Changes in Isoperoxidases during Cold Treatment of Dormant Pear Embryo

The number of isoperoxidases and the intensity of certain isozymes increased with increasing periods of stratification of pear (Pyrus communis cv. Bartlett) embryos. The presence of GA₃ or kinetin during stratification enhanced the activity of certain isoperoxidases, and these enhancements were blocked in the presence of ABA which by itself had an inhibitory effect. Enhancement in isoperoxidases of pear embryos during stratification was inhibited by 6-methylpurine and cycloheximide; and in the presence of either of these two inhibitors, stratification failed to release the dormancy. Pear embryos germinated for 3 days showed changes in the pattern of isoperoxidases depending on the length of stratification.     

Rejuvenation of Sequoia sempervirens in Vitro: Changes in Isoesterases and Isoperoxidases

Rejuvenation, as evidenced by restored rooting competence, ofSequoia sempervirens by a series of repeated grafting of adultshoot tips onto juvenile rootstocks in vitro was confirmed.Complete restoration of rooting competence was achieved aftergrafting repeatedly five times. The rejuvenation was correlatedwith a disappearance of adult-associated esterase and peroxidaseisozymes and an appearance of isoesterases and isoperoxidasesthat were characteristic of juvenile-phase shoots. These esteraseand peroxidase isozymes could serve as markers to assist phasechangeinvestigations. (Received May 8, 1995; Accepted November 6, 1995)     

Alkaloid Changes in Tobacco Seeds during Germination

Nicotine, nornicotine, anabasine, and anatabine, normally found in growing and mature tobacco (Nicotiana tabacum L.) plants, were extracted and quantified from mature tobacco seeds and young tobacco seedlings. The rate of net alkaloid disappearance and accumulation in tobacco seedlings was related to phases of germination.     

Impact of in vitro Cultivation Conditions on Stress Responses and on Changes in Thylakoid Membrane Proteins and Pigments of Tobacco during ex vitro Acclimation

Four physiologically and phenotypically diversified tobacco (Nicotiana tabacum L. cv. Samsun) plantlet variants had been generated by cultivation on media either lacking or containing sucrose (0 and 3 %, m/v) under two different photon flux densities (PFD), 50 µmol m^–2 s^–1 (LL) and 200 µmol m^–2 s^–1 (HL). Plantlets were transferred into soil without any pre-acclimation and grown either under PFD of 200 µmol m^–2 s^–1 or 700 µmol m^–2 s^–1. Sucrose feeding in vitro resulted in reduced degree and duration of wilting after transfer. The highest readiness for ex vitro acclimation was found in 3 % HL plants, in which changes of photosynthetic apparatus and stress responses were the smallest. On the contrary, the steepest decline of Fv/Fm ratio on the first day after transplantation, doubled chlorophyll content and almost tripled D1/LHC 2 ratio after 7 d of ex vitro growth under 700 µmol m^–2 s^–1 characterized 0 % HL plants, which had suffered chronic photoinhibition in vitro. Remarkably high abscisic acid content at the end of in vitro cultivation and during acclimation as well as increased synthesis of both D1 and LHC 2 proteins even at the end of analyzed acclimation period were found only in 0 % LL plants. Increase of D1/LHC 2 ratio and chlorophyll contents demonstrate that in vitro developed leaves of all plant variants are able to acclimate to new environment. The most surprising result in the whole study is the drop of D1 protein synthesis in all plants on the 3^rd day. Five times decline of photoprotection level of xanthophylls in plants after ex vitro transfer into the same PFD showed stress character of in vitro cultures.     

Isolation of Tobacco Isoperoxidases Accumulated in Cell-Suspension Culture Medium and Characterization of Activities Related to Cell Wall Metabolism

All of the most important guaiacol-type peroxidase (POX) isoforms accumulated in the culture medium of BY-2 tobacco (Nicotiana tabacum L. cv Bright Yellow 2) cells have been isolated. Five basic and two acidic isoforms were found. The four major isoforms (B2, B3, P1, and P2), all strongly basic, have been purified to homogeneity and partially sequenced. B2 and B3 are new isoforms showing high homology to only one POX isolated so far. Amino acid sequencing and specific activities indicated that basic isoPOXs constitute two pairs of strictly related isoforms (P1/P2 and B2/B3). Their specific activities measured in the presence of different substrates, as monolignols and NAD(P)H, indicated possible specialized functions in cell wall metabolism. Only P-type POXs were able to oxidize indoleacetic acid. Variations in pH could play a regulatory role by changing the relative contribution of different isoforms to total POX activity. Apart from cell culture medium, polyclonal antibodies obtained against P1 and P2 detected P1 in roots and in lower parts of stems. Immunocytochemical labeling indicated that P-type POXs were expressed in stem phloem and in phloem and epidermal cells of roots.     

Changes in Cell and Organ Shape during Early Development of the Ocular Lens

It is currently fashionable to attribute changes in organ shapeduring development to the actions of microtubules and microfilamentson individual cells of the organ in question. In the case ofthe eye lens it has been proposed that cellular elongation underthe influence of microtubules and/or apical contraction by microfilamentsare responsible for the remodeling of the originally low cuboidalectoderm into the tall and wedge-shaped presumptive lens cells.Invagination of the lens is thought to follow automatically.These ideas cannot account for certain observations on lensmorphogenesis, such as the relatively fixed diameter of theorgan rudiment during early development, which is incompatiblewith the supposed contraction of the rudiment. We found that the area of contact between presumptive lens andoptic cup does become fixed after a few hours of "induction."There is a remarkable correlation in time between this fixation,and the process of lens cell elongation and increase in lenscell density. We calculated that the latter two can, in fact,be accounted for by population pressure caused by continuedcell division within the defined area of the lens rudiment.A mathematical model along these lines was developed, whichexplains lens invagination on the basis of cell number and size,extent of the area of contact between ectoderm and optic cup,and cell population doubling times. We hypothesize that the prevention of lateral cell spreadingwithin the lens territory, after the contact area becomes fixed,is a function of the build-up in extracellular materials inthis area during the "induction period." Both lens rudimentand presumptive retina contribute to this extracellular matrix.     

Glycosaminoglycans and Epithelial Organ Formation

The concept that extracellular matrix materials are involvedin the morphogeuetic process is supported by substantial indirectevidence. Essential morphogenetically active materials are obscurewith regard to their nature, their mode of action, and whetherthey are causally involved in tissue interactions. Studies are presented indicating that glycosaminoglycans arecomponents of embryonic epithelial basal laminae, and that materialswithin the basal lamina which are, at least in part, glycosaminoglycanare required for establishing and maintaining braching epithelialmorphogenesis. The tissue of origin and molecular nature ofbasal laminar glycosaminoglycan are described and speculationsare made regarding its possible mode of action in the contextof a model for branching morphogenesis.     

Changes in Stigma-Specific Lipids of Tobacco Plant during Flower Development

Tobacco stigma contained multiacyl glycerides having -hydroxyfatty acids in their molecules. These compounds were not detectedin other organs; leaf, pith, root, petal, ovary, anther andseed. The content of multiacyl glycerides in stigma increaseduntil anthesis and then decreased. Three lipid fractions correspondingto triacylglycerol, diacylglycerol and polar lipid containedsignificant amounts of -hydroxy fatty acids, oleic acid--OHand linoleic acid--OH. The amount of the triacylglycerol fractionwas the largest. Although -hydroxy fatty acids were detectedin the polar lipid fraction, the compounds did not appear inthe fractions corresponding to phosphatidylcholine, phosphatidylethanolamine,phosphatidylinositol, phosphatidylglycerol, monogalactosyldiglyceride,digalactosyldiglyceride and sulfoquinovosyldiglyceride. The-hydroxy fatty acids were contained in both surface and cytoplasmiclipids of stigma at all stages of flower development. (Received September 16, 1982; Accepted December 20, 1982)     

Non-autotrophic CO2 Fixation during Shoot Formation in Tobacco Callus

Non-autotrophic carbon fixation has been studied during growthof tobacco callus cultured in dark under shoot-forming (SF)and non-shoot-forming (NSF) conditions. The enzymes involvedin malate metabolism—phosphoenolpyruvate carboxylase,malic dehydrogenase, glutamic-oxalacetic transaminase, and malicenzyme—increased sharply during the first 4 d of cultureparticularly in SF tissue. The activities of the enzymes studiedwere considerably greater in SF than in NSF tissue. There wasa dramatic increase in malate content in SF tissue during thefirst 4 d of culture. Subsequently malate was rapidly depletedduring the time of organogenesis. In NSF tissue there was acontinuous build-up of malate content throughout the cultureperiod. We suggest that malate derived from dark fixation ofCO₂ plays differing roles in NSF (callus) and SF tissues. Inthe former, malate acts primarily as an osmotic solute regulating,at least in part, cell expansion between successive cell divisions.In shoot-forming tissue, on the other hand, malate preferentiallyprovides NADPH for reductive biosynthesis.     

Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.     

Changes in Endogenous Gibberellin Contents during Growth of Cultured Tobacco Cells

Changes in the contents of endogenous gibberellins (GAs) wereexamined in three kinds of cultured tobacco cells; a crown gallcell and two cultured cells derived from normal tissue of Nicotianatabacum. The relative amounts of the GAs were analyzed by systematicchromatographic purifications followed by GC-SIM. In all thecell lines examined, the content of GAj was the highest duringthe logarithmic phase of growth, indicating that GA₁ has a physiologicalrole in the growth of dedifferentiated cells. ³ Present address: College of Agriculture, Chonnam NationalUniversity, Kwangju 500, Korea. (Received April 11, 1984; Accepted July 10, 1984)     

Changes in Nucleic Acids and Sugars of Tobacco Leaves during Maturity Stage and Flue-curing

Changes in the contents of nucleic acids and sugars in tobacco leaves during maturity stage and flue-curing were investigated. The content of RNA continued to decrease during both periods and is lower in lower leaves on the stalk. However, a temporary reverse trend was observed after topping. A sharp decrease after topping in ribonuclease activity was followed by an increase in RNA several days later, and thereafter soluble protein also increased temporarily in maturity stage.

Sucrose, glucose and fructose decreased temporarily after topping and then increased gradually in the latter stage of maturity, followed by an abrupt increase in the yellowing stage of flue-curing. Minor sugars, i.e. maltose, ribose, mannose, galactose and xylose were determined quantitatively in tobacco leaves. However, these sugars have not shown any remarkable changes during maturity stage and flue-curing.