Effects of pod removal on the transport and accumulation of abscisic Acid and indole-3-acetic Acid in soybean leaves

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the second most recently expanded trifoliolate leaf were determined during reproductive development of soybean (Glycine max [L.] Merr cv `Chippewa 64'). The concentration of ABA in leaves was constant during most of the seed filling period until the seeds began to dry. The concentration of IAA in the leaves decreased throughout development. Removal of pods 36 hours prior to sampling resulted in increased concentrations of ABA in leaves during the period of rapid pod filling but had little effect on the concentration of IAA in leaves. ABA appears to accumulate in leaves after fruit removal only when fruits represent the major sink for photosynthate.

ABA and IAA moving acropetally and basipetally in petioles of soybean were estimated using a phloem exudation technique. ABA was found to move mostly in the basipetal direction in petioles (away from laminae). IAA, primarily in the form of ester conjugate(s), was found to be moving acropetally (toward laminae) in petioles. The highest amount of IAA ester(s) was found in petiole exudate during the mid and late stages of seed filling. Removal of fruits 36 hours prior to exudation reduced the amount of IAA ester recovered in exudate, suggesting that fruits were a source of the IAA conjugate in petiole exudate.

     

The influence of the extraction procedure on yield of indole-3-acetic acid in plant extracts

Different types of plant material, including both dry and swollen maize kernels, swollen bean seeds, bean seedlings and dry rose seeds, were extracted by different methods and the yield of IAA was determined with the indolo-α-pyrone method. Extraction of dry maize kernels during short time experiments, varying from 3 to 24 h, gave the highest IAA yield when methanol was the extractant and a significant lower yield when diethyl ether or dichloromethane were used. The duration of the extraction period increased the yield with all the extractants. Progressive extractions for several days or weeks had little effect on the yield when 100% acetone was used in contrast to methanol and ether as extractants, which increased the yield during prolonged extraction. Extractions of tissue treated to 100°C for 1 h contradicted the hypothesis that IAA is enzymatically liberated during ether extraction. Water in the extractant solvents increased the yields. This was most pronounced when aqueous acetone was used instead of 100% acetone. Increased extraction temperature augmented the IAA yields. The yield of IAA from other types of tissue extracted with methanol for periods of 3 or 24 h was, however, independent of the duration of the extraction time. This indicates that some tissues contain less not easily extractable IAA than dry maize kernels. The terms “free” and “bound” IAA are discussed; they should be replaced by “easily extractable” and “not easily extractable” IAA. The results also show that IPyA in vitro can partly be converted to IAA during extraction and fractionation.     

The influence of kinetin on the endogenous content of indoleacetic acid in swelling seeds of Phaseolus, Zea and Pinus and young plants of Phaseolus

Treatment of different plant materials, seeds of Phaseolus vulgaris, Zea mays and Pinus silvestris and young plants of Phaseolus, with kinetin increased the level of extractable IAA. For seeds this increase was most pronounced in bean seeds, which contained the lowest amount of endogenous IAA and cytokinins, and lower in maize seeds with high endogenous content of IAA and cytokinins. – For young bean plants the kinetin treatment significantly increased the extractable amounts of IAA from all parts of the plant, hypocotyls, cotyledons, epicotyls and primary leaves, when the cut plants were placed for 24 h in kinetin solution. For plants sprayed with kinetin solution only the primary leaves showed a significantly higher level of extractable IAA, which could be explained by the fact that the plants were growing very close together, so that the primary leaves received most of the kinetin during spraying.     

Quantitation of indoleacetic Acid conjugates in bean seeds by direct tissue hydrolysis

Gas chromatography-selected ion monitoring-mass spectral analysis using [¹³C₆]indole-3-acetic acid (IAA) as an internal standard provides an effective means for quantitation of IAA liberated during direct strong basic hydrolysis of bean (Phaseolus vulgaris L.) seed powder, provided that extra precautions are undertaken to exclude oxygen from the reaction vial. Direct seed powder hydrolysis revealed that the major portion of amide IAA conjugates in bean seeds are not extractable by aqueous acetone, the solvent used commonly for IAA conjugate extraction from seeds and other plant tissues. Strong basic hydrolysis of plant tissue can be used to provide new information on IAA content.     

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.     

The Appearance of New Active Forms of Trypsin Inhibitor in Germinating Mung Bean (Vigna radiata) Seeds

Ungerminated seeds of mung bean contain a single major species (F) of trypsin inhibitor with five minor species (A-E) separable on diethylaminoethyl-cellulose. During germination the level of trypsin inhibitory activity decreases from 1.8 units/grams dry weight in ungerminated cotyledons to 1.2 units/grams in cotyledons from seeds germinated 5 days. This decrease is accompanied by major changes in the distribution of inhibitory activity among the inhibitor species. By 48 hours of germination, inhibitor F has largely disappeared with an accompanying rapid increase in inhibitor C. Similarly, though less rapidly, inhibitor E decreases while inhibitor A increases. A similar sequence of changes is found in vitro when purified inhibitor F is incubated with extracts from seeds germinated 96 hours. The combined in vivo and in vitro data suggest a conversion sequence of: F → E → C → A. The in vitro conversion is inhibited by phenylmethyl sulfonyl fluoride but not by iodoacetamide, indicating that at least the initial phases of inhibitor conversion are not catalyzed by the mung bean vicilin peptidohydrolase.     

Malate synthase activity in cotton and other ungerminated oilseeds: a survey

Extracts from several species and varieties of ungerminated cotton seeds plus homogenates from 18 other oilseeds (representing 11 different families) were examined for malate synthase and isocitrate lyase activity. Malate synthase activities in the various cotton seeds ranged from 35 to 129% of the units per dry seed weight found in Deltapine 16 cotton. For other oilseeds, the range was from 0.3 to 58% of Deltapine 16 cotton. Castor bean (Ricinus communis L.) had the least activity per mg dry weight (12-fold lower than the next lowest species), while Pima cotton (Gossypium barbadense L.) had the highest level (8.53 units). On a per seed basis, these values were 15 and 747 nanomoles per minute.     

Deoxyribonucleic Acid, Ribonucleic Acid, Protein and Uncombined Amino Acid Content of Legume Seeds During Embryogeny

The nucleic acid, protein and uncombined amino acid contentof seeds of soya-bean (Glycine max L. Merr.), garden pea (Pisumsativum L.), kidney bean (Phaseolus vulgaris L.) and peanut(Arachis hypogaea L.) were measured at various times duringseed formation in an effort to understand why the soya-beanhas nearly twice as much protein as the other legume seeds.In all these species the concentration of deoxyribonucleic acid,ribonucleic acid and uncombined amino acids decreased duringseed formation. The protein level of kidney bean was relativelyconstant during development whereas the protein levels of pea,peanut and soya-bean increased during development. The proteincontent of the soya-bean increased throughout development whereasthe protein increase in peanut took place early and that inpea took place later in development. The ratio of protein toribonucleic acid was highest in peanut, less in soya-bean, andlowest in pea and kidney bean. Similarly, the ratio of proteinto deoxyribonucleic acid was higher in kidney bean than in soya-bean.Soya-beans had a lower amino acid content than any of the otherseeds at all stages of development. These results indicate thatneither total deoxyribonucleic acid, ribonucleic acid nor uncombinedamino acid content is responsible for the higher protein contentof soya-beans.     

Free and conjugated indole-3-acetic Acid in developing bean seeds

The changes in conjugated indole-3-acetic acid (IAA) levels compared to the levels of free IAA have been analyzed during the development of bean (Phaseolus vulgaris L.) seed using quantitative mass spectrometry. Free and ester-linked IAA levels are both relatively high in the early stages of seed development but drop during seed maturation. Concomitantly, the amide-linked IAA becomes the major form of IAA present as the seed matures. In fully mature seed, amide IAA accounts for 80% of the total IAA. The total IAA pool in the seed is maintained at approximately the same level (150-170 nanograms/seed) once the level of free IAA has attained its maximum. Thus, the amount of amide IAA conjugates that accumulate in mature seed is closely related to the amounts of free and ester-linked IAA that disappeared from the rapidly growing seed. Analysis of developing bean pods, from which the seeds were taken for analysis, showed very low levels of both ester and amide-linked IAA conjugates. The pattern of changes seen in the levels of free and conjugated IAA in developing bean seed supports our prior hypothesis suggesting a role of IAA conjugates in the storage of the phytohormone in the seed.     

Amide-Linked Indoleacetic Acid Conjugates May Control Levels of Indoleacetic Acid in Germinating Seedlings of Phaseolus vulgaris

We have shown that amide-linked IAA (indole-3-acetic acid) conjugates accumulated to high levels during maturation of bean seeds (K. Bialek and J.D. Cohen [1989] Plant Physiol 91: 775-779). In the present study, we were interested in the fate of these and other IAA conjugates during seed germination. The content of amide-linked conjugates of IAA in cotyledons declined dramatically during the first hours of imbibition. The rate of decline slowed markedly during the period of the resumption of axis growth. The level of amide-linked IAA conjugates in cotyledons remained relatively high after almost 1 week of germination. The decline of IAA conjugates in cotyledons was followed by a steady increase in the content of both free and amide-linked IAA in the embryonic axes. Amide-linked IAA conjugates were also present in the axes cultured on agar after the cotyledons were removed, which suggests that de novo production of these IAA conjugates occurs in the axis of germinating bean seedlings. A comparison of relative amounts of free and conjugated IAA in the axes of intact seedlings and axes cultured on agar showed lower levels of free IAA and higher levels of conjugated IAA in much slower growing isolated axes. These results suggest a more general role for IAA conjugates in the control of seedling growth than simply to serve as a seed storage form of auxin.     

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims

Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination.

Methods

Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC.

Key Results

During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied.

Conclusions

The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.     

Concentrations of Abscisic Acid and Indole-3-Acetic Acid in Soybean Seeds during Development

Concentrations of abscisic acid (ABA) and indole-3-acetic acid (IAA) in seed parts were determined during reproductive development of soybean plants (Glycine max [L.] Merr. cv `Chippewa 64'). The concentration of ABA and IAA changed independently in individual seed parts with time. Measurement of the level of ABA and IAA in whole seeds masked the changes which occurred in individual seed tissues. The concentration of ABA was generally highest and that of IAA was generally lowest in the embryonic axis of soybean seeds. In the testa, the IAA concentration was generally highest while the ABA concentration was generally the lowest compared to other parts of the seed.     

Role of the testa in preventing cellular rupture during imbibition of legume seeds

Studies with the seeds of soybean, navy bean, pea, and peanut were made to determine the extent of leakage of intracellular enzymes during imbition. Embryos with intact testae from all four species were found to leak detectable activities of either intracellular enzymes of the cytosol (glucose-6-phosphate dehydrogenase) or enzymes found in both the cytosol and organelles (malate dehydrogenase, glutamate dehydrogenase, glutamate oxaloacetate transaminase, and NADP-isocitrate dehydrogenase) after 6 hours imbition at 25 C. Pea and peanut embryos with testae leaked considerably lower levels of activity for these enzymes than did those of soybean and bean. Leakage of mitochondrial marker enzymes (fumarase, cytochrome c oxidase, and adenylate kinase) was not detected from embryos with testae, suggesting that a differential diffusion of intracellular components out of cells occurred. Soybean and bean embryos without testae leaked high, and proportionally (per cent dry seed basis) similar, levels of all cytosol, cytosol-organelle, and mitochondrial marker enzymes and protein during imbibition, indicating that cell membranes were not differential to leakage and that they had ruptured. Pea and peanut embryos without testae leaked detectable activities of all cytosol and cytosol-organelle enzymes, although fumarase was the only detectable mitochondrial marker enzyme leaked, suggesting that some degree of differential leakage may have occurred in these species. The outermost layers of embryo cells of seeds without testae of all four species absorbed and sequestered the nonpermeating pigment Evan's blue after 5 to 15 minutes imbibition, indicating that membranes had ruptured. This occurred to a much lesser extent in seeds with intact testae. Both soybean and bean embryos without testae were observed to disintegrate during imbibition, whereas those of pea and peanut did not. These data indicate that seeds of certain legumes are susceptible to cellular rupture during imbibition when seed coats are damaged or missing.     

Influence of environmental,geographic, and seasonal variations in the chemical composition of Miconia species from Cerrado

Miconia albicans (Sw.) Triana and Miconia chamissois Naudin, commonly known as “pixirica” and “pixirica-açu”, two Melastomataceae species, are commonly found in Cerrado areas. M. albicans species grows in dry and humid habitats, so it has adapted to both conditions and M. chamissois species grows in humid environment only. In this work, we have investigated the content of triterpenes, flavonols and flavanones in three different Cerrado fragments of São Paulo State, involving distinct environments (dry and humid) and seasons (dry and rainy) to comprehend the complex interactions among plants and the seasonal, environmental conditions and geographic locations. The leaves plants materials were harvest in August 2016 (dry season) and November/December 2016 (rainy season) in Pirassununga, Pedregulho and Luíz Antônio in dry and humid environments. The contents of the standards rutin (R), quercetin (Q), miconioside B (B), matteucinol 7-O-β-apiofuranosyl(1 → 6)-β-glucopyranoside (matt), ursolic acid (AU), and oleanolic acid (AO) were determined by HPLC-DAD. The data were analyzed using nonparametric tests, Pearson's linear correlation, principal component analysis and hierarchical cluster analysis. The results demonstrated similar flavanone and triterpene production during the dry period, and similar flavonol and flavanone production in the rainy season, thus confirming a seasonal variation in the content of the compounds in the evaluated specimens. Our results also demonstrated intra- and inter-population variations in compounds patterns, M. albicans contained major amounts of the flavonols R and Q; their concentrations were higher in the rainy season. B and matt were the major compounds in M. chamissois; the matt concentration decreased in the rainy season. The triterpenes AU and AO occurred in both M. albicans and M. chamissois, but their production dropped during the rainy periods. Thus corroborating the adaptation of M. albicans and M. chamissonis to the high stress and low levels of resources presented in Cerrado.     

The release of proteinase inhibitors from legume seeds during germination

The seeds of twelve common species of legumes were examined for the release of proteinase inhibitor activity during germination. All species released inhibitory activity against bovine trypsin (EC 3.4.21.4), ranging from 1.0 unit per g dry wt. of seed in 24 hr for soybean (Glycine max), to 0.07 unit per g for broad beans (Vicia faba) and sugar pod peas (Pisum sativum). This release corresponds to approximately 1–13 % of the total trypsin inhibitory activity of the seed, with lentils (Lens culinaris) releasing the greatest percentage, and the scarlet runner bean (Phaseolus coccineus) the least. In most species the amount of inhibitor released increases until 24–48 hr of germination, and then remains roughly the same or decreases slightly by 72 hr of germination. Five species of legumes were also examined for the release of inhibitory activity against bovine chymotrypsin (EC 3.4.21.1). In each case chymotryptic inhibitory activity was released in a manner similar to the trypsin inhibitor.     

Dormancy Mechanism and the Effect of Scarification in the Germination of Halimium halimifolium Seeds

Germination of Halimium halimifolium seeds was very low during the experimental period. The scarification treatment enhanced the germination significantly at all the three temperatures tested, indicating that H. halimifolium seeds are endowed with complex dormancy mechanisms. The highest temperature (30°C) showed an adverse effect which can be correlated with a secondary dormancy at embryo level.The lipidic nature of H. halimifolium exotesta is probably the first factor causing dormancy in this species, acting both as a water and oxygen barrier. We can conclude that H halimifolium shows a tegumentary dormancy, which is presumably located at exotesta level.     

The effect of faba bean extrusion on the growth performance,nutrient utilization,metabolizable energy,excretion of sialic acids and meat quality of broiler chickens

Soybean meal in broiler chicken diets can partly be replaced by faba bean seeds. Unfortunately, high levels of antinutritional factors and resistant starch found in these seeds can have a detrimental impact on both broiler chickens’ performance and nutrient digestibility. It is, however, possible to increase the usefulness of faba bean for broiler nutrition by a technological process known as extrusion. In this study, the authors made and attempt to investigate the effect of different forms of faba bean seeds (raw or extruded) on broiler chicken performance, nitrogen-corrected apparent metabolizable energy values (AME_N), nutrient utilization and meat quality, as well as on the excretion of total and free sialic acids. In the trial, the total of 160 1-day-old male broiler chicks of the Ross 308 strain were used. Experimental birds were randomly assigned to two dietary treatments, each containing 10 replication (floor pens); one replication included nine birds. The first group contained 300 g/kg diet of raw faba bean seeds, whereas the second group – 300 g/kg diet of extruded faba bean seeds. The applied extrusion process was found to exert a positive impact and led to a decrease in phytic phosphorus, H, NDF, ADF and resistant starch content in studied faba bean seeds. Experimental birds fed diets containing extruded faba bean seeds were characterized by a lower feed intake (2299 g) and feed conversion ratios (FCRs) (1.52 g/g) in comparison with the other group (feed intake 2466 g; FCR 1.61 g/g). Extrusion of faba bean seeds improved dry matter retention, dietary AME_N value, apparent ileal digestibility of dry matter and starch, as well as most amino acids. In addition, the above-mentioned process resulted in a decrease in the excretion of total and free sialic acids. Extruded faba bean seeds failed to have a significant impact on broiler meat quality. It was concluded that application of extrusion can increase the use of faba bean seeds in broiler chicken nutrition.     

Bioaccumulation of Trace Mercury in Trophic Levels of Benthic, Benthopelagic, Pelagic Fish Species, and Sea Birds from Arvand River, Iran

In this study, concentration of mercury was determined in the trophic levels of benthic, benthopelagic, pelagic fish species, and river birds from Arvand River, located in the Khuzestan province in the lowlands of southwestern Iran at the head of the Persian Gulf. The order of mercury concentrations in tissues of the fish species was as follows: liver>gill>muscle and in tissues of the kingfisher species was as follows: feather>liver>kidney>muscle. Therefore, liver in fish and feather in kingfisher exhibited higher mercury concentration than the other tissues. There was a positive correlation between mercury concentrations in fish and kingfisher species with size of its food items. We expected to see higher mercury levels in tissues of female species because they are larger and can eat larger food items. The results of this study show that the highest mean mercury level were found in the kingfisher (Anas crecca), followed by benthic (Epinephelus diacanthus), benthopelagic (Chanos chanos), and pelagic fish (Strongylura strongylura). Mean value of mercury in fish species, S. strongylura were (0.61 μg g^?1 dry weight), C. chanos (0.45 μg g^?1 dry weight), E. diacanthus (0.87 μg g^?1 dry weight), and in kingfisher species A. crecca was (2.64 μg g^?1 dry weight). Significant correlation between mercury concentration in fish and kingfisher may be related to high variability of mercury in the fish.     

Indole-3-acetic acid protein conjugates: novel players in auxin homeostasis

Indole-3-acetic acid (IAA) is found in plants in both free and conjugated forms. Within the group of conjugated IAA there is a unique class of proteins and peptides where IAA is attached directly to the polypeptide structure as a prosthetic group. The first gene, IAP1, encoding for a protein with IAA as a prosthetic group, was cloned from bean (Phaseolus vulgaris). It was shown that the expression of IAP1 as a major IAA modified protein in bean seed (PvIAP1) was correlated to a developmental period of rapid growth during seed development. Moreover, this protein underwent rapid degradation during germination. Since further molecular analysis was difficult in bean, the IAP1 gene was transformed into Arabidopsis thaliana and Medicago truncatula. Expression of the bean IAP1 gene in both plant species under the control of its native promoter targeted protein expression to the seeds. In Arabidopsis no IAA was found to be attached to PvIAP1. These results show that there is specificity to protein modification by IAA and suggests that protein conjugation may be catalyzed by species specific enzymes. Furthermore, subcellular localization showed that in Arabidopsis PvIAP1 was predominantly associated with the microsomal fraction. In addition, a related protein and several smaller peptides that are conjugated to IAA were identified in Arabidopsis. Further research on this novel class of proteins from Arabidopsis will both advance our knowledge of IAA proteins and explore aspects of auxin homeostasis that were not fully revealed by studies of free IAA and lower molecular weight conjugates.     

Auxin production by the Klebsiella planticola strain TSKhA-91 and its effect on development of cucumber (Cucumis sativus L.) seeds

Capacity of Klebsiella planticola strain TSKhA-91 for synthesis of indole-3-acetic acid (IAA) and other auxins was studied. The qualitative and quantitative composition of these compounds depends on the presence of exogenous tryptophan and on the nitrogen source. The highest IAA yield was obtained at the stationary phase of growth. Addition of L-tryptophan to the medium resulted in a significant increase (up to 85.5 μg/mL) of auxin biosynthesis, especially in the presence of nitrates. Thin-layer chromatography revealed that the indole-3-acetamide pathway was not active in this strain. The biological activity of auxins was confirmed by assay with kidney bean cuttings; the height of root formation and root number increased 16- and 6-fold, respectively. Under conditions of low-temperature stress, protective effect of K. planticola TSKhA-91 on development of cucumber (Cucumis sativus L.) seeds and stimulation of germination and root formation by its seeds were shown.