in Hibiscus. Evidence suggests that the regular patterns of wall folding may result from a mechanism located in the cell wall. Wall collapse in dry seeds is explained as a means of coordinating wall and protoplasmic shrinkage during desiccation and is thought to be essential for preserving the structural integrity of the tissue by conserving intercellular communication and plasmalemma-cell wall association. Implications of these observations may relate to retention of viability in seeds.