Microtubules in maize leaf protoplasts in relation to donor tissue and in vitro culture

Summary Maize (Zea mays) leaf protoplasts were isolated from various leaves of two-week (4-leaf) seedlings and from sections of the third leaf blades. Microtubules (MTs) were visualized using immunofluorescence microscopy. Only freshly isolated protoplasts from the third and fourth leaf blades contained MTs, with protoplasts from the fourth leaf containing the most i.e. 13% of fourth-leaf protoplasts contained MTs. In general, protoplasts with fewer and smaller chloroplasts had more MTs. Initially 90–95% of protoplasts from basal portions of leaves had MTs but the percentage decreased slightly during culture particularly after 10 days. The antioxidant n-propyl gallate was beneficial in maintaining MT content. Few protoplasts from older sections intitially contained MTs but in all sections at least some protoplasts regained a significant MT content during culture (e.g., 10% of protoplast from the tip section possessed microtubules after 7 days of culture). Far fewer MTs were observed in individual leaf protoplasts than those isolated from suspension culture.Abbreviations BMS Black Mexican Sweet - MT microtubule - MtSB microtubule stabilizing buffer - PBS phosphate buffered saline     

Osmotic stress, endogenous abscisic acid and the control of leaf morphology in Hippuris vulgaris L

Abstract. Previous reports indicate that heterophyllous aquatic plants can be induced to form aerial-type leaves on submerged shoots when they are grown in exogenous abscisic acid (ABA). This study reports on the relationship between osmotic stress (e.g. the situation encountered by a shoot tip when it grows above the water surface), endogenous ABA (as measured by gas chromatography-electron capture detector) and leaf morphology in the heterophyllous aquatic plant, Hippuris vulgaris. Free ABA could not be detected in submerged shoots of H. vulgaris but in aerial shoots ABA occurred at ca. 40ng (g fr wt)⁻¹. When submerged shoots were osmotically stressed ABA appeared at levels of 26 to 40ng (g fr wt)⁻¹. These and other data support two main conclusions: (1) Osmotically stressing a submerged shoot causes the appearance of delectable levels of ABA. (2) The rise of ABA in osmotically stressed submerged shoots in turn induces a change in leaf morphology from the submerged to the aerial form. This corroborates the hypothesis that, in the natural environment, ABA levels rise in response to the osmotic stress encountered when a submerged shoot grows up through the water/air interface and that the increased ABA leads to the production of aerial-type leaves.     

Plant regeneration from cultured leaves of Lavandula latifolia Medicus: Influence of growth regulators and illumination conditions

Leaves were obtained from 4-week-old seedlings of Lavandula latifolia Medicus grown in vitro. Leaf explants were then cultured on MS medium supplemented with different concentrations and combinations of the auxins IAA or NAA with the cytokinin BA and maintained under three illumination conditions, 16h photoperiod, darkness or darkness followed by a photoperiod, to assess morphogenic responses. Irrespective of illumination conditions, bud regeneration was achieved only in media containing BA or BA/auxin combinations, with the best results being obtained in the presence of BA and 0.06 or 0.6 M IAA or NAA. A photoperiod of 16h appeared to yield the best response in terms of bud regeneration percentage. High auxin concentrations (6.0 or 11.0 M) inhibited bud differentiation, especially when explants were cultured in darkness. On the other hand, low auxin levels and photoperiod improved shoot development. Excised shoots were induced to form roots by transfer to hormone-free MS medium with macronutrients at half strength. The obtained plantlets were ultimately grown in the greenhouse.Abbreviations BA benzyladenine - BM basal medium - IAA indoleacetic acid - MS Murashige & skoog - NAA -naphthaleneacetic acid     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

The effect of glucosinolates on responses of young Phyllotreta nemorum larvae to non-host plants

All recorded host plants of Phyllotreta nemorum L. (Coleoptera: Chrysomelidae) contain glucosinolates and belong to the plant families Brassicaceae (Cruciferae), Resedaceae and Capparaceae. The acceptability of 56 plant species from 28 other plant families (non-hosts) for young larvae has been studied in the laboratory. None of these species were fully acceptable for initiations of leaf mines when intact untreated leaves were presented, and only one species, Malva silvestris L. (Malvaceae), was partially acceptable. The acceptability of some species increased when leaf discs were presented instead of intact leaves; but the highest percentages of mine initiations occurred in leaf discs treated with the glucosinolate, sinigrin. A stimulatory effect of sinigrin could be demonstrated in experiments with 7 plant species: Papaver dubium L., Papaver rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. and Lactuca sativa L. The majority of species remained unacceptable even after treatment with glucosinolates.The main causes for these differences between plant species are supposed to be differences in contents of deterrents and/or other stimulants for mine initiation. These possibilities are discussed in relation to the content of allelochemicals in acceptable plants and the position of these plants in botanical classifications.

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Zusammenfassung Phyllotreta nemorum L. ist ein oligophager Erdfloh, der an Cruciferen und anderen Glukosinolat-haltigen Pflanzenarten gebunden ist. Die Imagines fressen Löcher in die Blätter und die Weibchen legen ihre Eier in den Boden. Die Larven sind Blattminierer. Nach dem Schlüpfen im Boden klettern sie an die Pflanzen hoch, und die Einbohrung und der Anfang der Minierung erfolgt in eines der unteren Blätter der Wirtspflanze.Die Wirkung von Glukosinolaten auf die Einbohrung von Junglarven in Pflanzenarten, die keine natürliche Inhalt von Glukosinolaten haben, ist in Laborexperimenten untersucht worden. 56 Pflanzenarten aus 28 Familien wurden präsentiert teils als unbehandelte Blätter und teils als Glukosinolatbehandelte Blattscheiben. Unbehandelte Blätter von nicht-Glukosinolathaltigen Arten waren immer unbefriedigend für die Larven. Nur in eine Art, Malva silvestris L. war die Frequenz der Einbohrung ein bisschen höher als 10%. Eine signifikante Erhöherung der Anzahl eingebohrten Larven nach der Sinigrin-behandlung erfolgte in 7 Pflanzenarten: Papaver dubium L., P. rhoeas L., Fumaria officinalis L., Malva silvestris L., Pisum sativum L., Campanula latifolia L. und Lactuca sativa L. Doch blieben die meisten Pflanzenarten (84%) auch nach der Sinigrin-Behandlung unbesiedelt.Pflanzenarten, die nach der Sinigrin-Behandlung nicht besiedelt werden enthalten vielleicht frasshemmende Stoffe, oder ihnen fehlen noch weitere Frass-stimulierende Stoffe. Diese Möglichkeiten werden diskutiert in Zusammenhang mit den Inhalt von Allelochemikalien in besiedelten Pflanzenarten und mit ihrer taxonomischer Position.

     

The development of the sensory organs of the legs in the blowfly,Phormia regina

Summary The development of the sensory neurons of the legs of the blowfly,Phormia regina has been described from the third instar larva to the late pupa using immunohistochemical staining. The leg discs of the third instar larva contain 8 neurons of which 5 come to lie in the fifth tarsomere of the developing leg. Whereas 2 neurons persist at least to the late pupa, the other cells degenerate. The first neurons of gustatory sensilla arise in the fifth tarsomere at about 1.5 h after formation of the puparium. Most of these sensilla, however, appear within a short time period beginning at about 18 h. The femoral chordotonal sensory neurons first appear at the time of formation of the puparium, as a mass of cells situated in the distal femur. During later pupal development 2 groups of these cells come to lie at the femur-trochanter border, where they become the proximal femoral chordotonal organ of the adult; the remaining cells become the distal femoral chordotonal organ. Other scolopidial neurons appear later in development. The nerve pathways of the late pupal leg are established either by the axons of the cells that are present in the larval leg disc or by new outgrowing processes of sensory neurons. In the tibia, the initial direction of new outgrowth differs in different regions of the segment: proximal tibial neurons grow distally, while distal tibial neurons grow initially proximally.     

Anatomy of the hominoid wrist joint: Its evolutionary and functional implications

Observations on the behavior of living hominoids show generic differences in the use and posture of the wrist joint. Both orang-utans and hylobatids usually use the wrist in suspensory behaviors. However, orang-utans emphasize markedly adducted and flexed wrist postures, while hylobatids emphasize violent forearm and wrist rotation. African apes, especially the gorilla, use the wrist more frequently than other hominoids for terrestrial quadrupedal weight-bearing. Humans use the wrist less frequently for supportive purposes than do other hominoids. These behavioral differences correspond to structural specializations in the proximal carpal joint of each of the hominoid genera. Although each of the hominoid genera has apparently modified its proximal carpal joint best to serve its characteristic behaviors, all hominoids share a unique proximal carpal joint that permits approximately 160ℴ of forearm rotation. The hylobatid proximal carpal joint is specialized in exhibiting a marked development of those structures limiting forearm rotation, but it is in most respects the least derived— that is, closest to the nonhominoid anthropoids. Chimpanzees show a proximal carpal joint that is more generalized than those of the other great apes but more derived than that of hylobatids. The human and gorilla proximal wrist joints, on the other hand, show marked modifications for weight-bearing in terrestrial behaviors. Orang-utans have the most derived proximal carpal joint, which in many respects parallels that of the slow-climbing nonhominoid primates. The comparative anatomy and structural specializations of the wrist joint support (a) an early divergence of hylobatids from the common hominoid stock, (b) a common ancestry for gorillas and humans separate from the other hominoids, and (c) a long independent evolutionary period for orang-utans since their divergence from the common hominoid stock, or one that was marked by strong selection pressures for wrist specializations. Unfortunately, the generalized condition of the chimpanzee’s wrist joint and the very derived condition of the orang-utan wrist provide uncertain evidence as to which of the two was first to diverge from the common hominoid stock. Identification of hominoid wrist specializations as reflecting real phylogenetic relationships or parallelisms depends on how well the phytogeny inferred from wrist morphology accords with those arrived at from the study of other systems.     

Leaf age and larval performance of the leaf beetle Paropsis atomaria

ABSTRACT.

• 1 Larval performance of the leaf beetle Paropsis atomaria Oliver was determined for larvae raised on both new and mature leaves of Eucalyptus blakelyi Maiden. Larvae were transferred to mature leaves at different ages; control larvae stayed on new leaves through all instars.
• 2 Only larvae reared on new leaves through the third instar survived to pupate on mature leaves; developmental time was prolonged by 20% and pupal weight was reduced by 50% in these larvae compared with larvae reared entirely on new leaves. Almost all larvae died when transferred to mature leaves as first, second or third instars.
• 3 Low survival and slow development on mature leaves was mainly due to failure by larvae to feed. Larvae palpated leaves and could discriminate among leaf ages immediately, without biting into the leaf tissue.
• 4 New leaves had higher concentrations of oil and tannins than old leaves, while there were no significant differences in nitrogen concentrations in the two types of leaves. Mature leaves were more than 3 times tougher than new leaves, in terms of g mm^?2 of penetrometer force.
• 5 In drought years E. blakelyi may not produce sufficient new leaves to supply specialist herbivores with their preferred food resource. We infer that drought years reduce P. atomaria larval performance significantly, and influence the population dynamics of the insect.

     

Proteolysis of ribulose-1,5-bisphosphate carboxylase/oxygenase in leaves of Citrus explants throughout the annual cycle and its regulation by ethylene

Leaf senescence and ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBP carboxylase, EC 4.1.1.39) degradation in orange [ Citrus sinensis (L.) Osbeck cv. Washington Navel] explants have been investigated. Explants consisted of a segment of stem (ca 15 cm) and 5 mature leaves. In vitro RuBP carboxylase degradation was determined by culturing the explants in water for different periods of time (3 days usually) and quantifying the two RuBP carboxylase subunits in the extracts following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In vitro RuBP carboxylase degradation was estimated by autodigestion of leaf extracts and SDS-PAGE. The extent of in vivo RuBP carboxylase degradation in explants cultured under 16 h light/8 h dark photoperiod varied throughout the year and showed a cyclic behaviour correlated with the growth cycle of Citrus. The highest proteolytic activity both in vivo and in vitro was found in explants made from April to August coinciding with the maximum vegetative growth period of the tree.
Leaf senescence and abscission could be retarded significantly at any time of the year by maintaining the explants continuously in the dark. Treatment of the explants in the dark with a continuous flow of ethylene enhanced both leaf abscission and rate of RuBP carboxylase degradation, proportionally to ethylene concentration (0.1-0.6 ppm). Ethylene-induced senescence of Citrus leaf explants in the dark appears to be a convenient model system to study the regulation of the proteolytic degradation of RuBP carboxylase.