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)     

Synthesis of Cellulase during Abscission of Phaseolus vulgaris Leaf Explants

When abscission in leaf explants from Phaseolus vulgaris, cultivar Red Kidney, was allowed to proceed while the explants were in ²H₂O, a 1.25% increase in the buoyant density of cellulase in a cesium chloride gradient was observed. These data indicate that the increase in cellulase activity during abscission is a result of the synthesis of new protein. Two differentially soluble forms of cellulase are present in the abscission zone. The form which is soluble only in a high salt buffer seems more closely related to the abscission process than the form which is soluble in dilute buffer. The correlation between changes in pull force and increase in cellulase activity and the effects of several hormones on cellulase activity are discussed.     

Methylammonium Transport in Phaseolus vulgaris Leaf Slices

Methylammonium (as a nonmetabolized analog of ammonium) transport was studied in leaf slices of Phaseolus vulgaris L. var. `Hawkesbury Wonder.' The relationship of influx to external pH (6.0-10.5) shows that the influx at low external pH is a larger fraction of that at high external pH than would be expected from the pK_α of methylammonium and the assumption that only CH₃NH₂ is entering the cells. The relationship between methylammonium influx and external methylammonium concentration shows some evidence of saturation; this is a function of the transport system rather than of the (limited) methylammonium metabolism in the cells. The “equilibrium” concentration ratio for methylammonium between leaf slices and bathing medium is far higher than can be explained by the transport of CH₃NH₂ alone and the pH of the compartments involved. These three lines of evidence strongly suggest that there is an influx of CH₃NH₃⁺, possibly by a uniporter driven by the electrical potential of the cytoplasm with respect to the medium, as has been shown for other plant cells. Competitive inhibition of methylammonium influx by ammonium suggests that there is also an ammonium transport system. The significance of this for the recycling of N within the plant and for exchange of gaseous NH₃ between leaves and the atmosphere is discussed.     

Photosynthetic Response to Water Stress in Phaseolus vulgaris

Water stressed Phaseolus vulgaris L. plants were monitored to detect the relationships between net photosynthesis, transpiration, boundary layer plus stomatal resistance, mesophyll resistance, CO₂ compensation point, ribulose, 1,5-diphosphate carboxylase activity and leaf water potential. At full expansion, the first trifoliate leaves of greenhouse grown bean plants were subjected to water stress by withholding irrigation. Gas exchange and enzyme activity of the central trifoliolate leaflets were monitored as leaf water potential decreased. Although increased stomatal resistance appeared to be the primary causal factor of reduced net photosynthesis, increased mesophyll resistance and decreased ribulose 1,5-diphosphate carboxylase activity further documented the role of non-stomatal factors.     

Alterations in Endogenous Levels of Gibberellin-like Substances during Germination of Phaseolus vulgaris Seeds

The endogenous gibberellin-like substances were determined in mature dry and germinating bean seeds, Phaseolus vulgaris L. cv. Alabaster. Methanol extracts were partitioned against ethyl acetate and butanol at neutral and acid pH. Each phase was individually chromatographed on a silica gel column. The gibberellin activity was measured with the Tan-ginbozu dwarf rice micro-drop bioassay. Each extract was tested in two dilutions. Extracts from dry seeds showed the highest gibberellin activity, largely attributable to ethyl acetate-soluble substances. The activity was considerably reduced in extracts from seeds imbibed for 1 day. Gibberellin-like substances soluble in butanol appeared in extracts from seeds soaked for 1 or more days.     

ABA Levels and Effects in Chilled and Hardened Phaseolus vulgaris

Leaf abscisic acid (ABA) levels of chilled P. vulgaris weremeasured after 18 h chilling at 5°C, at a saturation deficitof 1.24 g m^–3 (SD), and after chilling in a water-saturatedatmosphere. Changes were also followed during a chill hardeningperiod of 4 d at 12°C, 2.1 g m^–3 SD. It was foundthat hardening resulted in an almost 5. fold increase in ABAlevels after 3 d at 12°C, and this decreased to approximatelycontrol levels on the fourth day. Subsequent chilling of hardenedplants produced no change in ABA levels from that of controlplants (22° C). In contrast, non-hardened plants chilledat 1.24 g m^–3 SD had ABA levels almost 3 times the levelof control plants. However, chilling in a water-saturated atmosphereresulted in a decrease in ABA levels. In addition, the response of leaf diffusion resistance (LDR)to exogenous ABA fed via the transpiration stream was measuredat 5 ° C and 22° C in hardened and non-hardened plants.Use of tritium-labelled ABA was made to calculate the stomatalsensitivity to ABA. It was found that exogenous ABA caused anincreased in LDR at 22°C in both hardened and non-hardenedplants. However, the sensitivity of the hardened plants to ABAwas greater in terms of rate of closure and amount of ABA requiredto close the stomata. At 5°C, however, ABA caused stomatalopening and the maintainance of open stomata in non-hardenedplants. In hardened plants, ABA caused stomatal closure at 5°C.These results are discussed in relation to the locking-openresponse of chilled P. vulgaris stomata. Key words: Chilling, Stomata, ABA, Phaseolus vulgaris     

Ochna pulchra Hook: Leaf Growth and Development Related to Photosynthetic Activity

Ochna pulchra Hook. is a deciduous broad-leaved tree in theMixed Bushveld vegetation of the Northern Transvaal. The growthand development of leaves taken from trees in the field werestudied from a stage shortly before bud break, in late spring,until they were fully expanded and at the peak of photosyntheticactivity. Leaf area was measured by photographing the leaf against a transparentmm² grid. Finally a constant relationship between leaf area(A) and the linear dimension of length (L) and breadth (B) wasestablished: A = b x LB, where coefficient b = 0.72. Transverse sections of the lamina of the youngest leaves showeda five-layered plate meristem with a few functional conductingelements in the midrib. During further leaf development, celldivision was followed by means of autoradiography using [³H]thymidine.It was most active during the week after bud break. Leaf cell increment following on cell division made the majorcontribution to leaf growth resulting in a lamina that was atleast 90% expanded 4 weeks after bud break. The histologicalchanges accompanying cell division were observed using lightand electron microscopy. Even in late stages of leaf development mature and differentiatingstomata occurred together, limited to the abaxial epidermisand the midrib. Scanning electron microscopy showed stomataldistribution, their increasing density and gradual opening.The structure of these sunken stomata could reduce the outwarddiffusion of water vapour and increase the diffusion resistanceto carbon dioxide. Carbon assimilation rates of the developing leaves were measuredusing an IRGA (infra-red gas analyser) and their chlorophyllvalues were calculated. Photosynthesis was first measured amonth after bud break when the leaves were fully expanded, over50 % of the stomata exposed and leaf mesophyll tissue differentiatedwith mature chloroplasts. Net photosynthetic rates and chlorophyllvalues peaked 1 month later. Ochna pulchra Hook., photosynthesis, leaf development, leaf area, stomata, chlorophyll, savanna     

Influence of Translocation of Photosynthetic Efficiency of Phaseolus vulgaris L

Measurements of net photosynthesis show that in Phaseolus vulgaris L. the cultivar Michelite-62 exceeds the cultivar Red Kidney in net CO₂ uptake by 23 to 31%. Data on translocation of pulse label indicate that export of a pulse of photosynthetically assimilated ¹⁴C from the source leaf of either M-62 or Red Kidney follows an exponential pattern and shows an initial rapid phase followed by a second slower phase. The steeper slope for both phases in M-62 suggests its rate of translocation of pulse label is higher than that of Red Kidney. Furthermore, only 38% of the ¹⁴C remains in the leaf of M-62 after 8 hours, while Red Kidney retains up to 60% of the label. Leaf autoradiographs obtained after pulse labeling demonstrate a much faster rate of vein loading in M-62 and are considered evidence for the higher translocation efficiency of M-62. These results provide evidence for a positive correlation between photosynthetic efficiency and translocation efficiency in M-62 and Red Kidney and give support to our hypothesis that translocation is one of the important physiological factors controlling the varietal differences in photosynthetic efficiency in Phaseolus vulgaris.     

Cytokinin Effects on Leaf Architecture in Phaseolus vulgaris L.

Treatment of expanding primary leaves of bean plants (Phaseolnsvulgaris L. cv. Limburgse vroege) with benzyladenine (BA) orkinetin at 0.5 mM for five consecutive days resulted in thickerleaves showing a significant decrease in intercellular air spacevolume. Compared with control plants, exposed mesophyll cellsurface area was lower per unit tissue volume, but unchangedwhen expressed per unit leaf surface area. Stomata of treatedplants were not fully closed in the dark and they did not openas wide as controls in the middle of the light period, suggestingthat the treatment resulted in impaired stomatal action. Allthe effects mentioned were more pronounced after treatment withBA, compared to kinetin. In spite of their magnitude, the observedchanges in leaf structure and function did not seem to havean important effect on total leaf diffusion resistance to carbondioxide during the course of the light period. Key words: Cytokinins, Leaf architecture     

Physiological Stress and Crystallites in Leaf Plastids of Phaseolus vulgaris L.

Crystalline inclusions were found in leaf plastids of Phaseolusvulgaris L. cultivar Limburg when excised plant parts were used.Removal of the root system induced crystalloid production afteran incubation period of optimal length. In agreement with thefindings of other authors physiological stress seems to be theunderlying condition of crystal formation in plastids.     

An Association Between Acid Invertase Activity and Cell Growth during Leaf Expansion in Phaseolus vulgaris L.

Changes in lamina area, dimensions of epidermal and palisadecells, acid invertase activity and content of sucrose and hexosein the primary leaves of Phaseolus vulgaris L. were determinedbetween emergence of the hypocotyl hook and the completion ofleaf expansion. Growth in area and thickness of the primaryleaf after emergence was attributable to the expansion of cellsalready present in the lamina at emergence. The major invertasein the expanding leaf was a readily soluble acid invertase;little insoluble invertase activity was detected. Soluble andinsoluble fractions of leaf homogenates contained little neutralinvertase activity. The specific activity of the soluble acidinvertase increased rapidly during the early stages of leafexpansion, reaching a peak at the time of most rapid cell enlargement(5 d after emergence) and then declining as the leaf matured.Highly significant positive correlations were found betweenenzyme specific activity and the rates of cell and leaf enlargement. The early, rapid phase of lamina expansion was characterizedby high concentrations of hexose sugar and low concentrationsof sucrose. As the rates of leaf cell enlargement declined theconcentration of hexose fell and that of sucrose increased.Between 5 d and 11 d after hypocotyl emergence, the hexose/sucroseratio in the primary leaf decreased approximately 10-fold asthe specific activity of acid invertase decreased. The results are discussed with reference to sources of carbonsubstrates for cell growth and to the sink/source transitionduring leaf development. Key words: Leaf expansion, Acid invertase, Hexose, Sucrose, Phaseolus     

Desiccation-Tolerant and Desiccation-Intolerant Stages during the Development and Germination of Phaseolus vulgaris Seeds

Embryonic axes of Phaseolus vulgaris undergo a transition fromdesiccation-intolerance to desiccation-tolerance in the courseof their development. Biochemical and ultrastructural observationsindicate that desiccation and rehydration during the early intolerantdevelopmental stages drastically reduces the metabolic and cellularintegrity of the axis. During the desiccation-tolerant stagesuch perturbations do not occur. Coincident with the acquisitionof desiccation-tolerance during development the seeds gain thecapacity to germinate upon subsequent rehydration. Drying presumablyacts to terminate developmental processes and to initiate themetabolic processes essential for the completion of germination.During germination of the mature axis, desiccation and rehydration,up to 12 h from the start of imbibition, does not affect subsequentseedling growth and development. But gradually desiccation-tolerancedecreases and metabolism is severely and irreversibly reducedby drying.     

Auxin Biosynthesis during Seed Germination in Phaseolus vulgaris

The relative roles of de novo biosynthesis of indoleacetic acid (IAA) and IAA conjugates stored in mature seeds (Phaseolus vulgaris L.) in supplying auxin to germinating bean seedlings were studied. Using ²H oxide and 2,4,5,6,7-[²H]l-tryptophan as tracers of IAA synthesis, we have shown that de novo biosynthesis of IAA, primarily from tryptophan, is an important source of auxin for young bean seedlings. New synthesis of IAA was detected as early as the second day of germination, at which time the seedlings began to accumulate fresh weight intensively and the total content of free IAA began to increase steadily. IAA conjugates that accumulate in large amounts in cotyledons of mature seeds may thus be considered to be only one of the possible sources of IAA required for the growth of bean seedlings.     

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.     

Comparison of the Activities and Some Properties of Pyrophosphate and ATP Dependent Fructose-6-Phosphate 1-Phosphotransferases of Phaseolus vulgaris Seeds

The distribution of pyrophosphate: fructose 6-phosphate phosphotransferase (PFP) and ATP: fructose-6-phosphate 1-phosphotransferase (PFK) was studied in germinating bean (Phaseolus vulgaris cv Top Crop) seeds. In the cotyledons the PFP activity was comparable with that of PFK. However, in the plumule and radicle plus hypocotyl, PFP activity exceeds that of PFK. Approximately 70 to 90%, depending on the stage of germination, of the total PFP and PFK activities were present in the cotyledons. Highest specific activity of both enzymes, however, occurred in the radicle plus hypocotyl (64-90 nanomoles·min·milligram protein). Fractionation studies indicate that 40% of the total PFK activity was associated with the plastids while PFP is apparently confined to the cytoplasm. The cytosolic isozyme of PFK exhibits hyperbolic kinetics with respect to fructose 6-P and ATP with K_m values of 320 and 46 micromolar, respectively. PFP also exhibits hyperbolic kinetics both in the presence and absence of the activator fructose-2,6-P₂. The activation is caused by lowering the K_m for fructose 6-P from 18 to 1.1 millimolar and that for pyrophosphate (PPi) from 40 to 25 micromolar, respectively. Levels of fructose 2,6-P₂ and PPi in the seeds are sufficient to activate PFP and thereby enable a glycolytic role for PFP during germination. However, the fructose 6-P content appears to be well below the K_m of PFP for this compound and would therefore preferentially bind to the catalytic site of PFK, which has a lower K_m for fructose 6-P. The ATP content appears to be at saturating levels for PFK.     

Leaf recovery responses during rehydration after water deficit in two bean (Phaseolus vulgaris L.) cultivars

Abstract

Our hypothesis was that recovery responses (RI and RII) upon rehydration, after 1 and 8 d of moderate (WDI) and severe water deficit (WDII), are evidence of tolerance in two commercial bean cultivars, Tacarigua (T cv) and VUL-73-40 (V cv). Recovery of leaf water (Ψw) and osmotic potentials (Ψs), and relative water content (LRWC), showed strong dependence on soil water potential (sΨw) followed by protein content; recovery connection between stomatal conductance and soil Ψw is showed. Chlorophyll (a + b), Ribulose-1,5-bisphosphate carboxylase oxygenase (Rubisco) activity, dry biomass (DM), and leaf area (LA) recovery were sensitive to WD intensity. Specific leaf area (SLA) and leaf density (D) recovery were less dependent on WD intensity and in time-dependent manner; V cv recovery was slower, showed faster recovery of Rubisco activity and DM due to slower recovery in SLA and D, which promoted it. Rubisco activity presented correlations with LRWC and Ψw at moderate and severe WD in both cultivars, and significant correlation with Ψs was observed in V cv. We conclude that recovery after rehydration reveals intrinsic tolerance to WD, due to an integration of metabolic and structural interactions, in responses to leaf water status components.     

Maintenance of High Photosynthetic Rates during the Accumulation of High Leaf Starch Levels in Sunflower and Soybean

Sunflower (cv. “Mammoth Greystripe”) and soybean (Merr. cv. “Amsoy 71”) leaves were exposed to continuous light for at least 52 hours in an attempt to determine the relationship between leaf starch levels and photosynthetic rates. Immature rapidly expanding and relatively mature slowly expanding sunflower leaves were studied. After 52 hours continuous light, the rapidly expanding leaves accumulated high starch levels (3.3 milligrams per square centimeter, 43% of dry weight) with only about a 10% decline from the initial photosynthetic rate of 42 milligrams CO₂ per square decimeter per hour. Under the same conditions, the slowly expanding leaves accumulated less starch, but the photosynthetic rate declined 30%. Soybean leaves, which were slowly expanding, accumulated less starch than sunflower leaves (2.1 milligrams per square centimeter, 34% of dry weight), and their photosynthetic rates declined only about 10% after 54 hours continuous light.     

Structure of Crystal Cells and Influences of Leaf Development on Crystal Cell Development and Vice Versa in Phaseolus vulgaris (Leguminosae)

The structure of cells with calcium oxalate crystals and their nelghbouring cells has been studied by light and transmission electron microscopy at different stages of bean leaf development. Plants were grown with varying calcium supply to identify a possible influence of calcium nutrition on cell structure. Crystals are formed inside the vacuole of already highly vacuolated cells of bundle sheath extensions. The membrane around the crystal vacuole is continuous with the plasmalemma. The crystal vacuole contains membraneous structures. In the fully expanded leaf the crystal becomes ensheathed by wall material. Chloroplasts of bundle sheath extension cells, with or without crystals, are smaller, with fewer membranes, and with much narrower stroma regions than those of the palisade parenchyma. There is a stage in the young leaf when only the bundle sheath extension cells without crystals have starch grains in their chloroplasts. As their number is lower in plants grown with high calcium supply this means that, in this case, less cells are competent for photosynthesis.     

Hypobaric Control of Ethylene-Induced Leaf Senescence in Intact Plants of Phaseolus vulgaris L

A controlled atmospheric-environment system (CAES) designed to sustain normal or hypobaric ambient growing conditions was developed, described, and evaluated for its effectiveness as a research tool capable of controlling ethylene-induced leaf senescence in intact plants of Phaseolus vulgaris L.

Senescence was prematurely-induced in primary leaves by treatment with 30 parts per million ethephon. Ethephon-derived endogenous ethylene reached peak levels within 6 hours at 26°C. Total endogenous ethylene levels then temporarily stabilized at approximately 1.75 microliters per liter from 6 to 24 hours. Thereafter, a progressive rise in ethylene resulted from leaf tissue metabolism and release. Throughout the study, the endogenous ethylene content of ethephon-treated leaves was greater than that of nontreated leaves.

Subjecting ethephon-treated leaves to atmospheres of 200 millibars, with O₂ and CO₂ compositions set to approximate normal atmospheric partial pressures, prevented chlorophyll loss. Alternately, subjecting ethephon-treated plants to 200 millibars of air only partially prevented chlorophyll loss. Hypobaric conditions (200 millibars), with O₂ and CO₂ at normal atmospheric availability, could be delayed until 48 hours after ethephon treatment and still prevent most leaf senescence. In conclusion, hypobaric conditions established and maintained within the CAES prevented ethylene-induced senescence (chlorosis) in intact plants, provided O₂ and CO₂ partial pressures were maintained at levels approximating normal ambient availability.

An unexpected increase in endogenous ethylene was detected within nontreated control leaves 48 hours subsequent to relocation from winter greenhouse conditions (latitude, 42°00″ N) to the CAES operating at normal ambient pressure. The longer photoperiod and/or higher temperature utilized within the CAES are hypothesized to influence ethylene metabolism directly and growth-promotive processes (e.g. response thresholds) indirectly.

     

Expression of Two Different Glutamine Synthetase Polypeptides during Root Development in Phaseolus vulgaris L

Immunoprecipitation and two-dimensional gel electrophoresis analysis of the glutamine synthetase (GS) polypeptides (α and β) during Phaseolus vulgaris root development shows that the α polypeptide is the main component of the enzyme in the embryo and in up to 5 day old roots. From 5 days on, the β polypeptide becomes the root predominant GS monomer. The α/β ratio of the in vitro translated GS polypeptides from the total polysomal RNA isolated at different root ages correlates with the α/β ratio observed in the root extracts. These results suggest that the two root GS polypeptides are encoded by different mRNA species in Phaseolus vulgaris.