Callose Deposition and Photoassimilate Export in Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Callose accumulated on sieve plates of phloem of white bean seedlings exposed to excess Co, Ni, or Zn. The callose deposits ranged in thickness and were most pronounced in midribs of unifoliate leaves and their subtending petioles. Lesser callose deposits were found in stems. Although translocation of ¹⁴C was reduced drastically in seedlings exposed to excess metal, no correlation was found between translocated ¹⁴C and the amount of callose in the petioles. It is concluded that the inhibition of phloem translocation in seedlings exposed to excess metal is due to effects other than callose deposition.     

Vein Loading in Seedlings of Phaseolus vulgaris Exposed to Excess Cobalt, Nickel, and Zinc

Vein loading in unifoliate leaves of white bean seedlings exposed to excess Co²⁺, Ni²⁺, or Zn²⁺ for 1 to 4 days was studied by incubating leaf discs in [¹⁴C]sucrose. The discs from plants exposed to metal exhibited an increased total uptake of radiosucrose but reduced vein loading. Differences between treatments due to infiltration of disc margins were eliminated by analyzing 7-millimeter discs cut from the center of incubated 14-millimeter discs. Uptake of radiosucrose was greater particularly in discs from seedlings exposed to excess Ni²⁺ and Zn²⁺. The effect increased as exposure of the seedlings to metal increased up to 4 days. Autoradiographs showed vein loading in control leaf tissues with most of the radiosucrose accumulating in minor veins and little remaining in the mesophyll. In discs from metal-treated plants, most of the sucrose remained in the mesophyll without accumulating preferentially in the minor veins. This effect was evident within 24 hours of exposure to excess metal and intensified with longer exposures to metal. The inhibition of vein loading was also evident in situ. Both the preferential accumulation of sucrose into the minor veins of control tissues and the accumulation into mesophyll of metal exposed tissues were sensitive to 2,4-dinitrophenol and the blockage of sulfhydryl groups. It is concluded that the inhibition of vein loading contributes markedly to the observed toxicological effects of reduced photoassimilate export and of accumulation of carbohydrates in fully expanded leaves of bean seedlings exposed to excess metal ions.     

Changes in Carbohydrate Levels in Red Kidney Bean (Phaseolus vulgaris L.) Exposed to Sulphur Dioxide

Red kidney bean (Phaseolus vulgaris L.) was exposed to variousconcentrations of SO² for 24 h (16 h light/8 h dark photoperiod)with continuous monitoring of photosynthetic and respiratoryactivity. Plants were harvested at the end of the dark periodinto samples of mature and immature leaf tissue, stems, androots for determination of sugar and starch levels. In all tissue samples the levels of total sugars were increasedby exposure to the lower concentrations of SO², but decreasedby the higher concentrations. Starch levels in leaves followeda similar trend. Increases in sugar and starch levels precededsymptoms of visible injury. Decreasing rates of photosynthesiswere correlated with increasing rates of respiration, the occurrenceof visible injury, and the depletion of sugar and starch levels.     

Photosynthesis, Carbohydrate Metabolism, and Export in Beta vulgaris L. and Phaseolus vulgaris L. during Square and Sinusoidal Light Regimes

Rates of photosynthesis, sucrose synthesis, starch accumulation and degradation were measured in sugar beet (Beta vulgaris L.) and bean (Phaseolus vulgaris L.) plants under a square-wave light regime and under a sinusoidal regime that simulated the natural daylight period. Photosynthesis rate increased in a measured manner in direct proportion to the increasing light level. In contrast to this close correspondence between photosynthesis and light, a lag in photosynthesis rate was seen during the initial hour under square-wave illumination. The leaf appeared to be responding to limits set by carbon metabolism rather than by gas exchange or light reactions. Under the sinusoidal regime starch degradation occurred during the first and last 2 hours of the photoperiod, likely in response to photosynthesis rate rather than directly to light level. Starch broke down when photosynthesis was below a threshold rate and accumulated above this rate. Under square-wave illumination, accumulation of starch did not begin until irradiance was at full level for an hour or more and photosynthesis was at or near its maximum. Under a sinusoidal light regime, sucrose synthesis rate comprised carbon that was newly fixed throughout the day plus that from starch degradation at the beginning and end of the day. Synthesis of sucrose from recently fixed carbon increased with increasing net carbon fixation rate while its formation from degradation of starch decreased correspondingly. The complementary sources of carbon maintained a relatively steady rate of sucrose synthesis under the changing daytime irradiance.     

Carboxyarabinitol 1-Phosphate Phosphatase from Leaves of Phaseolus vulgaris and Other Species

Carboxyarabinitol 1-phosphate (CA1P) phosphatase activity occurredin leaves of 10 species examined, with the highest activityin leaves of Phaseolus vulgaris. Enzyme was purified from P.vulgaris 1,580-fold to a final specific activity of 6.1 µmolmin^–1 (mg protein)^–1. Structural characteristicsof positive effectors and substrate analogs for the CA1P phosphatasereaction were examined. Positive effectors were compounds thatcontained one phosphate group in close proximity to a secondphosphate or a carboxyl group (e.g. 2-phosphoglycolate, pyrophosphate,3-phosphoglycerate, and carboxyethylphosphonic acid). Many ofthe activators are structurally quite similar to CA1P, but werenot used as substrates. In addition to the natural substrateCA1P, carboxypentitol and carboxyhexitol bisphosphates wereshown to be good substrates (e.g. carboxyarabinitol bisphosphateand carboxymannitol bis-phosphate). A substrate arabinitol configuration(R) was preferred at C-2, and reactivity was lost when a hydroxymethylgroup was substituted for the carboxyl group. Despite structuralsimilarities to positive effectors, none of the tested reactionsubstrates could activate the enzyme. (Received November 11, 1996; Accepted February 3, 1997)     

The Effect of Light, Temperature, and Anaesthetics on ATP Levels in the Leaves of Chenopodium rubrum and Phaseolus vulgaris

The effects of light, temperature, and anaesthetics on ATP levelsin the leaves of Chenopodium rubrum and of Phaseolus vulgarishave been determined by an UV-enzymic method. ATP levels were found to vary inversely with temperature. Moreover,the anaesthetics, ether and ohloroform, were found to elevateleaf ATP. The effect of light was to reduce ATP levels althougha tendency for increased ATP at high light intensities was detected. A correlation is deduced between elevated ATP level and decreasedcytoplasmic viscosity, for cold, light, and anaesthetics. On the basis that cold, and some anaesthetic substances, havebeen shown to release plants from dormancy, it is suggestedthat ATP levels may play a fundamental role in vernalizationand in the breaking of dormancy, and that the interaction oflight may also be on this basis. An explanation is suggested for these effects, and their widerimplications are discussed.     

Photomorphogenesis, Photosynthesis, and Early Growth of Primary Leaves of Phaseolus vulgaris

The response of leaf tissue to white, blue, red, and far-redlight has been examined. Leaves on plants grown in darknessshow increased cell number, cell volume, and area when exposedto long periods (up to 48 h) of low-intensity red, blue, orfar-red radiation. This is believed to be a photomorphogenicresponse which does not involve photosynthesis. Leaves fromplants exposed to white light during germination do not usuallyrespond to red, blue, or far-red light. An exception to thiswas found for leaf discs which showed a larger increase in areathan the dark controls following exposure to far-red light for24 h. Leaf tissue from light-grown plants responds to high-intensitywhite light, probably through photosynthesis. Discs cut fromdark-grown plants and cultured in white light grow equally wellin air and CO₂-free conditions. Application of the photosyntheticinhibitor DCMU reduces growth and chlorophyll formation, however. It is concluded that light, perhaps acting through the phytochromemechanism, has initially a number of morphogenic effects includinginitiation of development of the photosynthetic apparatus. Theresponses to photomorphogenically active radiation do not persistand light effects through photosynthesis are rapidly initiatedand dominate the later stages of leaf growth.     

Ribonuclease in Leaves of Phaseolus vulgaris during Maturation and Senescence

The levels of the enzyme ribonuclease (RNase) were determined in primary leaves of Phaseolus vulgaris in an attempt to correlate changes in RNA with maturation and senescence. RNase, RNA and chlorophyll levels increased in expanding and maturing tissue and subsequently declined in senescing tissue. Senescence of the primary leaves started with the onset of flowering. A simultaneous increase of RNA and RNase in maturing tissue and a decrease during senescence suggests that the enzyme activity is correlated with the rate of‘turnover’of RNA rather than the absolute levels of RNA present in the tissue.     

Appearance of Photochemical Activity in Isolated Chloroplasts from Far-red-illuminated Leaves of Phaseolus vulgaris

Chloroplast development was followed in intact bean leaves illuminatedwith far-red light by extracting chloroplasts at various timesto assay photosynthetic activities. Photochemical activity wasdetected in isolated chloroplasts prior to the times which werepreviously reported for intact leaf discs. Cyclic phosphorylationwas observed in isolated chloroplasts after 8 h of far-red illuminationwhile non-cyclic electron transport and phosphorylation weremeasurable after 12 and 16 h of illumination respectively. TheP/2e ratios were less than 0.5 after 24 h of far-red exposurebut approached a value of 1.0 by 60 h of illumination. Ammoniumchloride (10^–3 M) had little effect on electron transportin isolated chloroplasts until after 24 h of far-red illumination.Chlorophyll a accumulated slowly from the onset of far-red illuminationwhile chlorophyll b was not detected until after 48 h of far-redexposure. Leaf fresh weight increased four-fold over the 60h illumination period. Electron microscopy of isolated chloroplasts from far-red-illuminatedleaves indicated the presence of unfused primary thylakoidsby 12 h of exposure and prolamellar bodies throughout the entire60 h illumination period. Grana were not observed in isolatedchloroplasts nor were they induced by a 2 min exposure of thechloroplasts to 172 000 lx of white light. O₂ evolution in leaf discs of far-red-illuminated plants wasmeasurable after 16 h of illumination, attained a maximum valueby 36 h of far-red exposure, and then declined. Net CO₂ fixationwas observed in leaf discs after 8 h of far-red illuminationand the rates remained constant for an additional 16 h, beforeincreasing at least two-fold.     

Carbon Partitioning among Leaves, Fruits, and Seeds during Development of Phaseolus vulgaris L

Development of vegetative and floral buds was found to be a key factor in establishing the way carbon is distributed among growing leaves and fruits in Phaseolus vulgaris L. plants. Leaves emerged principally during a period 14 to 32 days after planting while flowers were produced during a 10- to 12-day period near the end of leaf emergence. Timing of anthesis established the sigmoidal time course for dry weight accumulated by the composite of all fruits on the plant. During the first 12 days following anthesis, fruit growth mainly consisted of elongation and dry weight accumulation by the pod wall. Thereafter, seed dry weight increased for about 1 week, decreased markedly for several days, and then increased again over the next 2 weeks. Accumulation of imported carbon in individual seeds, measured by steady-state labeling, confirmed the time course for dry weight accumulation observed during seed development. Seed respiration rate initially increased rapidly along with dry weight and then remained nearly steady until seed maturation. A number of developmental events described in the literature coincided with the different phases of diauxic growth. The results demonstrated the feasibility of relating current rates of carbon import in individual seeds measured with tracer ¹⁴C to the rates of conversion of imported sucrose and use of the products for specific developmental processes. The resulting data are useful for evaluating the roles of conversion and utilization of imported sucrose in regulating import by developing seeds.     

Effect of Rapid Changes in Sink-Source Ratio on Export and Distribution of Products of Photosynthesis in Leaves of Beta vulgaris L. and Phaseolus vulgaris L

Effects of increasing sink-source ratio on rate of translocation and net carbon exchange were studied by darkening all but one source leaf of Beta vulgaris L. or one primary leaf of Phaseolus vulgaris L. Rates of export of labeled material and patterns of its distribution among sinks were studied by means of GM detectors. Changes in export and import rates were compared with adjustments in starch, sucrose, and glucose levels in sugar beet source leaves before and during treatment.     

Zinc,Copper, Iron,and Selenium Levels in Brain and Liver of Mice Exposed to Acrylonitrile

The mechanism of toxicity of acrylonitrile (AN) has not been fully defined. The research described herein was undertaken to investigate the possible effects of AN on the levels of metallic elements in liver and brain of mice. Thirty-two mice were randomly assigned to four separate groups and treated intraperitoneal (i.p.) once daily for 1 week. Mice in the control group received normal saline, and mice in the three exposure groups received 5, 10, or 20 mg AN/kg b.w. Samples of brain and liver were collected immediately after decapitation. Tissue levels of trace elements (zinc, copper, iron) were determined with flame atomic absorption spectrophotometer or double channel atomic fluorescence absorption spectrophotometer (selenium). Mean brain weights of AN-treated mice were increased as a function of dose compared to controls, but there was no significant change in the ratio of liver/body weight in the four groups. While mean brain zinc decreased as a function of AN dosage, mean liver zinc of the low-dose group significantly increased (p < 0.05); mean liver copper in the medium-dose AN group was significantly higher compared to controls (p < 0.05); however, mean brain copper was increased, but the difference did not attain statistical significance in the three AN groups when compared with the controls (p > 0.05). Mean brain iron levels were significantly decreased in the middle-dose AN group (p < 0.05), but there were no consistent changes in liver iron. Tissue levels of selenium in brain and liver were similar for the control and AN treatment groups. AN induces significant and differential changes in the levels of zinc, copper, and iron in brain and liver. These changes likely play a pivotal role in mediating AN toxicity, most likely via changes in cellular redox status.     

The Distribution of Nickel, Copper, Zinc, and Iron in Tree Leaves

The distributions of nickel, copper, zinc, and iron in freshleaves from four tree species were studied using differentialoentrifugation of cell homogenates to fractionate the cell organellesand atomic absorption spectrophotometry to measure the concentrationsin the various fractions. Results from each species were closelysimilar. Approximately 90 per cent of the nickel was presentin the supernatant fraction while approximately 80 per centof the iron was present in the chloroplast fraction. Both copperand zinc had subcellular distributions similar to one anotherand contained appreciable amounts of the elements in both chloroplastand supernatant fractions. High-voltage paper electrophoretic separations of the solubleextracts of leaves showed that while copper, zinc, and ironexisted principally as anionic complexes, nickel existed asa cationio species. It was considered that this cationic formof nickel was unlikely to be an unchelated aquated cation.     

Invertase Activity, Carbohydrate Metabolism and Cell Expansion in the Stem of Phaseolus vulgaris L.

In the stem of Phaseolus vulgaris L. the specific activity ofacid invertase was highest in the most rapidly elongating internode.Activity of the enzyme was very low in internodes which hadcompleted their elongation, in young internodes before the onsetof rapid elongation, and in the apical bud. From shortly afterits emergence from the apical bud the elongation of internode3 was attributable mainly to cell expansion. Total and specificactivities of acid invertase in this internode rose to a maximumat the time of most rapid elongation and then declined. Transferof plants to complete darkness, or treatment of plants withgibberellic acid (GA₃), increased the rate of internode elongationand final internode length by stimulating cell expansion. Bothtreatments rapidly increased the total and specific activitiesof acid invertase in the responding internodes; peak activitiesof the enzyme occurred at the time of most rapid cell expansion. In light-grown plants, including those treated with GA₃, rapidcell and internode elongation and high specific activities ofacid invertase were associated with high concentrations of hexosesugar and low concentrations of sucrose. As cell growth ratesand invertase activities declined, the concentration of hexosefell and that of sucrose rose. In plants transferred to darkness,stimulated cell elongation was accompanied by a rapid decreasein hexose concentration and the disappearance of sucrose, indicatingrapid utilization of hexose. No sucrose was detected in theapical tissues of light-grown plants. The results are discussed in relation to the role of acid invertasein the provision of carbon substrates for cell growth. Key words: Cell expansion, Acid invertase, Hexose, Sucrose, Phaseolus     

Carbohydrate and organic acid composition of effective and ineffective root nodules of Phaseolus vulgaris

Dicarboxylic acid transport mutants of Rhizobium species are usually deficient in their ability to fix atmospheric dinitrogen. We report here a study comparing the physiology of root nodules on Phaseolus vulgaris L. cv. Goldie induced by an effective strain of Rhizobium leguminosarum biovar phaseoli and a C₄-dicarboxylic acid utilization mutant. The mutant, while able to form nodules, was ineffective in N₂ fixation. Carbohydrates and organic acids of roots and nodules formed by the 2 strains were monitored at 3-day intervals from 13 to 34 days after inoculation. Both carbohydrates and organic acids accumulated in ineffective nodules in comparison with the effective nodules. The concentration of malic acid was tenfold higher in ineffective nodules than in effective nodules. Other organic acids, i.e., lactate, malonate, ascorbate and gluconate, were also detected. Lactate and ascorbate were the only other organic acids accumulating in ineffective nodules. The most prevalent carbohydrates found in both types of nodules were sucrose, glucose and fructose. Myo-inositol was the only cyclitol detected in both types of nodules. Carbohydrates and organic acids were present in lower concentration in roots than in nodules, except for lactate. These compounds were not consistently detected in higher concentration in roots from plants inoculated with the mutant strain, as was the case in nodules.     

Leaf-Specific Antisense Inhibition of Starch Biosynthesis in Transgenic Potato Plants Leads to an Increase in Photoassimilate Export from Source Leaves during the Light Period

In an attempt to study the importance of starch synthesis inleaves with respect to sink-source interactions, we investigateddaily turnover of carbohydrates in leaves of transgenic potatoplants inhibited for ADP-glucose pyrophosphorylase (AGPase).Down-regulation of AGPase has been performed using two differentpromoters: the near-constitutive CaMV 35S promoter, and theSTLSI promoter which is active in photosynthetic cells only.Residual AGPase activity in leaves was between 6 and 30% inindividual transformants as compared to wild-type potato plants.We found that: (i) photosynthesis is not significantly alteredrelative to wild-type plants; (ii) levels of starch are markedlyreduced in leaves of transgenic plants; (iii) levels of solublesugars and malate are largely unaffected by the inhibition ofAGPase; (iv) the reduction of starch synthesis leads to a higherportion of assimilated carbon being transported from leavesto sink tissues during the light period; (v) altered leaf exportcharacteristics do not change tuber yield under greenhouse conditions.Collectively, these data demonstrate a striking flexibilityof the potato plant with respect to day/night rhythms of carbonexport from leaves and utilization by the major storage sinks,i.e. developing tubers. (Received November 1, 1994; Accepted March 2, 1995)     

Transpiration and Calcium Deposition by Unifoliate Leaves of Phaseolus vulgaris Differing in Maturity

Unifoliate leaves were individually enclosed in clear, plastic chambers for the 24 hour treatment periods and then sacrificed for Ca analysis. Two transpiration rates were obtained by passing dry air through the chambers tising flow rates of 160 and 260 cm³/min. A third rate was obtained by a combination of shade and the lower air flow rate. Neither the transpiration rate nor solution-Ca concentration (0.5mM and 2.5 mM of 0.1, and 0.5 strength Hoagland solution) altered the amount of Ca deposited in the unifoliate leaves of 22 day old bean plants (Phaseolus vulgaris). The transpiration rate per unit area of leaf remained constant for all ages studied (1l–20 days) and was 1.8, 2.7, 3.6 g H₂O per dm² day for the three different imposed conditions. A definite pattern of Ca deposition occurred. With all the transpiration rates there was a maximum rate of calcium deposition at 13 days of growth and a gradual decrease thereafter. When the Ca concentration of the nutrient solution was 20 μg/ml the daily Ca deposition in terms of water transpired by the unifoliate leaves exceeded this amount, except for the oldest leaf tested, and, the maximum Ca to water ratios were 250, 320, and 430 (μg Ca/g) in order of decreasing transpiration rates. The uptake of Ca against a concentration gradient and approximately the same total uptake regardless of transpiration rates and solution concentrations used, firmly suggest that Ca secretion into root-xylem elements from a surrounding low level Ca solution requires energy expenditure by the plant. A possible explanation was proposed for the decreased rate of Ca deposition by the unifoliate leaves subsequent to the 13th day.     

Concerning the Preparation of Chloroplasts Active in Hill and Photosynthetic Phosphorylation Activities from Leaves of Phaseolus vulgaris

When bean plants are cultivated under certain conditions, liberation of cell contents by grinding with mortar and pestle yields chloroplasts of low Hill and photosynthetic phosphorylation activities, while grinding in a high speed blendor yields active chloroplasts. The inactivity of plastids obtained with mortar and pestle is due to the presence of an inactivator in the homogenate. The culture conditions that determine whether bean leaves ground with mortar and pestle contain inactivator were not determined.     

Purification and properties of cellulase from Phaseolus vulgaris

Cotyledons of germinating kidney beans contain two forms of a carboxy methyl cellulase which can be separated by ammonium sulfate fractionation and isoelectric focusing. The two cellulases are similar in their molecular weight but differ in isoelectric points, pH and temperature optimum, pH and temperature stability and sensitivity to thiol inhibitors and metal ions. One cellulase (isoelectric point 4.8) has been purified 100-fold to give a major protein band on acrylamide gel electrophoresis.