The roles of inorganic nitrogen salts in maintaining phytochrome- and gibberellin A3-mediated germination control in skotodormant lettuce seeds

Skotodormant seeds of Lactuca sativa Grand Rapids imbibed in darkness for 10 days (10-day DS) germinated poorly upon terminal treatment with red light (R) or gibberellin A₃ (GA₃). Inorganic nitrogen salts in the imbibition solutions reduced seed skotodormancy. Ten-day DS seeds, imbibed in 25 mm salt solutions followed by terminal R, germinated 99% if imbibed in NH₄NO₃, 70% if imbibed in KNO₃ or NH₄Cl, and 55% if imbibed in NaNO₃. Seeds imbibed in higher salt concentrations germinated fully upon terminal R treatment. Seeds imbibed in 25 mm NH₄Cl or in 50 mm NH₄NO₃ germinated completely upon GA₃ treatment. Osmotic effects of imbibition media accounted for only part of the effect, since seeds imbibed in 50 mm CaCl₂ or NaCl germinated poorly following R or GA₃ treatment. Seeds imbibed in 500 mm polyethylene glycol (PEG) 1000 or mannitol solutions for 10 days still exhibited skotodormancy. Treatments of R or GA₃ did not stimulate germination in seeds imbibed in mannitol, but germination was complete if seeds were given 1-h acid immersion plus a water rinse before the terminal R or GA₃ treatment. Seeds imbibed in 50–500 mm PEG during 10-day DS germinated significantly better in response to terminal R. Terminal GA₃ significantly improved germination only in seeds imbibed at 500 mm PEG. P_fr appeared to function in mannitol-imbibed seed only after an acid treatment. Seed exposure to inorganic nitrogen salts during the 10-day DS maintained seed sensitivity to terminal R or GA₃ treatment. The depth of seed skotodormancy was related to the availability of inorganic nitrogen and also involved the levels of P_fr or endogenous GA₃.Abbreviations FR far red - DS dark storage - R red - GA₃ gibberellin A₃ - PEG polyethylene glycol - SHAM salicylhydroxamic acid - ANOVA analysis of variance - GLM general linear model - LSD least squares difference - P_fr far-red absorbing form of phytochrome     

Block-surface staining for differentiation of starch and cell walls in wheat endosperm.

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat (Triticum aestivum L.) caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca. 11 microns in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.     

Gibberellin-induced hydrolysis of endosperm cell walls in gibberellin-deficient tomato seeds prior to radicle protrusion

The weakening of the mechanical restraint of the endosperm layer in tomato (Lycopersicon esculentum Mill.) seeds, a prerequisite for germination, has been studied with the use of seeds of the gibberellin (GA)-deficientgib-1 mutant. Incubation ofgib-1 endosperms, including part of the testa, in 10 M GA₄₊₇, resulted within 12 h in the release of fructose, glucose, galactose and mannose into the incubation medium. Only small amounts of sugars diffused out of thegib-1 endosperms during incubation in water. Chemical hydrolysis of endosperm cell walls ofgib-1 seeds showed that they are mainly composed of mannose, and smaller quantities of glucose and galactose. Treatment with GA₄₊₇ induced in the endosperms the production of endo--mannanase activity that was not detectable during incubation in water, and also increased the activities of mannohydrolase and -galactosidase as compared with the water controls. No cellulase activity was found. It is concluded that in tomato seeds the weakening of endosperms prior to radicle protrusion is mediated by a GA-induced enzymatic degradation of the mannan-rich cell walls.Abbreviation GA(s) gibberellin(s)     

FTIR imaging of wheat endosperm cell walls in situ reveals compositional and architectural heterogeneity related to grain hardness.

Endosperm cell walls of cultivars of wheat (Triticum aestivum L.) selected for their endosperm texture (two soft and two hard) were analysed in situ by Fourier transform infrared (FTIR) microspectroscopy. FTIR imaging coupled with statistical analysis was used to map the compositional and structural heterogeneity within transverse sections from which cell contents had been removed by sonication. In the majority of grains analysed, two distinct populations of endosperm cells could be identified by spectral features that were related to cell morphology and age, regardless of cultivar. The main cell-wall component responsible for these differences was the polysaccharide arabinoxylan. In a few samples, this heterogeneity was absent, for reasons that are not understood, but this was not correlated to endosperm texture or growth conditions. Within the same population of endosperm cells, cell walls of hard endosperm could be distinguished from those of soft endosperm by their spectral features. Compared to hard cultivars, the peripheral endosperm of soft cultivars was characterised by a higher amount of polymer, whose spectral feature was similar to water-extractable arabinoxylan. In contrast, no specific compound has been identified in the central endosperm: structural differences within the polysaccharides probably contribute to the distinction between hard and soft cultivars. In developing grain, a clear difference in the composition of the endosperm cell walls of hard and soft wheat cultivars was observed as early as 15 days after anthesis.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. Grand Rapids

The timing of mobilisation of lipid, sucrose, raffinose and phytate in lettuce seeds (achenes) (cv. Grand Rapids) has been examined. These reserves (33%, 1.5%, 0.7%, 1.4% of achene dry weight, respectively) are stored mostly in the cotyledons. Except for a slight degradation of raffinose and increase in sucrose, there is no detectable reserve mobilisation during germination. The endosperm (8% of seed dry weight), which has thick, mannan-containing cell walls (carbohydrate, 3,4% of seed dry weight), is completely degraded within about 15h following germination. Mannanase activity increases about 100-fold during the same period and arises in all regions of the endosperm. Also during this period sucrose and raffinose are degraded and fructose and glucose accumulate in the embryo. The endosperm hydrolysis products are taken up by the embryo, and are probably used as an additional reserve to support early seedling growth. However, endosperm cell-wall carbohydrates, such as mannose, are not found as free sugars. Lipid and phytate are degraded in a later, second phase of mobilisation. Low levels of sucrose are present in the embryo, mostly in the cotyledons, and large amounts of fractose and glucose (14% of seedling dry weight at 3 days after sowing) accumulate in the hypocotyl and radicle. It is suggested that sucrose, produced in the cotyledons by gluco-neogenesis, is translocated to the axis and converted there to fructose and glucose.     

Characterization of a small proteolytic enzyme which lyses bacterial cell walls

Ensign, J. C. (University of Wisconsin, Madison), and R. S. Wolfe. Characterization of a small proteolytic enzyme which lyses bacterial cell walls. J. Bacteriol. 91:524-534. 1966.-An enzyme isolated from a myxobacter possesses both cell-wall lytic and proteolytic activity. The enzyme has been purified over 600-fold and is electrophoretically homogeneous upon cellulose acetate at several pH values and upon polyacrylamide gel columns. A single peak was obtained upon ultracentrifugation and density gradient centrifugation. Based upon Sephadex gel filtration, a molecular weight of 8,700 was determined for the enzyme. Albumin and casein were extensively degraded by the enzyme, with approximately one-third of the peptide bonds present in these proteins being hydrolyzed. The enzyme lyses cell walls by hydrolyzing peptide bonds in the glycosaminopeptide.     

Investigation of ferulate deposition in endosperm cell walls of mature and developing wheat grains by using a polyclonal antibody

A polyclonal antibody has been raised against ferulic acid ester linked to arabinoxylans (AX). 5-O-feruloyl-α-l-arabinofuranosyl(1→4)-β-d-xylopyranosyl was obtained by chemical synthesis, and was coupled to bovine serum albumin for the immunization of rabbit. The polyclonal antibody designated 5-O-Fer-Ara was highly specific for 5-O-(trans-feruloyl)-l-arabinose (5-O-Fer-Ara) structure that is a structural feature of cell wall AX of plants belonging to the family of Gramineae. The antibody has been used to study the location and deposition of feruloylated AX in walls of aleurone and starchy endosperm of wheat grain. 5-O-Fer-Ara began to accumulate early in aleurone cell wall development (beginning of grain filling, 13 days after anthesis, DAA) and continued to accumulate until the aleurone cells were firmly fixed between the starchy endosperm and the nucellus epidermis (19 DAA). From 26 DAA to maturity, the aleurone cell walls changed little in appearance. The concentration of 5-O-Fer-Ara is high in both peri- and anticlinal aleurone cell walls with the highest accumulation of 5-O-Fer-Ara at the cell junctions at the seed coat interface. The situation is quite different in the starchy endosperm: whatever the stage of development, a low amount of 5-O-Fer-Ara epitope was detected. Contrary to what was observed for aleurone cell walls, no peak of accumulation of feruloylated AX was noticed between 13 and 19 DAA. Visualization of labelled Golgi vesicles suggested that the feruloylation of AX is intracellular. The distribution of (5-O-Fer-Ara) epitope is further discussed in relation to the role of ferulic acid and its dehydrodimers in cell wall structure and tissue organization of wheat grain.     

Apple-fruit xyloglucans: a comparative study of enzyme digests of whole cell walls and of alkali-extracted xyloglucans.

Apple cell walls or alkali-extracted xyloglucans were digested with an endo-glucanase from Trichoderma viride and the resulting oligosaccharides were isolated by chromatography on Bio-Gel P-4. Three main oligosaccharides were present in similar proportions, and their structures were shown to be [Xyl(Glc)]3-Glc, [Xyl(Glc)]2-(FucGalXyl)Glc-Glc, and XylGlc-(GalXyP)Glc-(FucGalXyl)Glc-Glc. Each non-reducing-end Glc was 6-linked, each reducing-end Glc was 4-substituted, and each other Glc was 4,6-disubstituted. The Xyl was either terminal or 2-substituted, the Fuc was terminal, and the Gal was either terminal or 2-substituted. The 1H-NMR spectra of the oligosaccharides extracted directly from the cell wall showed that they are not acetylated. Other oligosaccharides, notably GalXyl3Glc4, Xyl2Glc4, and Xyl2Glc3, were present in smaller proportions in the digest of the cell walls.     

Preparation and evaluation of an enzyme which degrades yeast cell walls

Summary A method for the production and preparation of an enzyme which degrades yeast cell walls from a species of aRhizoctonia (tentatively identified asR. solani) was established on a commercial scale. The production of crude enzyme powder, having a lytic activity of 100 units/mg, in batches of 80 kg is feasible.The enzyme preparation was evaluated for industrial use. When yeast cells were treated with this enzyme, the digestibility of feed yeast was improved 1.4–2 fold in vitro; the efficiency of a mechanical disintegrator in extracting cellular substances was increased 35–50%; the release of soluble glucans having widely varying degrees of polymerization was induced; the extraction of cellular protein by alkali was facilitated 2–3 fold; an 80% release of cell-bound invertase was induced and 2–3 times more yeast extract could be prepared.Studies on Fungal Enzymes Active in Hydrolysing Yeast Cell Wall (VII)     

Mannan transglycosylase: a novel enzyme activity in cell walls of higher plants

Mannan transglycosylase is a novel cell wall enzyme activity acting on mannan-based plant polysaccharides in primary cell walls of monocotyledons and dicotyledons. The enzyme activity was detected by its ability to transfer galactoglucomannan (GGM) polysaccharides to tritium-labelled GGM-derived oligosaccharides generating tritium-labelled GGM polysaccharides. Mannan transglycosylase was found in a range of plant species and tissues. High levels of the enzyme activity were present in flowers of some kiwifruit (Actinidia) species and in ripe tomato (Solanum lycopersicum L.) fruit. Low levels were detected in mature green tomato fruit and activity increased during tomato fruit ripening up to the red ripe stage. Essentially all activity was found in the tomato skin and outermost 2 mm of tissue. Mannan transglycosylase activity in tomato skin and outer pericarp is specific for mannan-based plant polysaccharides, including GGM, galactomannan, glucomannan and mannan. The exact structural requirements for valid acceptors remain to be defined. Nevertheless, a mannose residue at the second position of the sugar chain and the absence of a galactose substituent on the fourth residue (counting from the non-reducing end) appear to be minimal requirements. Mannan-based polysaccharides in the plant cell wall may have a role analogous to that of xyloglucans, introducing flexibility and forming growth-restraining networks with cellulose. Thus mannan transglycosylase and xyloglucan endotransglycosylase, the only other known transglycosylase activity in plant cell walls, may both be involved in remodelling and refining the cellulose framework in developmental processes throughout the life of a plant.Abbreviations EBM Endo--mannanase - GGM galactoglucomannan - GGMO Galactoglucomannan-derived oligosaccharide - G₂M₅ Di-galactosyl mannopentaitol - M₂–M₅ Mannobiitol to mannopentaitol oligosaccharides - SK+OP Skin plus outer pericarp - XET Xyloglucan endotransglucosylase - XG Xyloglucan     

An enzyme activity capable of endotransglycosylation of heteroxylan polysaccharides is present in plant primary cell walls

Heteroxylans in the plant cell wall have been proposed to have a role analogous to that of xyloglucans or heteromannans, forming growth-restraining networks by interlocking cellulose microfibrils. A xylan endotransglycosylase has been identified that can transglycosylate heteroxylan polysaccharides in the presence of xylan-derived oligosaccharides. High activity was detected in ripe fruit of papaya (Carica papaya), but activity was also found in a range of other fruits, imbibed seeds and rapidly growing seedlings of cereals. Xylan endotransglycosylase from ripe papaya fruit used a range of heteroxylans, such as wheat arabinoxylan, birchwood glucuronoxylan and various heteroxylans from dicotyledonous primary cell walls purified from tomato and papaya fruit, as donor molecules. As acceptor molecules, the enzyme preferentially used xylopentaitol over xylohexaitol or shorter-length acceptors. Xylan endotransglycosylase was active over a broad pH range and could perform transglycosylation reactions up to 55 °C. Xylan endotransglycosylase activity was purified from ripe papaya fruit by ultrafiltration and cation exchange chromatography. Highest endotransglycosylase activity was identified in fractions that also contained high xylan hydrolase activity and correlated with the presence of the endoxylanase CpaEXY1. Recombinant CpaEXY1 protein transiently over-expressed in Nicotiana benthamiana leaves showed both endoxylanase and xylan endotransglycosylase activities in vitro, suggesting that CpaEXY1 is a single enzyme with dual activity in planta. Purified native CpaEXY1 showed two- to fourfold higher endoxylanase than endotransglycosylase activity, suggesting that CpaEXY1 may act primarily as a hydrolase. We propose that xylan endotransglycosylase activity (like xyloglucan and mannan endotransglycosylase activities) could be involved in remodelling or re-arrangement of heteroxylans of the cellulose-non-cellulosic cell wall framework.     

Chemical composition of Streptococcus mutans cell walls and their susceptibility to Flavobacterium L-11 enzyme

The susceptibility to a cell wall lytic L-11 enzyme from Flavobacterium sp. and the quantitative and/or qualitative composition of the cell walls of some strains of cariogenic Streptococcus mutans and a non-cariogenic strain of Streptococcus mitis were determined. The purified cell walls of S. mutans strains HS-1 (serotype a), BHT (b), NCTC10449 (c), C67-1 (c), C67-25 (c), OMZ 176 (d), MT703 (e), MT557 (f), OMZ65 (g), and AHT (g), and S. mitis CHT contained glutamic acid, alanine, and lysine as well as muramic acid and glucosamine as a peptidoglycan component. Besides these amino acids, significant amounts of threonine were detected in strains HS-1, OMZ65, and AHT cell walls, and considerable amounts of aspartic acid and/or threonine as well as several other amino acids in OMZ176, OMZ65, and CHT cell walls. Rhamnose was a common special component of the cell walls of S. mutans strains BHT, NCTC10449, MT703, B2 (e), MT557, and AHT, and S. mitis CHT. An additional sugar component, glucose, was detected in the cell walls of all of these strains except BHT, and galactose was found in BHT, AHT, and CHT cell walls. Galactosamine was present in S. mitis CHT cell walls. Varying amounts of phosphorus were detected in the cell walls of all the strains examined. The cell walls of all these streptococcal strains except MT703, 6715, and AHT were susceptible to the lytic action of the L-11 enzyme to various extents. No consistent relationship was observed between the amino acid and sugar composition of these cell walls and their susceptibility to the L-11 enzyme. The chemical composition of these cell walls is discussed in terms of the serological classification of S. mutans.     

Changes in germination and respiratory potential of embryos of dormant Grand Rapids lettuce seeds during long-term imbibed storage,and related changes in the endosperm

Grand Rapids lettuce (Lactuca sativa L.) seeds were stored in an imbibed state for up to two years. Embryos dissected from stored seeds showed a progressive loss with time in their ability to germinate on polyethylene glycol (PEG) solutions. Little germination of dissected embryos from one-month imbibed seeds occurred on-6 bar PEG but only after four months of storage did the dissected embryos fail to germinate on-4 bar PEG. After two years storage 30% of dissected embryos still were able to germinate on-2 bar PEG. This loss of germination potential, which may be a symptom of the development of an embryo dormancy, could be reversed by N⁶-benzyladenine (BA) and red light (R) applied together or separately to dissected embryos. Two weeks of chilling of 12-month imbibed seeds restored sensitivity to R and a 48-h BA pretreatment prior to R resulted in germination rates similar to those of seeds emerging from primary dormancy. There was loss of embryo control of endo--mannanase activity after two weeks of storage even though the endosperms themselves retained their capacity for enzyme synthesis for six more weeks. Eventually, then, endo--mannanase synthesis is not possible because of inherent changes in both the embryo and endosperm, although each tissue undergoes changes at its own rate. Oxygen uptake by embryos dissected from two-month imbibed seeds did not increase to the same extent as embryos dissected from freshly imbibed seeds. In intact seeds germinating from a skotodormant state, oxygen uptake increased at a time coincident with radicle protrusion, but did not achieve the levels of uptake of those seeds germinating from a primary dormant state. The decline in uptake of oxygen by secondary dormant seeds is the result of a lowered respiratory capability of the embryo itself, rather than of changes in permeability of the surrounding structures.Abbreviations BA N⁶-benzyladenine - Pfr active (far-redabsorbing) form of phytochrome - R red light - PEG polyethylene glycol     

Identification of a lytic enzyme of Clostridium acetobutylicum that degrades choline-containing pneumococcal cell walls

Abstract A protein that degrades pneumococcal walls containing choline, but not ethanolamine, in the teichoic acids has been isolated and purified from supernatants obtained from cultures of Clostridium acetobutylicum . The analyses of the degradation products of [³H]choline-labeled cell walls treated with this enzyme indicated that the purified protein, showing an apparent M _r of 115 000, is an N-acetylmuramyl- l -alanine amidase. Our results also suggest that C. acetobutylicum contains choline in its cell wall.     

Degradation of the endosperm cell walls of Lactuca sativa L., cv. grand rapids in relation to the mobilisation of proteins and the production of hydrolytic enzymes in the axis,cotyledons and endosperm

The timing of changes in total nitrogen and soluble amino nitrogen content, and in the activities of proteinase (pH 7.0), isocitrate lyase, catalase, phytase, phosphatase (pH 5.0), -galactosidase and -mannosidase were studied in extracts from the cotyledons, axis and endosperms of germinating and germinated light-promoted lettuce seeds. The largest amount of total nitrogen (2.7% seed dry weight) occurs within the cotyledons, as storage protein. As this decreases the total nitrogen content of the axis increases and the soluble amino nitrogen in the cotyledons and axis increases. Proteinase activity in the cotyledons increases coincidentally with the depletion of total nitrogen therein. Enzymes for phytate mobilisation and for gluconeogenesis of hydrolysed lipids increase in activity in the cotyledons as the appropriate stored reserves decline. Beta-mannosidase, an enzyme involved in the hydrolysis of oligo-mannans released by the action of endo--mannase on mannan reserves in the endosperm, arises within the cotyledons. This indicates that complete hydrolysis of mannans to the monomer does not occur within the endosperm. Mobilisation of all cotyledon reserves occurs after the endosperm has been degraded, providing further evidence that the endosperm is an early source of food reserves for the growing embryo.Abbreviations HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid - EDTA ethylenediamine tetraacetic acid disodium salt - TCA trichloroacetic acid Part 2 of a series, of which the first was published in Planta 139, 1–8 (1978)     

A technique for cytological preparations following enzymatic digestion of pollen mother cell walls.

A technique is presented for making meiotic chromosome preparations with enzymatic digestion of pollen mother cell walls with cellulase. This technique has given excellent chromosome definition from early pachytene stages on. Such chromosome preparations were found useful for in situ nucleic acid hybridisation studies on plant chromosomes.     

A gene (sleB) encoding a spore cortex-lytic enzyme from Bacillus subtilis and response of the enzyme to L-alanine-mediated germination.

The Bacillus subtilis sleB gene, which codes for the enzyme homologous to the germination-specific amidase from Bacillus cereus, was cloned and its nucleotide sequence was determined. Sequence analysis showed that it had an open reading frame of 918 bp, coding for a polypeptide of 305 amino acids with a putative signal sequence of 29 residues. Enzyme activity was not found in germination exudate of B. subtilis spores, which differs from the case of B. cereus enzyme. A B. subtilis mutant with an insertionally inactivated sleB gene revealed normal behavior in growth and sporulation. However, the sleB mutant was unable to complete germination mediated by L-alanine.     

Sequential solvent extraction and structural characterization of polysaccharides from the endosperm cell walls of barley grown in different environments

The objective of this study was to examine the composition and molecular structure of the endosperm cell walls (CW) derived from barley grain grown in three environments in Canada, and differing in grain hardness, protein, and total β-glucan contents. The endosperm CW were isolated from barley, cv. Metcalfe, grown in Davidson, SK (Sample A), Hythe, AB (sample B), and Hamiota, MB (sample C). The CW were sequentially extracted with water at 65 ^oC, saturated Ba(OH)₂, again with water at 25 ^oC, and 1 M NaOH, resulting in fractions designated WE65, BaE, Ba/WE, and NaE, respectively. The monosaccharide analysis indicated the presence of β-glucans, arabinoxylans, and small amounts of arabinogalactans, glucomannans, and xyloglucans. Cellulose was detected in the CW remnants. The CW of sample A, exhibiting a lower grain hardness than sample B, contained the lowest amount of β-glucans, but the highest amount of arabinoxylans and the mannose-containing polysaccharides. The CW of sample C, characterized by very high protein content in the grain, contained the highest amount of β-glucans and the lowest amount of other polysaccharides. Polysaccharides in the CW of sample B, exhibiting the highest grain hardness, were characterized by the highest weight average molecular weights (M_w). β-Glucans in the CW of Sample B showed the highest ratio of DP3/DP4 and the longest cellulosic fragments in the polymeric chains. Of the three barley samples, arabinoxylans in the endosperm CW of sample A exhibited the lowest degree of branching, the highest amount of unsubstituted Xyl residues, and the highest ratio of singly to doubly substituted Xylp. The highest water solubility of the CW of sample C was associated with the highest concentration of β-glucans, the lowest DP3/DP4 ratio, and the lowest M_w of the polymeric constituents. Arabinoxylans with the lowest amount of doubly substituted but the highest amount of unsubstituted xylose residues and long sequences of unsubstituted xylan regions were found in the NaE fractions. The NaE fractions showed a high ratio of →4)-Glcp-(1→ to →3)-Glcp-(1→ linkages and some →4)-Manp-(1→ linkages, indicating a high level of long cellulosic regions in β-glucan chains and the presence of glucomannans.