Superoxide production by thylakoids during chilling and its implication in the susceptibility of plants to chilling-induced photoinhibition

Factors influencing the rate of superoxide (O ₂ ^- ) production by thylakoids were investigated to determine if increased production of the radical was related to injury induced by chilling at a moderate photon flux density (PFD). Plants used were Spinacia oleracea L., Cucumis sativus L. and Nerium oleander L. grown at either 200° C or 45° C. Superoxide production was determined by electron-spin-resonance spectroscopy of the (O ₂ ^- )-dependent rate of oxidation of 2-ethyl-1-hydroxy-2,5,5-trimethyl-3-oxazolidine (OXANOH) to the corresponding oxazolidinoxyl radical, OXANO ·. For all plants, the steady-state rate of O ₂ ^- production by thylakoids, incubated at 25° C and 350 mol photon · m^–2 · s^–1 (moderate PFD) with added ferredoxin and NADP, was between 7.5 and 12.5 mol · (mg chlorophyll)^–1 · h^–1. Incubation at 5° C and a moderate PFD, decreased the rate of O ₂ ^- production 40% and 15% by thylakoids from S. oleracea and 20° C-grown N. oleander, chillinginsensitive plants, but increased the rate by 56% and 5% by thylakoids from C. sativus and 45° C-grown N. oleander, chilling-sensitive plants. For all plants, the addition of either ferredoxin or methyl viologen increased the rate of O ₂ ^- -production at 25° C by 75–100%. With these electron acceptors, lowering the temperature to 5° C caused only a slight decrease in O ₂ ^- production. In the absence of added electron acceptors, thylakoids produced O ₂ ^- at a rate which was about 45% greater than that when ferredoxin and NADP were present. The addition of 3-(3,4-dichlorophenyl)-1,1-dimethylurea reduced O ₂ ^- production under all conditions tested. The results show that the rate of O ₂ ^- production increases in thylakoids when the rate of electron transfer to NADP is reduced. This could explain differences in the susceptibility of thylakoids from chilling-sensitive and chilling-insensitive plants to chilling at a moderate PFD, and is consistent with the proposal that O ₂ ^- production is involved in the injury leading to the inhibition of photosynthesis induced under these conditions.Abbreviations Chl chlorophyll - DCMU 3-(3,4-dichlorophen-yl)-1,1-dimethylurea - Fd ferredoxin - MV methyl viologen - 20°oleander Nerium oleander grown at 20° C - 45°-oleander N. oleander grown at 45° C - OXANOH 2-ethyl-1-hydroxy-2,5,5-tri-methyl-3-oxazolidine - PFD photon flux density (photon fluence rate) - TEMED tetramethyl ethylenediamine We would like to thank R.T. Furbank, R.S.B.S., Australian National University, Canberra, A.C.T., and C.B. Osmond, now of Duke University, Durham, N.C., USA, for the gift of ferredoxin, R.A.J.H. was supported by a Commonwealth Postgraduate Research Award.     

Studies on the biosynthesis of tropane alkaloids in Datura stramonium L. transformed root cultures

The relative contributions made by the l-arginine/agmatine/N-carbamoylputrescine/putrescine and the l-ornithine/putrescine pathways to hyoscyamine formation have been investigated in a transformed root culture of Datura stramonium. The activity of either arginine decarboxylase (EC 4.1.1.19) or ornithine decarboxylase (EC 4.1.1.17) was suppressed in vivo by using the specific irreversible inhibitors of these activities, dl--difluoromethylarginine or dl--difluoromethylornithine, respectively. It was found that suppression of arginine decarboxylase resulted in a severe decrease in free and conjugated putrescine and in the putrescine-derived intermediates of hyoscyamine biosynthesis. In contrast, the suppression of ornithine decarboxylase activity stimulated an elevation of arginine decarboxylase and minimal loss of metabolites from the amine and alkaloid pools. The stimulation of arginine decarboxylase was not, however, sufficient to maintain the same potential rate of putrescine biosynthesis as in control tissue. It is concluded that (i) in Datura the two routes by which putrescine may be formed do not act in isolation from one another, (ii) arginine decarboxylase is the more important activity for hyoscyamine formation, and (iii) the formation of polyamines is favoured over the biosynthesis of tropane alkaloids. An interaction between putrescine metabolism and other amines is also indicated from a stimulation of tyramine accumulation seen at high levels of dl--difluoromethylornithine.Abbreviations ADC arginine decarboxylase - DFMA dl--dif-luoromethylarginine - DFMO dl--difluoromethylornithine - MPO N-methylputrescine oxidase - ODC ornithine decarboxylase - PMT putrescine N-methyltransferase We are indebted to Dr. E.W.H. Bohme of Merrell Dow Research Laboratories (Cincinnati, Ohio, USA) for kind gifts of DFMO and DFMA and to Dr. M.J.C. Rhodes for helpful advice and discussion.     

Regulation of chloroplast metabolism in leaves: Evidence that NADP-dependent glyceraldehydephosphate dehydrogenase,but not ferredoxin-NADP reductase,controls electron flow to phosphoglycerate in the dark-light transition

P₇₀₀ is rapidly, but only transiently photooxidized upon illuminating dark-adapted leaves. Initial oxidation is followed by a reductive phase even under far-red illumination which excites predominantly photosystem (PS) I. In this phase, oxidized P₇₀₀ is reduced by electrons coming from PSII. Charge separation in the reaction center of PSI is prevented by the unavailability of electron acceptors on the reducing side of PSI. It is subsequently made possible by the opening of an electron gate which is situated between PSI and the electron acceptor phosphoglycerate. Electron acceptors immediately available for reduction while the gate is closed corresponded to 10 nmol · (mg chlorophyll)^–1 electrons in geranium leaves, 16 nmol · (mg chlorophyll)^–1 in sunflower and 22 nmol · (mg chlorophyll)^–1 in oleander. Reduction of NADP during the initial phase of P₇₀₀ oxidation showed that the electron gate was not represented by ferredoxin-NADP reductase. Availability of ATP indicated that electron flow was not hindered by deactivation of the thylakoid ATP synthetase. It is concluded that NADP-dependent glyceraldehydephosphate dehydrogenase is completely deactivated in the dark and activated in the light. The rate of activation depends on the length of the preceding dark period. As chloroplasts contain both NAD- and NADP-dependent glyceraldehydephosphate dehydrogenases, deactivation of the NADP-dependent enzyme disconnects chloroplast NAD and NADP systems and prevents phosphoglycerate reduction in the dark at the expense of NADPH and ATP which are generated by glucose-6-phosphate oxidation and glycolytic starch breakdown, respectively.Abbreviations Chl chlorophyll - P₇₀₀ electron donor pigment in the reaction center of photosystem I Cooperation of the Institute of Botany of the University of Würzburg with the Institute of Astrophysics and Atmospheric Physics of the Estonian Academy of Sciences in Tartu was supported by the Deutsche Forschungsgemeinschaft and the Estonian Academy of Sciences. This work was performed within the Sonderforschungsbereich 251 of the University of Würzburg.     

Determination for inflorescence development is a stable state,separable from determination for flower development in Pisum sativum L. buds

Terminal meristems of Pisum sativum (garden pea) transit from vegetative to inflorescence development, and begin producing floral axillary meristems. Determination for inflorescence development was assessed by culturing excised buds and meristems. The first node of floral initiation (NFI) for bud expiants developing in culture and for adventitious shoots forming on cultured meristems was compared with the NFI of intact control buds. When terminal buds having eight leaf primordia were excised from plants of different ages (i.e., number of unfolded leaves) and cultured on 6-benzylaminopurine and kinetin-supplemented medium, the NFI was a function of the age of the source plant. By age 3, all terminal buds were determined for inflorescence development. Determination occurred at least eight nodes before the first axillary flower was initiated. Thus, the axillary meristems contributing to the inflorescence had not formed at the time the bud was explanted. Similar results were obtained for cultured axillary buds. In addition, meristems excised without leaf primordia from axillary buds three nodes above the cotyledons of age-3 plants gave rise to adventitious buds with an NFI of 8.3 ±0.3 nodes. In contrast seed-derived plants had an NFI of 16.5 ±0.2. Thus cells within the meristem were determined for inflorescence development. These findings indicate that determination for inflorescence development in P. sativum is a stable developmental state, separable from determination for flower development, and occurring prior to initiation of the inflorescence at the level of meristems.     

Oscillations in photosynthesis are initiated and supported by imbalances in the supply of ATP and NADPH to the Calvin cycle

Oscillations in the rate of photosynthesis of sunflower (Helianthus annuus L.) leaves were induced by subjecting leaves, whose photosynthetic apparatus had been activated, to a sudden transition from darkness or low light to high-intensity illumination, or by transfering them in the light from air to an atmosphere containing saturating CO₂. It was found that at the first maximum, light-and CO₂-saturated photosynthesis can be much faster than steady-state photosynthesis. Both Q_A in the reaction center of PS II and P₇₀₀ in the reaction center of PS I of the chloroplast electron-transport chain were more oxidized during the maxima of photosynthesis than during the minima. Maxima of P₇₀₀ oxidation slightly preceded maxima in photosynthesis. During a transition from low to high irradiance, the assimilatory force F_A, which was calculated from ratios of dihydroxyacetone phosphate to phosphoglycerate under the assumption that the reactions catalyzed by NADP-dependent glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase and triosephosphate isomerase are close to equilibrium, oscillated in parallel with photosynthesis. However, only one of its components, the calculated phosphorylation potential (ATP)/(ADP)(P_i), paralleled photosynthesis, whereas calculated NADPH/NADP ratios exhibited antiparallel behaviour. When photosynthetic oscillations were initiated by a transition from low to high CO₂, the assimilatory force F_A declined, was very low at the first minimum of photosynthesis and increased as photosynthesis rose to its second maximum. The observations indicate that the minima in photosynthesis are caused by lack of ATP. This leads to overreduction of the electron-transport chain which is indicated by the reduction of P₇₀₀. During photosynthetic oscillations the chloroplast thylakoid system is unable to adjust the supply of ATP and NADPH rapidly to demand at the stoichiometric relationship required by the carbonreduction cycle.Abbreviations PGA 3-phosphoglycerate - DHAP dihydroxyacetone phosphate - P₇₀₀ electron-donor pigment in the reaction enter of PS I - Q_A quinone acceptor in the reaction center of PS II This work received support from the Estonian Academy of Sciences, the Bavarian Ministry of Science and Art and the Sonderforschungsbereich 251 of the University of Würzburg. We are grateful for criticism by D.A. Walker, Robert Hill Institute, University of Sheffield, U.K. and by Mark Stitt, Institute of Botany, University of Heidelberg, FRG.     

Electron-microscopic cytochemical localization of diamine and polyamine oxidases in pea and maize tissues

An electron-microscopic cytochemical method was used to localize diamine oxidase (DAO) in pea and polyamine oxidase (PAO) in maize (Zea mays L.). The method, based on the precipitation of amine-oxidase-generated H₂O₂ by CeCl₃, was shown to be specific for DAO and PAO and permitted their localization in plant tissues with a high degree of resolution. Both enzymes are localized exclusively in the cell wall. Both DAO- and PAO-activity staining is most intense in the middle lamellar region of the wall and in cells exhibiting highly lignified walls. The oxidases could provide H₂O₂ for peroxidase-mediated cross-linking reactions in the cell wall and may, in this capacity, play a role in the regulation of plant growth.Abbreviations AG 1-aminoguanidine - AT 3-amino-1,2,4-triazole - -HEH -hydroxyethylhydrazine - DAO(s) diamine oxidase(s) - PAO(s) polyamine oxidase(s) - Put putrescine - Spd spermidine - Spm spermine The authors wish to thank Nancy Piatczyc for the technical assistance with electron-microscopy studies. We are grateful to Dr. Stanley J. Roux, University of Texas at Austin, for providing us with samples of maize cell-wall exudates. This work was supported by grants to R.D.S from the National Aeronautics and Space Administration (NAGW-1049 and NAGW-1382).     

Rates of corolla growth in tobacco determined with the plastochron index

The growth of vegetative and reproductive shoots of Nicotiana tabacum L. cv. Xanthi is analyzed with the plastochron index to estimate the relationship between corolla growth and time. The plastochron of leaves 9 through 20 declines steadily at each successive node. The flower plastochron increases steadily during the growth of an individual cyme, with the most distal flower to open having the longest plastochron. Variation in the flower plastochron is the result of variation in the rate of flower initiation, not the growth rate of individual flowers. The corolla has an extended phase of approximately constant relative growth in length (between 0.2 · d^–1 and 0.3 · d^–1) until a peak of growth (0.5 · d^–1) 2–3 d before anthesis. Corollas also have periodic peaks and troughs of growth that are low in amplitude (0.1 · d^–1), but persist throughout most of corolla development. The pattern of corolla expansion contrasts strongly with earlier reports of the pattern of tobacco leaf growth.Abbreviations PI plastochron index - PR plastochron ratio - RGR relative growth rate in length The authors thank: Drs. T. Sage and E.G. Williams for the considerable time and space they invested; the members of Dr. R. Wyatt's laboratory for allowing us to use their computer facilities; A. Tull and M. Smith for their care taken in the green-house. This research was supported by U.S. Department of Agriculture grant GAM-89-01056 and by National Science Foundation grant DCB-87-15799.     

The changing sensitivity to auxin of the plasma-membrane H+-ATPase: Relationship between plant development and ATPase content of membranes

The auxin sensitivity of the plasma-membrane H⁺-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H⁺-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H⁺-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H⁺-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA enzyme-linked immunosorbent assay - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T.     

Cytology of induced systemic resistance of cucumber to Colletotrichum lagenarium

The infection of cucumber leaves by Colletotrichum lagenarium was studied using cytological methods. Its progress in untreated plants was compared with that in plants in which systemic resistance had been induced by pre-infecting the first true leaf with the same fungus. In induced plants, a reduction of fungal development was observed at the leaf surface, in the epidermis, and in the mesophyll. On the leaf surface, formation of appressoria was slightly reduced. In the epidermis, enhanced formation of papillae beneath appressoria, and possibly increased lignification of entire cells, correlated with reduced development of infection hyphae. Papillae contained callose, identified by staining with aniline-blue fluorochrome and digestion with -1,3-glucanase, as a main structural component. In the mesophyll, reduced fungal development provided evidence for the existence of an additional induced defence reaction. The results imply that preinfection elicited a systemic, multicomponent defence reaction of the host plant against the fungus.Dedicated to the memory of Professor H. Grisebach