Induction of defense-related enzymes in soybean leaves by class IId bacteriocins (thuricin 17 and bacthuricin F4) purified from Bacillus strains

We have recently discovered a new class of bacteriocin (class IId) which stimulates plant growth in a way similar to Nod factors. Nod factors have been shown to provoke aspects of plant disease resistance. We investigated the effects of bacteriocins [thuricin 17 (T17) and bacthuricin F4 (BF4)] on the activities of phenylalanine ammonia lyase (PAL), guaiacol peroxidase (POD), ascorbate peroxidase (APX), superoxide dismutase (SOD), and polyphenol oxidase (PPO). Bacteriocin solutions were fed into the cut stems of soybean (Glycine max L. Merr. cv. OAC Bayfield) seedlings at the first trifoliate stage. PAL activity in T17 treated leaves was the highest at 72 h after treatment and was 75.5% greater than the control at that time. At 72 h after treatment POD activities in T17 and BF4 treated leaves increased by 72.7 and 91.3%, respectively, as compared with the control treatment. APX activity was 52.3 and 49.6% respectively, greater than the control in T17 and BF4 treated leaves at 72 h after treatment. SOD activity in T17 treated leaves was the highest at 72 h after treatment and was 26.0% greater than the control at that time. SOD activity was 70.5 and 60.2% greater, respectively, than the control in T17 and BF4 treated leaves, at 72 h. Using PAGE we found that one APX isozyme (28 kDa isoform) showed the strongest induction in all bacteriocin treated leaves at 72 h. Activity of the seven SOD isozymes was increased by both bacteriocins, relative to the control treatment. The 33 kDa PPO isozyme was induced strongly by both bacteriocins, relative to the control treatment. These results indicate that class IId bacteriocins can act as an inducer of plant disease defense-related enzymes and may be acting through mechanisms similar to Nod factors.     

Induction and development of somatic embryos from cell suspension cultures of soybean

Glycine max (L.) Merr. (soybean) andGlycine soja Sieb. and Zucc. cell suspension cultures were grown and used as inoculum sources for growing callus on agar-solidified nutrient media. Concentrations and chemical forms of the growth regulators in liquid and solidified media were altered in an attempt to achieve in vitro plant regeneration. Numerous embryoids, particularly ofG. soja, were produced on basal nutrient media supplemented with 100 ppm casein hydrolysate, 0.1 μM abscisic acid, 2.25 μM 2,4-dichlorophenoxyacetic acid, and 15 μM adenine or 0.46 μM kinetin. Often the roots of the embryoids elongated. This was enhanced in the presence of an inhibitor of gibberellin synthesis (1 to 20 μM Amo 1618). Callus recovered from aG. soja suspension culture produced one shoot structure when grown on a solid medium containing 0.2 μM Amo 1618 and 80 μM glutathione. The shoot structure consisted of two distinct buds, one producing two leaves. The shoot did not develop into a plant. Although regeneration of soybean plants was not achieved, these observations suggest that it may be achievable. The investigations reported in this paper (no. 81-3-100) were performed in connection with a project of the Kentucky Agriculture Experimental Station and the paper is published with the approval of the Director.     

Cytological studies of triploids and their progeny from male-sterile (ms1) soybean

Summary Triploids (2n=3X=60) were obtained from genetic male-sterile (ms1 ms1) soybean [Glycine max (L.) Merr.] plants. Meiosis, pollen fertility, and chromosome number of their progeny were studied. Studies of meiosis in fertile and sterile triploids revealed no distinguishable differences in chromosome associations. Male-sterile plants formed coenocytic microspores characteristic of the ms1 mutant. Restitution of some dyad and tetrad nuclei were observed in male-sterile plants. Chromosomes of the triploids tended to occur in trivalents during diakinesis and metaphase I (MI), but multivalents, bivalents, and univalents also were observed. Average types and frequencies of chromosome associations per cell in diakinesis and MI from 542 pollen mother cells were 0.004 IX + 0.06 VI + 0.002 V + 0.005 IV + 16.99 III + 1.79 II + 5.03 I. Some secondary associations, nonhomologous pairing, and aberrant nucleolar distributions occasionally were observed. Such behavior support the hypothesis of duplicated genomes and the polyploid origin of soybean. Pollen fertility in male-fertile triploid plants (Ms1 ms1 ms1) varied from 57% to 82%, with an average of about 71%. Chromosome numbers of progenies obtained from these fertile triploids varied from 2n=40 to 2n=71, and exhibited a near-random distribution, with the majority (about 60%) being between 56 and 65. Progenies of the fertile triploids gave segregation ratios for the ms1 allele, which confirmed the Ms1 ms1 ms1 genotype.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-11672 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA, Project 2471     

Mechanical impedance increases aluminium tolerance of soybean (Glycine max) roots

The effect of aluminium (Al) on root elongation was studied in solution culture and sand culture. Compared to solution culture, in sand culture a ten times higher Al supply was necessary to inhibit root elongation to a comparable degree. This was due to a much lower Al uptake into the 5 mm root tips in sand culture. Fe concentrations in root tips were also lower in sand culture. Ca concentrations were higher and less depressed by Al, whereas Mg and K concentrations were not affected by the culture substrate. Regressions of Al concentrations in root tips versus inhibition of root elongation by Al revealed root damage at lower Al concentrations in sand culture. The effect of culture substrate on Al tolerance was independent of N source and could also be shown in flowing solution culture with and without sand. The results indicate that mechanical impedance in sand culture decreased Al uptake. This may be due to enhanced exudation of organic complexors thus reducing activites of monomeric Al species.     

Effects of quercetin and enhanced UV-B radiation on the soybean (Glycine max) leaves

The possible ameliorative effects of quercetin on soybean [Glycine max (L.) Merr.] leaves exposed to UV-B radiation were conducted in greenhouse. The symmetrical leaves supplied with quercetin solution (0.2%, 1%) were exposed to UV-B radiation (0, 3.5, 6.5 kJ m⁻² d⁻¹). 0.2% quercetin ameliorated leaf photosynthesis, improved leaf water content (LWC), and decreased lipid oxidation. The unfavorable effect on photosynthetic parameter was displayed in 1% quercetin treatment. The effect of quercetin on phenylalanine ammonia lyase (PAL) activity varied with the quercetin concentration, UV-B radiation intensity and leaf development. In the later development polyphenol oxidase (PPO) activity was increased significantly by quercetin treatments. We suggested that quercetin with suitable concentration could serve as UV-B protective agent partly due to its antioxidant capacity.     

Characterization of a cytoplasmically inherited yellow foliar mutant (cyt-Y 3) in soybean

Summary Genetic analysis of a yellow foliar mutant in soybean (Glycine max L. Merr.) showed maternal inheritance of the mutant phenotype designatedcyt-Y ₃. The mutant was grown beside normal green sibs (cyt-G ₃) under three different photosynthetic photon flux densities (PPFD), and samples were collected to determine pigment content and for electron microscopy analyses of plastid ultrastructure. The plastid ultrastructure ofcyt-Y ₃ appeared normal at low PPFD and the carotenoid level ofcyt-Y ₃ was also normal, but the chlorophyll content was only approximately one-third that ofcyt-G ₃. Under medium and high PPFD,cyt-Y ₃ plastids lacked a structured thylakoid, and total chlorophyll content was only 28% and 1% of normal, respectively; the carotenoid levels ofcyt-Y ₃ also dropped to 33% and 2% of normal, respectively. These data indicate that the effect of high PPFD oncyt-Y ₃ might result from a deficiency in a plastid membrane protein. The resulting changes in membrane configuration could then interfere with the accumulation or stabilization of chlorophylls and carotenoids, thereby resulting in the subsequent photooxidation of both at medium and high PPFD. This mutant could be useful in the study of thylakoid biosynthesis and pigment stabilization, or could provide a source of conditionally identifiable plastids for organelle segregation studies.This is a joint contribution of North Central Region, USDA-ARS., and Journal Paper No. J-11124 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, Project 2471. Mention of a trademark of proprietary product by Iowa State University or the USDA does not imply its approval to the exclusion of other products that may also be suitable     

A non-destructive field assay for soybean nitrogen fixation by acetylene reduction

Summary A system for employing open-ended root chambers to measurein situ acetylene reduction rates under field conditions is described. Gas mixtures containing about 2 mbar acetylene were continuously flowed through the chambers providing a continuous record of acetylene reduction. These chambers have been used to measure acetylene reduction rates of soybeans during three growing seasons. The system has proved to be reliable with a high degree of precision. The large amount of plant-to-plant variability observed in N₂ fixation research has been confirmed by the data collected with this system. However, such variability in physiological studies can be reduced by using a non-destructive system to compare the response of an individual plant with its rates before treatment.     

Oxidation of the enzymes involved in nitrogen assimilation plays an important role in the cadmium-induced toxicity in soybean plants

Cadmium causes oxidative damage and hence affects nitrogen assimilation. In the present work we tested the relationship between the inactivation of the enzymes involved in nitrogen assimilation pathway (glutamine synthetase (GS)/glutamate synthase (GOGAT)) and the protein oxidation in nodules of soybean (Glycine max L.) plants under Cd²⁺ stress. Therefore, the effect of Cd²⁺ and reduced gluthatione (GSH) on GS and GOGAT activities, and protein abundance and oxidation were analyzed. Under the metal treatment, amino acids oxidative modification occurred, evidenced by the accumulation of carbonylated proteins, especially those of high molecular weight. When Cd²⁺ was present in the nutrient solution, although a decrease in GS and GOGAT activities was observed (17 and 52%, respectively, compared to controls), the protein abundance of both enzymes remained similar to control nodules. When GSH was added together with Cd²⁺ in the nutrient medium, it protected the nodule against Cd²⁺ induced oxidative damage, maintaining GS and GOGAT activities close to control values. These results allow us to conclude that the inactivation of the nitrogen assimilation pathway by Cd²⁺ in soybean nodules is due to an increment in GS and GOGAT oxidation that can be prevented by the soluble antioxidant GSH. Section Editor: H. Schat     

Hydrolysis of organic phosphates by corn and soybean roots

Because of the importance of organic phosphates as sources of P for plants, this work was performed to study the hydrolysis of nine organic phosphates by sterile, intact corn (Zea mays L.) and soybean (Glycine max L.) roots. Results showed that the rates of hydrolysis ofp-nitrophenyl phosphate (PNP) in buffered solutions by roots of three varieties of corn and three varieties of soybean ranged from 13 to 22 μmol PO₄−P g⁻¹ root h⁻¹ and from 2.1 to 2.2 μmol PO₄−P 0.1 g⁻¹ root h⁻¹, respectively. The average rate of hydrolysis of PNP in nonbuffered solutions was 2- to 3-fold lower for corn roots and 6- to 10-fold lower for soybean roots as compared with those obtained with buffered solutions. The orthophosphate released from hydrolysis of organic P compounds in buffered solutions during a 48-h incubation of corn roots showed that the maximum rate of hydrolysis of PNP was 4 to 6 times greater than the commonly used substrates: α- and β-glycerophosphates, phenolphthalein diphosphate, and glucose-6-phosphate. The rates of hydrolysis of glucose-6-phosphate and glucose-1-phosphate were similar and about 6- to 12-fold lower than that of PNP. Phosphoethanolamine and phosphocholine were hydrolyzed slightly, ando-carboxyphenyl phosphate was not hydrolyzed. The rates of hydrolysis of organic P compounds in nonbuffered solutions by corn and soybean roots were 1 to 3 and 1 to 10 times lower than those in buffered solutions, respectively. The trends in rates of hydrolysis by soybean roots of buffered organic P substrates were similar to those observed with corn roots, with the exception of glucose-1-phosphate and phosphoethanolamine.     

Exclusion of inefficient Bradyrhizobium japonicum serogroups by soybean genotypes

Soybean [Glycine max (L.) Merr.] forms a symbiosis with serogroups of Bradyrhizobium japonicum that differ in their dinitrogen fixing abilities. The objectives of this study were to identify soybean genotypes that would restrict nodulation by relatively inefficient serogroups indigenous to a large portion of the southeastern USA, and then characterize the nodulation responses of selected genotypes with specific bradyrhizobial strains under controlled conditions. From field screening trials followed by controlled single and competitive inoculations of serogroups USDA 31, 76 and 110, twelve soybean genotypes out of 382 tested were identified with varying levels of exclusion abilities. Soybean nodule occupancies and nodulation characteristics were influenced by plant genotype, environment (i.e. field or greenhouse), bradyrhizobial serogroup, and location of nodules (i.e. tap or lateral root). The cultivar Centennial sustains high seed yields even though it nodulates to a high degree with the inefficient serogroup USDA 31. In contrast, data from the released cultivars Braxton, Centennial and Coker 368 indicate that they may have been selected to exclude the inefficient serogroup USDA 76 from their tap root nodules, possibly contributing to high seed yield.     

Effects of arsenic on phosphorus content in different organs and chlorophyll fluorescence in primary leaves of soybean

The effect of arsenic (32 – 96 μM) on the phosphorus content and Chl fluorescence was studied in soybean (Glycine max Merril) grown in the nutrient solution with and without phosphorus. The increased concentration of As led to the decrease in P content in plant organs. Parameters of Chl fluorescence of soybean leaves in the presence of these As concentrations did not show significant changes.     

The frequency of polyembryonic seedlings and polyploids from ms1 soybean

Summary Seed from homozygous recessivems ₁ genetic male-sterile soybean (Glycine max (L.) Merr.) plants was studied for frequencies of polyembryonic seedlings and different levels of polyploidy among abnormal seedlings from six different source populations: Amesms ₁ (Ams), North Carolinams ₁ (NCms), Tonicams ₁ (Tms), Urbanams ₁ (Ums), and F₄ generation seed obtained from crosses ofms ₁ to two chromosome interchange lines (Ams x Clark T/T and Ums x KS-172-11-3). Frequencies of polyembryony observed in Tms, Ums, Ams, NCms, F₄ seed from Ams x Clark T/T, and F₄ seed from Ums x KS-172-11-3 were 3.6%, 2.4%, 3.1%, 2.5%, 2.2% and 0.1%, respectively. Frequencies of abnormal seedlings from these six sources varied from 1.7% (Ums X KS-172-11-3) to 16.8% (Ams X Clark T/T). Frequencies of polyploids among the abnormal seedlings ranged from 6.8% in Ums x Ks-172-11-3 to 66.7% in Tms. On average, the frequency of polyploid individuals from monoembryonic seedlings was 1.22%. Chromosome number of these seedlings varied from 20 to 200. Variation of the frequencies of polyembryonic seedlings and polyploid progeny among abnormal seedlings suggested that the mechanism(s) controlling the characters of polyembryony and formation of polyploids was associated with thems ₁ gene and was affected by other gene(s) or environmental factors.Joint contribution: Agricultural Research Service, US Department of Agriculture, and Journal Paper No. J-11255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA. Project 2471     

Inheritance of two independent isozyme variants in soybean plants derived from tissue culture

Summary Soybean [Glycine max (L.) Merr.] plants were regenerated via somatic embryogenesis from nine soybean cultivars. Our objective was to identify and characterize genetically novel mutations that would further our understanding of the soybean genome. Variant isozyme patterns were observed in two independent tissue culturederived lines. Genetic analyses were conducted on these two isozyme variants, and they were heritable. No variant isozyme patterns were evident in control (parental) soybean lines. In the cultivar BSR 101, a mutation of Aco2-b (aconitase) to a null allele was detected. The Aco2-bn mutant, Genetic Type T318, had not been previously observed in soybean. In the Chinese cultivar Jilin 3 (PI 427.099), a chlorophyll-deficient plant was identified that also lacked two mitochondrial malate-dehydrogenase (Mdh null) isozyme bands. These two mutant phenotypes, chlorophyll-deficient and Mdh null, were found to cosegregate. The Jilin 3 mutant, Mdh1-n (Ames 1) y20 (Ames 1) Genetic Type T317, was allelic to three chlorophyll-deficient, Mdh1 null mutants [Mdh1-n (Ames 2) y20 (Ames 2) (T323), Mdh1-n (Ames 3) y20 (Ames 3) (T324), and Mdh1-n (Ames 4) y20 (Ames 4) (T325)] previously identified from a transposon-containing soybean population, and to a chlorophyll-deficient, Mdh1 null mutant [Mdh1-n (Urbana) y20 (Urbana) k2, Genetic Type T253] which occurred spontaneously in soybean. The recovery of two isozyme variants from progeny of 185 soybean plants regenerated from somatic embryogenesis indicates the feasibility of selection for molecular variants.     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

Ferulic acid uptake by soybean root in nutrient culture

Ferulic acid uptake by soybean root in nutrient culture was investigated by the depletion method at different concentrations, temperatures and pH. Results showed that soybean roots absorbed this compound at greater rates in the concentrations between 0.05-mM and 1.0-mM and it was concentration dependent. Ferulic acid uptake was unaffected at pH 4.5 or 6.0 but reduced at pH 7.0. At pH 6.0, uptake rates decreased significantly with increasing temperature of nutrient solution.     

Nod Bj-V (C18:1, MeFuc) production by Bradyrhizobium japonicum (USDA110, 532C) at suboptimal growth temperatures

Nod factors (Lipo-chitooligosaccharides, or LCOs) act as bacteria-to-plant signal molecules that modulate early events of the Bradyrhizobium-soybean symbiosis. It is known that low root zone temperature inhibits the early stages of this symbiosis; however, the effect of low soil temperature on bacteria-to-plant signaling is largely uninvestigated. We evaluated the effect of low growth temperatures on the production kinetics of Nod factor (LCO) by B. japonicum. Two strains of B. japonicum, 532C and USDA110, were tested for ability to synthesize Nod Bj-V (C(18:1), MeFuc) at three growth temperatures (15, 17 and 28 degrees C). The greatest amounts of the major Nod factor, Nod Bj-V (C(18:1), MeFuc), were produced at 28 degrees C for both strains. At 17 and 15 degrees C, the Nod factor production efficiency, per cell, of B. japonicum 532C and USDA110 was markedly decreased with the lowest Nod factor concentration per cell occurring at 15 degrees C. Strain 532C was more efficient at Nod factor production per cell than strain USDA 110 at all growth temperatures. The biological activity of the extracted Nod factor was unaffected by culture temperature. This study constitutes the first demonstration of reduced Nod factor production efficiency (per cell production) under reduced temperatures, suggesting another way that lower temperatures inhibit establishment of the soybean N(2) fixing symbiosis.     

Photoautotrophic growth of soybean cells in suspension culture IV. Free amino acid pools and the effect of nitrogen on chlorophyll levels

A photoautotrophic soybean suspension culture was used to study free amino acid pools during a subculture cycle. Free amino acid analysis showed that the intracellular concentrations of asparagine, serine, glutamine, and alanine reached peaks of 200, 10, 9 and 7 mM, respectively, at specific times in the 14-day subculture cycle. Asparagine and serine levels peaked at day 14 but glutamine level rose quickly after subculture, peaking at day three and then declined gradually. Roughly similar patterns were found in the conditioned culture medium although the levels were 1000-fold lower than those found in cells. Photoautotrophic (SB-P) and photomixotrophic (SB-M) cultures were quantitatively similar with regard to free asparagine and serine but not glutamine or free ammonia. Heterotrophic (SB-H) cells had 81–85% less free asparagine on day seven than did SB-M or SB-P cells. Hence, similar to the phloem sap of a soybean plant, asparagine, glutamine, alanine and serine were the predominant amino acids in photoautotrophic soybean cell cultures. Varying the amount of total nitrogen in culture medium for two subcultures at 10, 25, 50, and 100% Of normal levels showed that growth was inhibited only at the 10 and 25% levels but that growth on medium containing 50% of the normal nitrogen was as good as that on 100% nitrogen. Moreover, cellular chlorophyll content correlated exceptionally well with initial nitrogen content of the medium. Thus, the photosynthesis of SB-P cells was not limited by chlorophyll content. SB-P cells grown for two subcultures on 10% nitrogen contained very low free amino acid levels and only 1% of the free ammonia levels found in cells growing on a full nitrogen complement.Abbreviations SB-P photoautotrophic soybean cells (no sucrose, high CO₂, high light) - SB-M photomixotrophic soybean cells (1% w/v sucrose, high light) - SB-H heterotrophic soybean cells (3% sucrose, dark)     

Carbohydrate and lipid status in soybean roots influenced by ferulic acid uptake

The objective of this research was to investigate how the allelochemical ferulic acid affects the carbohydrate and lipid contents of soybean roots cultivated in nutrient culture. The results presented revealed that ferulic acid has significant effects on carbohydrates by the increase in xylose, fructose and sucrose and decrease in glucose, after 24 h treatment of roots. Ferulic acid increased the contents of saturated and unsaturated fatty acids of the polar and non-polar lipid fractions. The results may contribute as additional data to explain allelopathic effects caused by ferulic acid.     

Photosynthetic acclimation and photosynthate partitioning in soybean leaves in response to carbon dioxide enrichment

Photosynthetic rates and photosynthate partitioning were studied in three-week-old soybean [Glycine max (L.) Merr. cv. Williams] plants exposed to either ambient (35 Pa) or elevated (70 Pa) CO₂ in controlled environment chambers. Ambient CO₂-grown plants also were given a single 24 h treatment with 70 Pa CO₂ 1 d prior to sampling. Photosynthetic rates of ambient CO₂-grown plants initially increased 36% when the measurement CO₂ was doubled from 35 to 70 Pa. Photosynthetic rates of the third trifoliolate leaf, both after 1 and 21 d of elevated CO₂ treatment, were 30 to 45% below those of ambient CO₂-grown plants when measured at 35 Pa CO₂. These reduced photosynthetic rates were not due to increased stomatal resistance and were observed for 2 to 8 h after plants given 1 d of CO₂ enrichment were returned to ambient CO₂. Initial and total ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco) activities, percent activation, Rubisco protein, soluble protein and leaf chlorophyll content were similar in all CO₂ treatments. Quantum yields of photosynthesis, determined at limiting irradiances and at 35 Pa CO₂, were 0.049±0.003 and 0.038±0.005 mol CO₂ fixed per mol quanta for ambient and elevated CO₂-grown plants, respectively (p<0.05). Leaf starch and sucrose levels were greater in plants grown at 70 than at 35 Pa CO₂. Starch accumulation rates during the day were greater in ambient CO₂-grown plants than in plants exposed to elevated CO₂ for either 1 or 21 d. However, the percentage of C partitioned to starch relative to total C fixed was unaffected by 1 d of CO₂ enrichment. The above results showed that both photosynthetic and starch accumulation rates of soybean leaflets measured at 35 Pa CO₂ were temporarily reduced after 1 and 21 d of CO₂ enrichment. The biochemical mechanism affecting these responses was not identified.Abbreviations SLW- specific leaf weight (g m^–2) - Rubisco- ribulose 1,5-bisphosphate carboxylase/oxygenase - Rul- 5bisP, ribulose 1,5 bisphosphate - DAP- days after planting - SAR- starch accumulation rate - C_i- intercellular CO₂ concentration