Ferritin in bean leaves with constant and changing iron status

Normal green leaves contain low levels of ferritin which stores 5 to 10% of the total iron (approximately 350 iron atoms/molecule). Chlorotic leaves do not have measurable amounts of ferritin, whereas iron-loaded leaves contain high levels of well-filled ferritin (1,500 to 2,500 iron atoms/molecule). The role of ferritin during a transient iron surplus in leaves was investigated. It is suggested that a short-term overdose of iron transported into the leaf is largely stored in or near the vessels in such a form that it can be quickly mobilized for export. Iron that reaches the mesophyll cells in an overdose situation is stored in ferritin and, when released, is most likely used for the leaf cells themselves and not for export.     

In-vitro translation of messenger-RNA from developing bean leaves. Evidence for the existence of stored messenger-RNA and its light-induced mobilisation into polyribosomes

Poly(A)-containing messenger RNA was purified from polyribosomes isolated from the primary leaves of 7-day-old dark-grown seedlings of Phaseolus vulgaris var. Masterpiece. Analysis of the messenger RNA on 2.4% polyacrylamide gels showed that it consists of a heterogeneous population of molecules with an average molecular weight of 500,000. The nucleotide composition of the RNA was 16.0% cytidylic acid, 39.4% adenylic acid, 21.3% guanylic acid and 23.2% uridylic acid. Based on the degree of resistance of the RNA to digestion with ribonucleases A and T₁ the average length of the poly(A) sequence was calculated to be 120 nucleotides. No significant differences in mobility in polyacrylamide gels, nucleotide composition or polyadenylic acid content were found between the poly(A)-containing mRNA from polyribosomes of primary leaves of dark-grown plants and those given a 16 h white light treatment. Purified poly(A)-containing mRNA was shown to direct the incorporation of [³⁵S]methionine into proteins in an in vitro protein-synthesising system from wheat germ. The protein products were fractionated according to molecular size by electrophoresis in 15% polyacrylamide/urea/SDS gels and the protein bands were detected by fluorography. Messenger RNAs directing the synthesis of three polypeptides with molecular weights of 34,000, 32,000 and 25,000 were detected in polyribosomes of plants following white light treatment. These messenger RNAs were absent, or present in much lower amounts, in polyribosomal messenger RNA from leaves of dark-grown plants, although they were present in total cell poly(A)-containing RNA. This indicates that certain messenger RNAs may be stored in the dark and that light stimulates these RNAs to engage in polyribosome formation. Continuous far-red (730 nm) irradiation for 4 h also caused the appearance of these messenger RNAs in the polyribosomes although 5 min red light followed by 4 h darkness had little effect. This suggests that phytochrome acting in the high energy mode, may be the photoreceptor responsible for initiating the response.Abbreviations mRNA messenger-RNA - rRNA ribosomal RNA - oligo (dT) oligo (deoxythymidylic acid) - poly(A) polyadenylic acid - EDTA ethylenediamine-tetra-acetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulphonic acid - SDS sodium dodecyl sulphate     

Immunocytochemical localization of reserve protein in the endoplasmic reticulum of developing bean (Phaseolus vulgaris) cotyledons

The ultrastructure of the storage parenchyma cells of the cotyledons of developing bean (Phaseolus vulgaris L.) seeds was examined in ultrathin frozen sections of specimens fixed in a mixture of glutaraldehyde, formaldehyde and acrolein, infused with 1 M sucrose, and sectioned at-80° C. Ultrastructural preservation was excellent and the various subcellular organelles could readily be identified in sections which had been stained with uranyl acetate and embedded in Carbowax and methylcellulose. The cells contained large protein bodies, numerous long endoplasmic reticulum cisternae, mitochondria, dictyosomes, and electron-dense vesicles ranging in size from 0.2 to 1.0 m. Indirect immunolabelling using rabbit immunoglobulin G against purified phaseolin (7S reserve protein), and ferritin-conjugated goat immunoglobulin G against rabbit immunoglobulin G was used to localize phaseolin. With a concentration of 0.1 mg/ml of anti-phaseolin immunoglobin G, heavy labeling with ferritin particles was observed ober the protein bodies, the cisternae of the endoplasmic reticulum, and the vesicles. The same structures were lightly labeled when the concentration of the primary antigen was 0.02 mg/ml. Ferritin particles were also found over the Golgi bodies. The absence of ferritin particles from other organelles such as mitochondria and from areas of cytoplasm devoid of organelles indicated the specificity of the staining, especially at the lower concentration of anti-phaseolin immunoglobulin G.Abbreviations ER endoplasmic reticulum - IgG immunoglobulin G     

Separation of cell enlargement and division in bean leaves

A method is presented for inducing cell enlargement in intact leaves and leaf strips of Phaseolus vulgaris L. without the complication of cell division. Primary bean leaves complete cell division and stop growing after 10 d in dim red light. Transfer to white light induces expansion (50% in 24 h) which is entirely the consequence of cell enlargement. Leaf strips from red-light-grown seedlings placed in white light and provided external solutes (10 mM KCl+10 mM sucrose) expand at the same rate as intact leaves in the light. This system makes possible future investigation of the mechanism of leaf cell enlargement.     

Photoinhibition of photosynthesis in intact bean leaves: role of light and temperature,and requirement for chloroplast-protein synthesis during recovery

Photoinhibition of photosynthesis was induced in intact leaves of Phaseolus vulgaris L. grown at a photon flux density (PFD; photon fluence rate) of 300 mol·m^-2·s^-1, by exposure to a PFD of 1400 mol·m^-2·s^-1. Subsequent recovery from photoinhibition was followed at temperatures ranging from 5 to 35°C and at a PFD of either 20 or 140 mol·m^-2·s^-1 or in complete darkness. Photoinhibition and recovery were monitored mainly by chlorophyll fluorescence emission at 77K but also by photosynthetic O₂ evolution. The effects of the protein-synthesis inhibitors, cycloheximide and chloramphenicol, on photoinhibition and recovery were also determined. The results demonstrate that recovery was temperature-dependent with rates slow below 15°C and optimal at 30°C. Light was required for maximum recovery but the process was light-saturated at a PFD of 20 mol·m^-2·s^-1. Chloramphenicol, but not cycloheximide, inactivated the repair process, indicating that recovery involved the synthesis of one or more chloroplast-encoded proteins. With chloramphenicol, it was shown that photoinhibition and recovery occurred concomitantly. The temperature-dependency of the photoinhibition process was, therefore, in part determined by the effect of temperature on the recovery process. Consequently, photoinhibition is the net difference between the rate of damage and the rate of repair. The susceptibility of chilling-sensitive plant species to photoinhibition at low temperatures is proposed to result from the low rates of recovery in this temperature range.Abbreviations and symbols Da Dalton - Fo, Fm, Fv instantaneous, maximum, variable fluorescence emission - PFD photon flux density - PSII photosystem II - photon yield C.I.W.-D.P.B. Publication No. 871     

Proton excretion and cell expansion in bean leaves

Light-induced expansion of Phaseolus vulgaris L. leaf cells is accompanied by increased cell-wall plasticity. The possibility that leaf-cell walls are loosened by excreted protons has been investigated. First, light causes acidification, detected at the leaf surface, within 5–15 min. Growth starts 10–20 min after exposure to light. Second, exogenous acid induces loosening of isolated leaf cell walls. Third, infiltration of the tissue with a neutral buffer inhibits light-induced growth. Fourth, fusicoccin stimulates growth of as well as H⁺ excretion by bean leaf cells, without light. These findings show that the acid-growth theory is applicable to light-induced growth of leaf cells, and indicate that light-induced proton excretion initiates cell enlargement in leaves.Abbreviations FC fusicoccin - RL red light - WEx wall extensibility - WL white light     

The relationship between steady-state gas exchange of bean leaves and the levels of carbon-reduction-cycle intermediates

The relationship between the gas-exchange characteristics of attached leaves of Phaseolus vulgaris L. and the pool sizes of several carbon-reduction-cycle intermediates was examined. After determining the rate of CO₂ assimilation at known intercellular CO₂ pressure, O₂ pressure and light, the leaf was rapidly killed (<0.1 s) and the levels of ribulose-1,5-bisphosphate (RuBP), 3-phosphoglyceric acid (PGA), fructose-1,6-bisphosphate, fructose-6-phosphate, glucose-6-phosphate, glyceraldehyde-3-phosphate, and dihydroxyacetone phosphate were measured. In 210 mbar O₂, photosynthesis appeared RuBP-saturated at low CO₂ pressure and RuBP-limited at high CO₂ pressure. In 21 mbar (2%) O₂, the level of RuBP always appeared saturating. Very high levels of PGA and other phosphate-containing compounds were found with some conditions, especially under low oxygen.Abbreviations and symbols C₁ intercellular CO₂ pressure - PGA 3-phosphoglyceric acid - RuBP ribulose-1,5-bisphosphate - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase     

Wall yield threshold and effective turgor in growing bean leaves

The rate of cell enlargement depends on cell-wall extensibility (m) and on the amount of turgor pressure (P) which exceeds the wall yield threshold (Y). The difference (P-Y) is the growth-effective turgor (P _e). Values of P, Y and P _ehave been measured in growing bean (Phaseolus vulgaris L.) leaves with an isopiestic psychrometer, using the stress-relaxation method to derive Y. When rapid leaf growth is initiated by light, P, Y and P _eall decrease. Thereafter, while the growth rate declines in maturing leaves, Y continues to decrease and P _eactually increases. These data confirm earlier results indicating that the changes in light-stimulated leaf growth rate are primarily controlled by changes in m, and not by changes in P _e. Seedlings incubated at 100% relative humidity have increased P, but this treatment does not increase growth rate. In some cases Y changes in parallel with P, so that P _eremains unchanged. These data point out the importance of determining P _e, rather than just P, when relating cell turgor to the growth rate.Abbreviations and symbols FC fusicoccin - m wall extensibility - P turgor pressure - P _e effective turgor - RH relative humidity - Y yield threshold - _w water potential - _s osmotic potential     

The relationship between stomatal resistance and abscisic-acid levels in leaves of water-stressed bean plants

Leaf water potentials of Phaseolus vulgaris L. plants exposed to a -3.0 bar root medium were reduced to between -7 and -9 bars within 25 min and remained constant for the next several hours. This treatment led to considerable variation between leaves in both abscisic-acid (ABA) content and R_s, although the two were well correlated after a 5-h treatment. There was an apparent 7-fold increase in leaf ABA levels necessary to initiate stomatal closure when plants were exposed to a -3.0 bar treatment, but when plants were exposed to a -5.0 bar stress R_s values increased prior to any detectable rise in ABA levels. To explain these seemingly contradictory results, we suggest that the rate of ABA synthesis in the leaf, rather than the total ABA content, determines the status of the stomatal aperture.Abbreviations ABA abscisic acid - PEG polyethylene glycol - R_s stomatal diffusion resistance of lower leaf surface - leaf water potential     

Distribution of thiophene derivatives in different organs of Tagetes patula seedlings grown under various conditions

An intensive accumulation of thiophene derivatives occurs during the first days of development in Tagetes seedlings. After that, the thiophene content (related to dry weight) decreases to reach low values until the twelfth day of growth. High performance liquid chromatography analyses performed on cotyledons, on hypocotyls, and on roots of normal-grown and of partly-etiolated seedlings showed that bithienylbutinen, the major compound in hypocotyls and roots, is accumulated earlier than the other thiophene derivatives. The synthesis of acetoxybutinylbithiophene seems to be light-induced. This compound is not found in cotyledons. Hydroxybutinylbithiophene is synthesized specifically in the roots, -terthiophene in cotyledons. The results obtained indicate a high intensity of thiophene metabolism in these particular organs during seedling development.     

The compartmentation of ethylene in developing cotyledons of Phaseolus vulgaris L.

Isolated cotyledons of Phaseolus vulgaris L. cv. Canadian Wonder accumulated ¹⁴C₂H₄ (0.7–1 l l^-1) from air to give partition coefficients of 1 to 4, which greatly exceeded the value obtained with steam killed cotyledons (0.05) and with water (0.11). After ¹⁴C₂H₄ treatment, 98% of the ¹⁴C in the tissue remained as ¹⁴C₂H₄. The labelled ethylene accumulated by cotyledons was released only slowly (1–10% h^-1) either in an air stream or into toluene. Heating to 60°C for 2 h, but not freezing and thawing, caused the immediate release of ¹⁴C₂H₄ from the tissue. Propylene and vinyl chloride competitively inhibited the accumulation of ¹⁴C₂H₄.Cotyledons emanated endogenous ethylene at a very low rate but after heating (although not freezing and thawing) 13 nl of ethylene per g fresh mass were released within minutes. It was concluded that french bean cotyledons hold ethylene in a compartmented form in sufficient amount to account for at least 200 h of emanation.Abbreviation PPO diphenyloxazole     

The effect of light on the production of ethylene from 1-aminocyclopropane-1-carboxylic acid by leaves

White light inhibits the conversion of 1-amino-cyclopropane-1-carboxylic acid (ACC) in discs of green leaves of tobacco (Nicotiana tabacum L.) and segments of oat (Avena sativa L.) leaves by from 60 to 90%. Etiolated oat leaves do not show this effect. The general nature of the effect is shown by its presence in both a mono- and a dicotyledon. Since the leaves have been grown and pre-incubated in light, yet can produce from 2 to 9 times as much ethylene in the dark as in the light, it follows that the light inhibition is fully reversible. The inhibition by light is about equal to that exerted in the dark by CoCl₂; it can be partly reversed by dithiothreitol and completely by mercaptoethanol. Thus the light is probably acting, via the photosynthetic system, on the SH group(s) of the enzyme system converting ACC to ethylene.Abbreviation ACC 1-aminocyclopropane-1-carboxylic acid     

Light-stimulated cell expansion in bean (Phaseolus vulgaris L.) leaves

Cell expansion in dicotyledonous leaves is strongly stimulated by bright white light (WL), at least in part as a result of light-induced acidification of the cell walls. It has been proposed that photosynthetic reactions are required for light-stimulated transport processes across plasma membranes of leaf cells, including proton excretion. The involvement of photosynthesis in growth and wall acidification of primary leaves of bean has been tested by inhibiting photosynthesis in two ways: by reducing chlorophyll content of intact plants with tentoxin (TX) and by treating leaf discs with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU). Exposure to bright WL stimulated growth of intact leaves of TX-treated plants. Discs excised from green as well as from TX-or DCMU-treated leaves also responded by growing faster in WL, as long as exogenous sucrose was supplied to the photosynthetically inhibited tissues. The WL caused acidification of the epidermal surface of intact TX-leaves, but acidification of the incubation medium by mesophyll cells only occurred when photosynthesis was not inhibited. It is concluded that light-stimulated cell enlargement of bean leaves, and the necessary acidification of epidermal cell walls, are mediated by a pigment other than chlorophyll. Light-induced proton excretion by mesophyll cells, on the other hand, may require both a photosynthetic product (or exogenous sugars) and a non-photosynthetic light effect.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1 -dimethylurea - OC osmotic concentration - RL red light - TX tentoxin - WL white light We thank Dr. G.E. Templeton, University of Arkansas, Fayetteville, USA, for initially supplying us with TX, and also Dr. Stephen O. Duke, Southern Weend Science Laboratory, Stoneville, Miss., USA, for suggesting this compound for our experiments. We are grateful to Professor E. Ballio for his generous gift of fusicoccin.     

On the origin of the yolk protein ferritin in snails

Summary The iron storage protein, ferritin, is the major yolk protein in freshwater snails. In this report we show by in vitro labelling experiments that yolk ferritin of the snails Lymnaea stagnalis L. and Planorbarius corneus L. is an exogenous protein synthesized in the midgut gland and secreted into the hemolymph. Gonad and mantle tissue are inactive in the synthesis of yolk ferritin, but, together with the midgut gland, they synthesize another ferritin type (soma ferritin) which is not released into the hemolymph and which may be a housekeeping ferritin. Soma ferritin and yolk ferritin are not in a precursor/product relationship since subunits of both ferritins are synthesized as primary translation products in rabbit reticulocyte lysate programmed with poly (A)⁺ RNA from midgut gland and gonad. Results suggest that both ferritins are synthesized on different mRNAs (and possibly on different genes) so they may be regulated in a different way.     

Effect of light on the nucleotide composition of rRNA of wheat seedlings

Both qualitative and quantitative differences in the minor nucleotide constituents of rRNA from normally grown and from etiolated wheat plants (Triticum aestivum L.) were established. Using different degradation methods and separation techniques the 18S+26S RNA of 8-day-old wheat seedlings grown in the light was found to contain 5-methylcytidine, 3-methylcytidine, 5-methyluridine, 3-methyluridine, 5-carboxymethyluridine, 1-methyladenine, N-methyladenine, 5-hydroxymethylcytidine, O²-methyluridine, O²-methylcytidine, pseudouridine, O²-methylpseudouridine, N²,N²-dimethylguanine, 1-methylguanine, ribothymidine and some unknown minor constituents. On the other hand, there were only a few minor nucleotides in the rRNA of etiolated wheat seedlings. Cycloheximide, a cytoplasmic protein synthesis inhibitor, simulated etiolation in that it reduced the number of minor nucleotides in rRNA, whereas chloramphenicol, a chloroplast protein synthesis inhibitor, had no significant effect on the minor nucleotide content of rRNA. This finding suggests that illumination may cause de novo synthesis of cytoplasmic modifying enzymes leading to the formation of highly modified rRNAs.Abbreviations m⁶A N⁶-methyladenine - m¹A 1-methyladenine - 5hmc 5-hydroxymethylcytidine - Cm O²-methylcytidine - m⁵C 5-methylcytidine - m³C 3-methylcytidine - m¹G 1-methylguanine - m ₂ ² G N², N²-dimethylguanine - pseudouridine - m O²-methylpseudouridine - Um O²-methyluridine - m³U 3-methyluridine - m⁵U 5-methyluridine - cm⁵U 5-carboxymethyluridine - rT ribothymidine - Pur purine - Pyr pyrimidine - RNase ribonuclease - UV ultra violet - p phosphate     

Ethylene evolution by rust-infected,detached bean (Phaseolus vulgaris L.) leaves susceptible and hypersensitive to Uromyces phaseoli (Pers.) Wint.

Ethylene production in leaves of susceptible and hypersensitive varieties of beans has been followed after inoculation with Uromyces phaseoli. Four different states of ethylene evolution are distinguishable: (1) 13 h after inoculation and concomitant to the penetration of the fungal mycelium through the stomata, all varieties show an outburst of ethylene with significant differences between the three varieties. (2) After 36 h postinoculation, in all three varieties ethylene evolution is scarcely higher than in noninfected leaves. (3) Starting 59 h after inoculation, only in the hypersensitive variety 765 (which shows the lowest ethylene production after 13 h), a second, very strong ethylene outburst is observed. (4) From 125 h after inoculation, significant ethylene production is not observed in any variety. At this time, characteristic symptoms are expressed in susceptible leaves (differentiation of uredosori) and in the hypersensitive variety 765 (large brown necrotic spots); no macroscopic symptoms are observed in the hypersensitive variety 814, which exhibits the strongest ethylene outburst 13 h after inoculation. The capacity for ethylene formation after mechanical wounding (point freezing) is almost identical in healthy leaves of all three varieties. This capacity is still preserved after the first ethylene outburst 36 h after infection.This work was supported by the Deutsche Forschungsgemeinschaft, by the Kleinwanzlebener Saatzucht AG (Einbeck, FRG) and by a NATO fellowship to P.M.     

Loss of capacity for acid-induced wall loosening as the principal cause of the cessation of cell enlargement in light-grown bean leaves

Cell enlargement in primary leaves of bean (Phaseolus vulgaris L.) can be induced, free of cell divisions, by exposure of 10-d-old, red-light-grown seedlings to white light. The absolute rate of leaf expansion increases until day 12, then decreases until the leaves reached mature size on day 18. The cause of the reduction in growth rate following day 12 has been investigated. Turgor calculated from measurements of leaf water and osmotic potential fell from 6.5 to 3.5 bar before day 12, but remained constant thereafter. The decline of growth after day 12 is not caused by a decrease in turgor. On the other hand, Instron-measured cell-wall extensibility decreased in parallel with growth rate after day 12. Two parameters influencing extensibility were examined. Light-induced acidification of cell walls, which has been shown to initiate wall extension, remained constant over the growth period (days 10–18). Furthermore, cells of any age could be stimulated to excrete H⁺ by fusicoccin. However, older tissue was not able to grow in response to fusicoccin or light. Measurements of acid-induced extension on preparations of isolated cell walls showed that as cells matured, the cell walls became less able to extend when acidified. These data indicate that it is a decline in the capacity for acid-induced wall loosening that reduces wall extensibility and thus cell enlargement in maturing leaves.Abbreviations and symbols FC fusicoccin - P turgor pressure - RL red light - WEx wall extensibility - WL white light - P _w leaf water potential - P _s osmotic potential     

Phytochrome and potassium uptake by mung bean hypocotyl sections

Uptake of potassium (K) and ⁸⁶rubidiumlabelled potassium (⁸⁶Rb) by sub-hypocotyl hook sections of Phaseolus aureus L. was inhibited by red light. The effect was reversible with far red light. Using short exposures of high irradiance the effect on ⁸⁶Rb-labelled K uptake was observed after 5 min. The response showed no specificity for a particular anion. Uptake of ⁸⁶Rb-labelled K by sections cut immediately below the cotyledons was enhanced by red light after 10 min incubation and was also far red reversible. These results are interpreted as a rapid phytochrome-induced change in membrane properties resulting in modified K uptake.Abbreviations P Phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - R red light - F far red light     

Changing cell-wall compositions in hypocotyls of dark-grown bean seedlings

Hypocotyls of dark-grown 6-day-old seedlings of Phaseolus vulgaris L. proved to be sufficiently homogeneous to permit studies relating the rate of cell elongation to the composition of the primary cell walls. Whereas the levels of cellulose and uronic acids remained practically constant during and after cell extension, all other components showed major or minor changes. Cell-wall protein, as such, decreased by more than 50%, but indications are that hydroxyproline-rich glycoprotein increased with a decreasing rate of cell elongation, concomitant with a rise in the degree of arabinosylation of wall-bound hydroxyproline. As cell elongation slowed down, non-cellulosic glucose accumulated, presumably in the form of a -(1–4)glucan closely associated with cellulose. These findings confirm the notion that the primary cell wall is a highly dynamic structure.     

Properties of Bacillus anthracis spores prepared under various environmental conditions

Bacillus anthracis makes highly stable, heat-resistant spores which remain viable for decades. Effect of various stress conditions on sporulation in B. anthracis was studied in nutrient-deprived and sporulation medium adjusted to various pH and temperatures. The results revealed that sporulation efficiency was dependent on conditions prevailing during sporulation. Sporulation occurred earlier in culture sporulating at alkaline pH or in PBS than control. Spores formed in PBS were highly sensitive towards spore denaturants whereas, those formed at 45°C were highly resistant. The decimal reduction time (D-10 time) of the spores formed at 45°C by wet heat, 2 M HCl, 2 M NaOH and 2 M H₂O₂ was higher than the respective D-10 time for the spores formed in PBS. The dipicolinic acid (DPA) content and germination efficiency was highest in spores formed at 45°C. Since DPA is related to spore sensitivity towards heat and chemicals, the increased DPA content of spores prepared at 45°C may be responsible for increased resistance to wet heat and other denaturants. The size of spores formed at 45°C was smallest amongst all. The study reveals that temperature, pH and nutrient availability during sporulation affect properties of B. anthracis spores.