Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Natural Inhibitor for Volatile C6-Aldehyde Formation from C18-Unsaturated Fatty Acids

The homogenate of tea seed cotyledons contained an inhibitor for C₆-aldehyde formation from linoleic acid and linolenic acid by isolated tea chloroplasts. Seed homogenates of other plants, such as soybean, kidney bean, cucumber, Japanese radish and rice, also contained the inhibitor for C₆-aldehyde formation. The inhibitor from tea seed and cucumber seed inhibited C₆-aldehyde formation by the homogenate of cucumber hypocotyl. Hydroperoxides of linoleic acid detected were reduced when the tea seed inhibitor was added to the reaction mixture, but the enzyme activities of lipoxygenase and hydroperoxide lyase were not inhibited. This means that the inhibitor is a decomposer of fatty acid hydroperoxides as an intermediate of C₆-aldehyde formation. The tea seed inhibitor was formed during the seed ripening and it was stable during the seed germination. These findings obtained here suggest that the inhibitor is widely present in plant seeds and inhibits C₆-aldehyde formation by a variety of plant tissues.     

Volatile C6-Aldehyde Formation via Hydroperoxides from C18-Unsaturated Fatty Acids in Etiolated Alfalfa and Cucumber Seedlings

1. Etiolated seedlings of alfalfa and cucumber evolved n-hexanal from linoleic acid and cis-3-hexenal and trans-2-hexenal from linolenic acid when they were homogenized.

2. The activities for n-hexanal formation from linoleic acid, lipoxygenase and hydro-peroxide lyase were maximum in dry seeds and 1~2 day-old etiolated seedlings of alfalfa, and in 6~7 day-old etiolated seedlings of cucumber.

3. n-Hexanal was produced from linoleic acid and 13-hydroperoxylinoleic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. cis-3-Hexenal and trans-2-hexenal were produced from linolenic acid and 13-hydroperoxylinolenic acid by the crude extracts of etiolated alfalfa and cucumber seedlings. But these extracts, particulariy cucumber one, showed a high isomerizing activity from cis-3-hexenal to trans-2-hexenal.

4. When the C₈-aldehydes were produced from linoleic acid and linolenic acid by the crude extracts, formation of hydroperoxides of these C₁₈-fatty acids was observed.

5. When 9-hydroperoxylinoleic acid was used as a substrate, trans-2-nonenal was produced by the cucumber homogenate but not by the alfalfa homogenate.

6. As the enzymes concerned with C₆-aldehyde formation, lipoxygenase was partially purified from alfalfa and cucumber seedlings and hydroperoxide lyase, from cucumber seedlings. Lipoxygenase was found in a soluble fraction, but hydroperoxide lyase was in a membrane bound form. Alfalfa lipoxygenase catalyzed formation of 9- and 13-hydroperoxylinoleic acid (35: 65) from linoleic acid and cucumber one, mainly 13-hydroperoxylinoleic acid formation. Alfalfa hydroperoxide lyase catalyzed n-hexanal formation from 13-hydroperoxylinoleic acid, but cucumber one catalyzed formation of n-hexanal and trans-2-nonenal from 13- and 9-hydroperoxylinoleic acid, respectively.

7. From the above results, the biosynthetic pathway for C₆-aldehyde formation in etiolated alfalfa and cucumber seedlings is established that C₆-aldehydes (n-hexanal, cis-3-hexenal and trans-2-hexenal) are produced from linoleic acid and linolenic acid via their 13-hydroperoxides by lipoxygenase and hydroperoxide lyase.     

Volatile Products of the Lipoxygenase Pathway Evolved from Phaseolus vulgaris (L.) Leaves Inoculated with Pseudomonas syringae pv phaseolicola

Activation of the "lipoxygenase pathway" in plants gives rise to a series of products derived from fatty acids. Analysis by gas chromatography-mass spectroscopy of volatile products produced by Phaseolus vulgaris (L.) cv Red Mexican leaves during a hypersensitive resistance response (HR) to the plant pathogenic bacterium Pseudomonas syringae pv phaseolicola showed evolution of several lipid-derived volatiles, including cis-3-hexenol and trans-2-hexenal, which arise from the 13-hydroperoxide of linolenic acid. These compounds were not produced in detectable amounts by buffer-inoculated leaves, nor did they evolve to such a high degree during comparable stages of the susceptible response. The absence of trans-2,cis-6-nonadienal, a product expected from 9-hydroperoxide of linolenic acid, suggests that lipid peroxidation during the HR proceeded primarily enzymically via bean lipoxygenase, which produces the 13-hydroperoxide, and not via autoxidative processes. The effects of trans-2-hexenal, cis-3-hexenol, and traumatic acid on P.s pv phaseolicola were investigaed. trans-2-Hexenal appeared to be highly bactericidal at low concentrations, whereas cis-3-hexenol was bactericidal only at much higher concentrations. Traumatic acid appeared to have no effect on P.s. pv. phaseolicola at the concentrations tested. These results demonstrate that during plant defense responses against microbial attack, several lipid-derived compounds are produced by the plant, some of which possess antimicrobial activity and conceivably are involved in plant disease resistance. The time of production of these substances, in amounts that would be expected to be antibacterial in vitro, correlated with a slowing down of the growth rate of bacteria in the leaves and was seen at a time before the accumulation of isoflavonoid phytoalexins in the host.     

Sodium Recirculation and Loss from Phaseolus vulgaris L.

In a split-root experiment, ²²Na was supplied to Phaseolus vulgarisL. roots emerging from the stem, 2.5 cm above the main roots.Sodium exported from these upper roots was translocated a shortdistance upward in the stem and downward to the main roots.Most of the ²²Na arriving in the main roots was lost to themedium. Sodium loss from P. vulgaris roots into KCI or NaCl was similarand was not affected by oligomycin. The results confirm a previous hypothesis regarding the mechanismof sodium exclusion from the tops of sodium non-accumulatorplants. Phaseolus vulgaris L., bean, sodium transport     

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.     

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.     

Isozymes of Glutamine Synthetase in Phaseolus vulgaris L. and Phaseolus lunatus L. Root Nodules

The glutamine synthetase (GS) isozymes in the plant fraction of nodule extracts from 62 cultivars of Phaseolus vulgaris L. and one cultivar of Phaseolus lunatus L. were analyzed by polyacrylamide gel electrophoresis. All P. vulgaris nodule extracts displayed two GS activity bands: a nodule-specific band (GS_n1) and a band (GS_n2) similar to the single band (GS_r) present in root extracts. In nodule extracts of P. lunatus, the GS_n1 band was detected, but the GS_n2 band was barely detectable. In contrast to P. vulgaris, the GS_n2 band and the GS_r band of P. lunatus appeared to be different. The electrophoretic mobility of the GS_n1 band in P. vulgaris was governed by both the plant cultivar and the development stage of the nodule. In nodule extracts of P. vulgaris and P. lunatus, the zone of GS_n1 activity coincided with six to nine distinct protein bands as revealed after treatment of gels, which had previously been stained for GS activity, with Coomassie blue. All these protein bands were shown to consist of polypeptides of identical molecular weight (approximately 47,000 daltons) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results indicate that P. vulgaris continuously generates isozymes of GS_n1 of increasing electrophoretic mobility during the course of nodule development.     

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.     

Dihydrozeatin riboside,a minor cytokinin from the leaves of Phaseolus vulgaris L.

Dihydrozeatin riboside has been identified in the leaves of decapitated bean plants by Sephadex LH20 chromatography and combined gas chromatography-mass spectrometry. The relationship between the cytokinins isolated and identified from this system and those previously reported in Phaseolus is discussed.Abbreviations DHZ dihydrozeatin - DHZOG dihydrozeatin-O--D-glucoside - DHZR dihydrozeatin riboside - GCMS combined gas chromatography-mass spectrometry - GLC gas-liquid chromatography - TIC total ion current - TMS trimethylsilyl - Z zeatin - ZR zeatin riboside     

Regeneration in Phaseolus vulgaris L. Promotive role of N6-benzylaminopurine in cultures from juvenile leaves

Plants were regenerated from cultured young leaves of Phaseolus vulgaris L. cv. Kinghorn. For inducing shoot regeneration the expiant had to consist of the petiole and a portion of the lamina, and N⁶-benzylaminopurine (BAP) had to be present in the culture medium. Furthermore, the frequency of shoot regeneration increased more than seven-fold if donor seedlings were raised on a medium containing 5 M BAP, followed by culture of the leaf explants on a medium containing 20 M BAP. Regenerated shoots developed roots on basal (hormone-free) medium and the resulting plantlets could be transplanted to soil.Abbreviations BAP N⁶-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) medium This research was supported by operating grants from the Research Board Grants Program of the University of Guelph and the Natural Sciences and Engineering Research Council of Canada to PKS. Technical and photographic assistance from Sangeeta Saxena and Jean Gerrath is gratefully acknowledged.     

The Development of Flower Primordia of Phaseolus vulgaris (L.) Savi.

The first-formed flower primordium on determinate varieties(plants with terminal inflorescences) of Phaseolus vulgarishas been identified and located. Irrespective of the numberof leaves on the main stem, it is formed directly in the axilof the uppermost leaf. The physiological significance of thisis discussed.     

Partial purification of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

The solubilised ethylene-binding site (EBS) of Phaseolus vulgaris L. cotyledons is an asymmetrical protein with a sedimentation coefficient of 2 S and a Stoke's radius of 6.1 nm (determined by ultracentrifugation on isokinetic gradients and gel-permeation chromatography, respectively). The molecular weight and frictional ratio were calculated as 52 000–60 000 and 2.37–2.48, respectively. The EBS has an isoelectric point at between pH 3–5, determined by isoelectric focussing and exhibits a negative charge at pH 8 during non-denaturing electrophoresis. The electrical charge on the EBS is shielded; the EBS does not bind to anion-exchange media under the experimental conditions reported here, is not precipitated by ammonium sulphate and does not precipitate at its isoelectric pH. The EBS preferentially partitions into detergent phases. The results indicate that the EBS is a hydrophobic protein complexed with detergent in aqueous solution. The techniques used to characterise the EBS also resulted in varying degress of purification.Abbreviations EBS ethylene-binding site - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)-ethyl]glycine - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol     

The influence of aluminium availability on phosphate uptake in Phaseolus vulgaris L. and Phaseolus lunatus L.

Aluminium toxicity is one of the major limiting factors of crop productivity on acid soils. High levels of available aluminium in soil may induce phosphorus deficiency in plants. This study investigates the influence of Aluminium (Al) on the phosphate (P_i) uptake of two Phaseolus species, Phaseolus vulgaris L. var. Red Kidney and Phaseolus lunatus L. The two bean species were treated first with solutions of Al at different concentrations (0, 25, 50 and 100 μM, pH 4.50) and second with solutions of P_i (150 μM) at pH 4.50. The higher the Al concentration the higher the Al concentration sorbed but P. vulgaris L var. Red Kidney adsorbed significantly more Al than P. lunatus L. Both species released organic acids: P. vulgaris L var. Red Kidney released fumaric acid and P. lunatus L. fumaric and oxalic acids which could have hindered further Al uptake.The two bean species showed a sigmoid P_i uptake trend but with two different mechanisms. P. vulgaris L var. Red Kidney showed a starting point of 3 h whereas P. lunatus L. adsorbed P_i immediately within the first minutes. In addition, P. vulgaris L var. Red Kidney presented significantly higher P_i uptake (higher uptake rate ‘k’ and higher maximum adsorption ‘a’ of the kinetic uptake model). The Al treatments did not significantly influence P_i uptake. Results suggest that P. lunatus L. might adopt an external Al detoxification mechanism by the release of oxalic acid. P. vulgaris L var. Red Kidney on the other hand seemed to adopt an internal detoxification mechanism even if the Al sorbed is poorly translocated into the shoots. More detailed studies will be necessary to better define Al tolerance and/or resistance of Phaseolus spp.     

Late Transplanting and the Yield of Phaseolus vulgaris L.

The prediction that very high seed yields of dry beans (Phaseolusvulgaris L.) would be produced by the delayed transplantingof large plants has been tested in a factorial experiment withfour dates of transplanting and eight plant populations. Therewere significant differences in yield between transplantingdates and between population densities, and there was a significantdate-density interaction. At low plant densities (up to about30 plants m^–2) the three transplanted treatments yieldedless than the hand-sown controls, and late transplanting yieldedless than early. At the highest density the situation was reversed;all three transplanted treatments out-yielded the controls andlate transplanting tended to out-yield plants transplanted early.The biggest yield was 340 g seed m^–2 from a transplantedcrop grown at 35 plants m^–2. The data on yield fitted a modified rectangular hyperbola ofthe form where y is yield per unit area, p is the number of plants perunit area, t is the number of days between sowing and transplanting,and B_o, n, m, and p are arbitrary parameters. This equationaccounted for 91 per cent of the variation in yield with t andp. It is suggested that late transplanting had adverse effects,due to transplanting ‘shock’ and which were mostmarked at low plant densities; and beneficial effects, ascribableto an effect on plant ‘plasticity’, which were mostmarked at high plant densities. Possible physiological mechanismsof these effects are discussed. Phaseolus vulgaris, yield, density, transplanting     

Protein Breakdown and Formation of Protease in Attached and Detached Cotyledons of Phaseolus vulgaris L

In contrast to earlier reported results of similar experiments in peas, in which almost no increase in protease activity occurred in incubated detached cotyledons, we report here an increase in protease activity in both attached and detached bean cotyledons. Detached bean cotyledons showed continually increasing protease activity up to the 12th day, while that in attached cotyledons declined after 6 days. The free amino acid level in detached cotyledons reached a maximum at the 11th day; protease formation leveled off after 50% of the original seed protein was digested. These data suggest that high free amino acid levels may inhibit protease formation.     

Purification and Characterization of a Polygalacturonase-Inhibiting Protein from Phaseolus vulgaris L

Homogeneous endo-polygalacturonase (PG) was covalently bound to cyanogen-bromide-activated Sepharose, and the resulting PG-Sepharose conjugate was utilized to purify, by affinity chromatography, a protein from Phaseolus vulgaris hypocotyls that binds to and inhibits PG. Isoelectric focusing of the purified PG-inhibiting protein (PGIP) showed a major protein band that coincided with PG-inhibiting activity. PGIP formed a complex with PG at pH 5.0 and at low salt concentrations. The complex dissociated in 0.5 m Na-acetate and pH values lower than 4.5 or higher than 6.0. Formation of the PG-PGIP complex resulted in complete inhibition of PG activity. PG activity was restored upon dissociation of the complex. The protein exhibited inhibitory activity toward PGs from Colletotrichum lindemuthianum, Fusarium moniliforme and Aspergillus niger. The possible role of PGIP in regulating the activity of fungal PG's and their ability to elicit plant defense reactions are discussed.     

Five primary trisomics from translocation heterozygote progenies in common bean,Phaseolus vulgaris L.

Summary Twelve distinct phenotypic groups of plants were isolated from nondisjunction progenies of 11 translocation heterozygote stocks. All the plants in these phenotypic groups originated in the light weight seed class. Five of the 12 phenotypic groups of plants have been verified as primary trisomics. They are all phenotypically distinguishable from each other and from disomics. One of the five primary trisomic groups, puckered leaf, was directly recovered as a primary trisomic from the original translocation heterozygote progenies. Three of the five trisomics — weak stem, dark green leaf, and convex leaf — originated first as tertiary trisomics. The related primary trisomics were isolated later from progenies of selfed tertiary trisomics. The fifth group, chlorotic leaf, originated at a low frequency among the progenies of three other trisomics: puckered leaf, convex leaf, and dark green leaf. The chlorotic leaf did not set seed under field conditions. The remaining four groups — puckered leaf, dark green leaf, convex leaf, and weak stem — are fertile, though sensitive to high temperature conditions. The transmission rate of the extra chromosome on selfing ranges from 28% to 41%. Physical identification of the extra chromosome has not been achieved for any of the five trisomic groups. Two trisomic groups, dark green leaf and convex leaf, have produced tetrasomics at low frequency. The phenotypes of these two tetrasomics are similar to the corresponding trisomics but more exaggerated.Fla. Agr. Expt. Stn. Journal Series No. 7137