Cytoplasmic membrane senescence in bean cotyledons

Distinguishable patterns of cytoplasmic membrane senescence in cotyledon tissue of Phaseolus vulgaris have been elucidated by examining the behavior of four microsomal enzymes—NADH-cytochrome C reductase, NADPH-cytochrome C reductase, glucose-6-phosphatase and 5′-nucleotidase during germination. For young cotyledon tissue, specific activities for the phosphatases were similar for rough and smooth microsomal fractions, but both cytochrome C reductases were 2–3 times more concentrated in the smooth fraction. These proportionalities changed with increasing age. As senescence becomes more intense the enzyme activities change independently of one another. These changes do not appear to be influenced by the presence or absence of ribosomes on the membranes. Parallel analyses of phospholipid levels in the isolated fractions revealed that loss of microsomal enzyme activity correlates with an ultimate dismantling of the membranes into their macromolecular constituents. The data have been interpreted as indicating that functionally distinct membranes or regions of the same membrane are differentially sensitive to senescence.     

Contributions of cytoplasmic factors to in vitro cellular senescence.

Mass populations of normal human lung fibroblasts were enucleated by centrifugation at greater than or equal to 25,000 g in 4 mug/ml cytochalasin B. The 1 per cent of cells that did not enucleate where rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99 per cent lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1 per cent surviving iodoacetate-treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life-spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.     

In vitro senescence of human keratinocyte cultures

Human keratinocytes have been serially cultivated in low (0.015 mM) and high (1.8 mM) calcium containing medium. The calcium concentration of the growth medium significantly influenced the cell growth period in vitro. Cells grown in low calcium medium underwent 35-40 population doublings over 16-17 passages, while cells grown in high calcium medium ceased to proliferate after 20 population doublings over 7 passages. Changing the keratinocytes from one in vitro environment to the other drastically altered the lifespan in culture of populations derived from the same primary tissue. The degree of DNA methylation of human keratinocytes was shown to decrease with age in both high and low calcium culture conditions but does not appear to be associated with differentiation.     

Differential changes in the steady state levels of thylakoid membrane proteins during senescence in Cucumis sativus cotyledons

Chloroplast structure and function is known to alter during foliar senescence. Besides, the alterations in the structural organisation of thylakoid membranes changes in the steady state levels of thylakoid membrane proteins occur due to leaf ageing. We monitored temporal changes in some of the specific proteins of thylakoid membrane protein complexes by western blotting in the Cucumis sativus cotyledons as a function of the cotyledon age. We observed that the levels of D1 and D2 proteins of photosystem II started declining at the early stages of senescence of Cucumis cotyledons and continued to decline with the progress of cotyledon age. Similarly the level of Cyt f of Cyt b6/f complex declined rapidly with progress of senescence in these cotyledons. The reaction centre proteins of photosystem I were relatively found to be more stable than that of photosystem II reaction centre proteins reflecting possibly the disorganisation of photosystem II prior to photosystem I. The 33 kDa extrinsic protein (MSP) of oxygen evolving complex, the LHCII apoprotein and the beta-subunit of ATPsynthase showed the declined levels with the progress of cotyledon age. However, the extents of loss of these proteins were not as high as the reaction centre proteins of photosystem II and the Cyt f. These results provide that during senescence, proteins of thylakoid membranes degrade in a specific temporal sequence and thereby affect the temporal photochemical functions in Cucumis sativus cotyledons.     

In vitro adventitious budding on Pinus pinaster cotyledons and needles

Adventitious budding can be induced on two types of Pinus pinaster Ait. organs. Cotyledons (10-mm-long), derived from 8 to 10-day-old seedlings, show morpho-genetic response when an appropriate mineral solution is used (NH₄⁺/K⁺= 1). Of the various cytokinin concentrations added to this optimal mineral medium, 0.8 μM BAP (6-benzylaminopurine), with 5 n M NAA (1-naphtaleneacetic acid), promoted organogenesis best. Buds were induced from outer mesophyll layers.
Short shoots and the elongating needles (70-mm-long) were collected from cuttings of a mature tree (10-years-old). These cuttings benefited from physiological advantages of a well-developed root system (by heating the substrate). In order to stimulate in vitro organogenesis, they had been sprayed every week from March to May with 10 μ M BAP.
When cultivated in the presence of 10 μ M BAP and 25 n M NAA, 79% of the explants produced buds from dome-shaped meristematic cell clusters that pre-existed at the top of the short shoots. Moreover, among these, 42% gave rise to adventitious buds induced from proliferating mesophyll cells at the needle base. The morphologies of the two kinds of shoots were similar. Adventitious budding on these two different explants should allow vegetative multiplication of selected seedlings and elite trees.     

In vitro plantlet regeneration from cotyledons of the tree-legume Leucaena leucocephala

Two plant regeneration methods applicable to Leucaenaleucocephala were developed. In the first method, involvingorganogenesis via callus formation, cotyledon, hypocotyl and root segments wereinitiated on MS medium containing different concentrations ofN⁶-benzyladenine (BA), 2,4-dichlorophenoxyacetic acid (2,4-D), andnaphthaleneacetic acid (NAA). Both compact (type I) and friable (type II) calliwere obtained from the cotyledon and hypocotyl explants treated with differentconcentrations of the growth regulators. Shoots were generated only from thefriable calli formed from the cotyledon explants. The calli formed from thehypocotyl explants did not generate shoots and the root explants died withoutforming callus. Cotyledon explants from 3–4 day old seedlings showedmaximum callus induction compared to those from older seedlings. In a secondmethod involving direct organogenesis, excised cotyledons were cultured on 1/2MS medium containing 10–35 mg l^–1N⁶-benzyladenine (BA) for 7–14 days. Transfer of thecotyledonsto regeneration medium containing low BA resulted in callus formation andsubsequent shoot regeneration from the base of the excised cotyledon explants,with up to 100% frequency. Regenerated shoots rooted best on a basal mediumcontaining no growth regulators.     

In vitro studies on the anatomy and morphology of bud regeneration in melon cotyledons

Summary The anatomy and morphology of bud regeneration were investigated in melon (Cucumis melo L.) cv. Galia, which regenerates in vitro only by direct organogenesis from the cotyledon explant. Explants were cut from the cotyledon proximal to the apex from 3-d-old in vitro seedlings. After 3 d on Murashige and Skoog medium with N⁶-benzyladenine, cell division can be observed in the epidermal layer on the adaxial side in the center of the explant, near the most proximal (wounded) cut edge. Over the next week, the area of the meristem increases laterally. Additional cell layers are added to the meristematic area by cell division in the epidermis. In places the epidermis remains active in cell division. Alongside those active areas there are zones where the epidermis has become inactive, although the subepidermal layers continue to divide. In transverse section, the explant now has small protuberances on the adaxial surface. After 10 d on cytokinin-containing medium, the first signs of development are visible on the adaxial surface adjacent to the proximal cut edge. The protuberances observed after 10 d are neither primordia nor buds, although some meristematic bulges are observed. The first regenerated shoot buds are observed histologically after 15 d, by which time the surface has many protuberances and some small leaves. The first shoot is found by histology after 22 d. By this time the surface is covered with protrusions and leaves, mostly without accompanying buds. The leaves may be produced from the protrusions initially visible after 10 d.     

In vitro shoot regeneration from cotyledons of immature ovules ofImpatiens platypetala Lindl.

Summary An efficient and reproducible protocol has been developed for in vitro shoot regeneration from cotyledonary explants derived by germinating immature ovules ofImpatiens platypetala Lindl. ‘TR6-27-2’. Cotyledonary explants were cultured on a modified Murashige and Skoog (MS) agar-solidified medium containing 7.5g · liter^−1 sucrose, 22.2µ M N⁶-benzyladenine (BA), and 0.54µM α-naphthaleneacetic acid (NAA). The induction of organogenic tissues occurred after 6 to 8 wk in culture. Exogenous auxin and cytokinin were essential for the induction of organogenic tissues and survival of explants, and BA was most effective for the induction of organogenic tissues, compared with other cytokinins tested. The addition of glutamine (500 mg · liter^−1) was also important for growth of organogenic tissues after induction and for reducing explant death during culture. The induction of organogenic tissue was also influenced by the type of cotyledon cultured and the age of the donor seedlings. On average, eight shoots per explant were induced from organogenic tissues larger than 0.5 cm in diameter 6 to 8 wk after transfer to a modified MS agar-solidified medium without NAA and BA reduced to 4.44µM. Shoots longer than 0.5 cm in length were successfully rooted 2 to 4 wk after transfer to a basal MS medium containing 30g · liter^−1 sucrose.     

In vitro shoot organogenesis from excised immature cotyledons and microcuttings production in Stone Pine

Adventitious buds were induced on isolated immature cotyledons of Pinus pinea L. in the presence of benzyladenine (BA). The response to different BA concentrations also depended upon the culture medium used (modified MS, SH and GD). A wide range of BA concentrations (5, 25 or 50 M) can be applied to the GD and SH media, which are the media with the lower nitrogen content, without damaging effects. In the MS medium, which has the highest nitrogen concentration, the range of BA that can be applied was narrower and the highest BA concentration was lethal. The addition of indolebutyric acid (0.05, 0.25 or 0.5 M) to the induction medium, decreased the response of cotyledons. The increase in the concentration of sucrose from 3% to 5% did not increase the number of responding cotyledons. The addition of activated charcoal (0.5 and 3 g l^-1) or indolebutyric acid (1.5 or 3 M) did not speed up the elongation of explants. Elongation of the buds produced shoots with two different phenotypes, each phenotype having a different multiplication rate.Abbreviations BA benzyladenine - GD Gresshoff & Doy medium - IBA indolebutyric acid - MS Murashige & Skoog medium - SH Schenk & Hildebrandt medium     

Changes in antioxidative enzymes in cucumber cotyledons during natural senescence: comparison with those during dark-induced senescence

Changes in 7 antioxidative enzymes in naturally senescent cotyledons of cucumber ( Cucumis sativus ) were investigated. The activities of superoxide dismutase (SOD; EC 1.15.1.1), catalase (EC 1.11.1.6), dehydroascorbate reductase (EC 1.8.5.1) and glutathione reductase (GR; EC 1.6.4.2) gradually decreased during the progression of senescence, while those of ascorbate peroxidase (APX; EC 1.11.1.11) and guaiacol peroxidase (GPX; EC 1.11.1.7) gradually increased. The activity of monodehydroascorbate reductase (MDAR; EC 1.6.5.4) was not significantly changed. Western blot analysis showed that the protein level of mitochondrial SOD gradually declined. The protein level of catalase transiently decreased and then increased in the later stages of senescence, despite the decrease in its activity. The overall behavior was markedly different from that found in cotyledons of artificially senescing seedlings transferred into darkness; the activities of SOD, catalase, APX, GPX and GR gradually increased.     

Influence of cytokinins on the methyl jasmonate-promoted senescence in Helianthus annuus cotyledons

Cotyledons of seven-day-old sunflower seedlings were pretreated withcytokinins prior to exposure to methyl jasmonate (MeJA). The rate of senescencewas then followed by measuring chlorophyll loss, electrolyte leakage, ethyleneproduction, and 1-aminocyclopropane-1-carboxylase (ACC) oxidase activity. MeJApromoted all these senescence parameters and the MeJA effects were partiallyblocked by cytokinin pretreatment. 8-azaguanine, which has been reported tohaveanticytokinin activity, blocked the ability of benzyl adenine (BA) to reversethe effect of MeJA on senescence. MeJA also increased SOD and catalaseactivity,decreased protein content. However, while the cytokinin BA more than overcamethe MeJA effect on protein content and SOD activity it did not antagonize theeffect of MeJA on catalase.     

Quantitative changes of fatty acids in soybean cotyledons during senescence and regreening

The quantity of total fatty acids in soybean cotyledons during aging, senescence and regreening has been studied. The greatest change in the fatty acid profile during the initial greening of the cotyledons (4–7 days after germination) was a 130% increase in the content of linolenate. Linoleate, as in the case of the other fatty acids, declined in the first 4 days and then increased by 7 days. Following the 10th day after germination, the quantity of palmitate, linoleate, and linolenate decreased continuously through senescence to 20–28% of the maximum quantity of each. When the cotyledons were regreened by removal of the epicotyl 15 or 16 days after germination, linolenate was present in quantities substantially higher than in the senescing cotyledon. On the 22nd day after germination, the quantity of linolenate in regreened tissue was 140% greater than that in senescing tissue of the same age. By contrast, the quantity of linoleate was only 30–40% greater in regreening tissue and the quantity of most of the other fatty acids was similar in both tissues. Similar changes in the quantity of chloroplast fatty acids were observed during this period. Removal of the epicotyl resulted in a higher level of chloroplast linolenate. During aging, the total chlorophyll and the number of chloroplasts reached a maximum on the 10th day and decreased rapidly during senescence. The amount of chlorophyll per chloroplast remained relatively constant during this period whereas the quantity of linolenate per chloroplast decreased during senescence. It is suggested that major structural changes observed in chloroplast membranes may be related to changes in fatty acid composition, but are not dependent on changes in chlorophyll concentration.     

In vitro synthesis of peroxisomal membrane polypeptides

Peroxisomal membranes containing predominantly integral peroxisome membrane polypeptides were obtained from a highly purified peroxisomal fraction. Following sodium dodecylsulfate polyacrylamide gel electrophoresis three polypeptides with apparent molecular weights of 69, 36, and 22 kDa were isolated and used to raise antibodies in rabbits. Cell-free synthesis of these polypeptides was carried out in an in vitro translational system derived from rabbit reticulocytes. By subjecting peroxisomal membranes to reductive methylation [14C]-radiolabeled mature membrane polypeptides were obtained. The comparison of the three mature integral peroxisome membrane polypeptides with their corresponding in vitro synthesis products revealed no size differences indicating the lack of recognizable presequences for these peroxisomal membrane polypeptides.     

In vitro folding of alpha-helical membrane proteins

For large-scale production, as required in structural biology, membrane proteins can be expressed in an insoluble form as inclusion bodies and be refolded in vitro. This requires refolding conditions where the native form is thermodynamically stable and where nonproductive pathways leading to aggregation are avoided. Examples of successful refolding are reviewed and general guidelines to establish refolding protocols of membrane proteins are presented.     

In vitro folding of alpha-helical membrane proteins

For large-scale production, as required in structural biology, membrane proteins can be expressed in an insoluble form as inclusion bodies and be refolded in vitro. This requires refolding conditions where the native form is thermodynamically stable and where nonproductive pathways leading to aggregation are avoided. Examples of successful refolding are reviewed and general guidelines to establish refolding protocols of membrane proteins are presented.     

In vitro cytotoxicity testing of coladerm membrane

Atpresent, biodegradable and biocompatible membranes based on collagen andglycosaminoglycans play an important role in substitutive medicine. Modernbiomaterials use a chemically modified collagen-based matrix for implants withprogrammable biodegradability as a substitute of buccal mucosa, skin,cartilage,etc. Besides the requirements for biocompatibility and biodegradability, themembranes must be also non-toxic. Therefore, cytotoxicity testing of thesematerials in vitro is an integral part of introducingnewlydeveloped types of membranes into clinical practice. As a biological model forthe tested COLADERM membrane, cell cultures from human embryonic fibroblasts(B-HEF-2) were used for both cytotoxicity testing as well as in tests to assessthe ability of cells to proliferate on this membrane. Along with the ability ofcells to grow on the surface and inside the membrane, immunohistochemicalexamination and scanning electron microscopy (SEM) were performed as well. Theobtained results have shown that the COLADERM membrane is non-toxic withsuitable structural and biological properties for clinical application as asubstitute of buccal mucosa following surgical ablation of malignant tissuesfrom the oral cavity.     

In vitro models of tail contraction and cytoplasmic streaming in amoeboid cells

We have developed a reconstituted gel-sol and contractile model system that mimics the structure and dynamics found at the ectoplasm/endoplasm interface in the tails of many amoeboid cells. We tested the role of gel-sol transformations of the actin-based cytoskeleton in the regulation of contraction and in the generation of endoplasm from ectoplasm. In a model system with fully phosphorylated myosin II, we demonstrated that either decreasing the actin filament length distribution or decreasing the extent of actin filament cross-linking initiated both a weakening of the gel strength and contraction. However, streaming of the solated gel components occurred only under conditions where the length distribution of actin was decreased, causing a self-destruct process of continued solation and contraction of the gel. These results offer significant support that gel strength plays an important role in the regulation of actin/myosin II-based contractions of the tail cortex in many amoeboid cells as defined by the solation-contraction coupling hypothesis (Taylor, D. L., and M. Fechheimer. 1982. Phil. Trans. Soc. Lond. B. 299:185-197). The competing processes of solation and contraction of the gel would appear to be mutually exclusive. However, it is the temporal-spatial balance of the rate and extent of two stages of solation, coupled to contraction, that can explain the conversion of gelled ectoplasm in the tail to a solated endoplasm within the same small volume, generation of a force for the retraction of tails, maintenance of cell polarity, and creation of a positive hydrostatic pressure to push against the newly formed endoplasm. The mechanism of solation-contraction of cortical cytoplasm may be a general component of the normal movement of a variety of amoeboid cells and may also be a component of other contractile events such as cytokinesis.     

In vitro studies of membrane protein folding.

The study of membrane protein folding is a new and challenging research field. Consequently, there are few direct studies on the in vitro folding of membrane proteins. This review covers work aimed at understanding folding mechanisms and the intermolecular forces that drive the folding of integral membrane proteins. We discuss the kinetic and thermodynamic studies that have been undertaken. Our review also draws on closely related research, mainly from purification studies of functional membrane proteins, and gives an overview of some of the successful methods. A brief survey is also given of the large body of mutagenesis and fragment work on membrane proteins, as this too has relevance to the folding problem. It is noticeable that the choice of solubilizing detergents and lipids can determine the success of the method, and indeed it appears that particular lipid properties can be used to control the rate and efficiency of folding. This has important ramifications for much in vitro folding work in that it aids our understanding of how to obtain and handle folded, functional protein. With this in mind, we also cover some relevant properties of model, lipid-bilayer systems.     

In vitro flowering and pod formation from cotyledons of groundnut (Arachis hypogaea L.)

Summary The response of groundnut cotyledons to the presence of various growth regulators in concentrations from 0.1 to 5 mg/l has been studied in detail using several genotypes of groundnut on two different media. Cotyledons with embryo axis, cultured on Blaydes' medium with cytokinins, produced shoots, in the axils of which 2–7 flower buds could be seen. The frequency of flower bud induction in general increased with increasing concentrations of cytokinins, the optimal levels being 3 mg/l of KN or 4 mg/l of BAP. Cotyledons without embryo axis, cultured on Blaydes' medium with BAP (0.5 mg/l), produced a cluster of flower buds directly, ranging in number from 8–28, without any vegetative growth. Excised embryo axes cultured on the same medium gave plantlets without flower buds. The growth regulators IAA, NAA, GA₃ and ABA failed to induce flower buds in independent treatments. However, lower concentrations of IAA and NAA in combination with cytokinins exerted a positive influence on flowering. The blooming of the flower buds was facilitated on media supplemented with low concentrations of cytokinins. Six percent of the induced flowers resulted in gynophore development and ultimately formed pods when cultured under complete dark conditions in modified MS medium supplemented with kinetin.     

In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls

A high-efficiency two-step culture procedure for direct somaticorganogenesis in loblolly pine (Pinus taeda L.) resulting inthe formation of multiple shoot structures induced on cotyledons andhypocotyls of mature zygotic embryos is described. Mature zygoticembryos of eight genotypes of loblolly pine were used as explants toinduce direct somatic organogenesis with this two-step culture method,involving the induction and the differentiation of direct adventitiousshoots. After mature zygotic embryos of eight genotypes of loblolly pinewere cultured on induction medium containing 2,4-dichlorophenoxyaceticacid (2,4-D) or -naphthaleneacetic acid (NAA), 6-benzyladenine(BA), and kinetin for 2–3 weeks, embryos were transferred todifferentiation medium. Adventitious shoot regeneration via directsomatic organogenesis with the frequency of 8.7–27.8% wasobtained from mature zygotic embryo cultures of the genotypes tested.The highest mean number of 32.6 adventitious shoots per mature zygoticembryo was produced from genotype La. The tissue culture protocol of invitro shoot regeneration via direct somatic embryogenesis was optimizedafter examining the periods of the induction culture, chillingtreatment, glutamine concentration, and basic medium levels. Rooting wasachieved on TE medium supplemented with 0.5 mg/l indole-3-butyric acid(IBA), 0.5 mg/l gibberellic acid (GA₃), and 1 mg/l6-benzyladenine (BA), and regenerated plantlets were established insoil. These results suggested that adventitious shoot regeneration viadirect somatic organogenesis could be useful for clonal micropropagationof some genotypes of loblolly pine and for establishing a transformationsystem of this coniferous species.