Hypocotyls Of Soybean Seedlings Research Articles

Characterization of fluorescence properties of wounds on soybean seedlings during healing process using excitation emission matrix and fluorescence imaging

To establish a simple and nondestructive method for measuring plant wound-healing ability, we characterized the fluorescence characteristics of wounds on hypocotyl of soybean seedlings during healing process. Wounds were manually created on the stem of soybean seedlings 7 days after sowing. The fluorescence time-series characteristics of the wounds were measured until 96 h after wounding using excitation emission matrix (EEM) and fluorescence images excited by wavelength of 365 nm. In the EEM of wounds, three main fluorescence peaks were observed, and the intensity decreased with time after wounding. The reddish color due to chlorophyll in fluorescence images also decreased with healing process. In addition, microscopic observation of the wounded tissue using a confocal laser microscope showed that the intensity of lignin or suberin like fluorescence increased with healing time, which might have blocked the excitation light. These results suggest that UV-excited fluorescence can be a new indicator of the healing ability of plant tissues.

Phomopsis (Diaporthe) Species as the Cause of Soybean Seed Decay in Korea

AbstractSoybeans are an important crop known to harbour a complex of Diaporthe and Phomopsis species. This complex has been reported to be involved in several soybean diseases, including Phomopsis seed decay. In this study, two species of Diaporthe/Phomopsis fungi from soybean plants were identified by morphological and molecular characterizations. Koch's postulates were confirmed by pathogenicity tests on hypocotyls of soybean seedlings. Phomopsis longicolla was found to be the most common and virulent pathogen to soybeans in Korea. Phomopsis sp., which was considered as a new soybean pathogen, might have been introduced from other plants given that similar strains of Phomopsis sp. have infected fruit trees in China, Japan and Portugal and vegetable plants in the United States.

Comparative Proteomic Analysis of Early-Stage Soybean Seedlings Responses to Flooding by Using Gel and Gel-Free Techniques

Gel-based and gel-free proteomics techniques were used to investigate early responses to flooding stress in the roots and hypocotyls of soybean seedlings. Proteins from 2-day-old soybean seedlings flooded for 12 h were extracted and analyzed. Two mass-spectroscopy-based proteomics analyses, two-dimensional fluorescence difference gel electrophoresis, and nanoliquid chromatography identified 32 from 17 spots and 81 proteins, respectively, as responsive to flooding stress. On the basis of the number and function of proteins identified, glycolysis and fermentation enzymes and inducers of heat shock proteins were key elements in the early responses to flooding stress. Analysis of enzyme activities and carbohydrate contents in flooded seedlings showed that glucose degradation and sucrose accumulation accelerated during flooding due to activation of glycolysis and down-regulation of sucrose degrading enzymes. Additionally, the methylglyoxal pathway, which is detoxification system linked to glycolysis, was up-regulated. Furthermore, two-dimensional polyacrylamide gel electrophoresis-based phosphoproteomics analysis showed that proteins involved in protein folding and synthesis were dephosphorylated under flooding conditions. These results suggest that translational and post-translational control during flooding possibly induces an imbalance in the expression of proteins involved in several metabolic pathways including carbohydrate metabolism that might cause flooding injury of soybean seedlings.

Proteomics approach for identifying osmotic-stress-related proteins in soybean roots

Osmotic stress can endanger the survival of plants. To investigate the mechanisms by which plants respond to osmotic stress, protein profiles from soybean plants treated with polyethylene glycol (PEG) were monitored by a proteomics approach. Treatment with 10% aqueous PEG reduced the lengths of roots and hypocotyls of soybean seedlings. Proteins from soybean roots were separated by two-dimensional polyacrylamide gel electrophoresis, and 415 proteins were detected by Coomassie brilliant blue staining. Thirty-seven proteins changed by PEG treatment were analyzed using Edman sequencing and peptide-mass fingerprinting method and this group included proteins involved in disease/defense. Seven proteins were selected for further experiments using the results of cluster analysis and statistical analysis of the abundance change. A comparison with the effects of other abiotic stresses showed that caffeoyl-CoA- O-methyltransferase and 20S proteasome alpha subunit A were decreased and increased by abiotic stresses, respectively. Expression analyses of these transcripts were also changed by PEG treatment. Caffeoyl-CoA- O-methyltransferase and 20S proteasome alpha subunit A may control the sensitivity of several regulatory genes specific to short exposure to osmotic stress.

Analysis of Plasma Membrane Proteome in Soybean and Application to Flooding Stress Response

The plasma membrane acts as the primary interface between the cellular cytoplasm and the extracellular environment. To investigate the function of the plasma membrane in response to flooding stress, plasma membrane was purified from root and hypocotyl of soybean seedlings using an aqueous two-phase partitioning method. Purified plasma membrane proteins with 81% purity were analyzed using either two-dimensional polyacrylamide gel electrophoresis followed by mass spectrometry and protein sequencing (2-DE MS/sequencer)-based proteomics or nanoliquid chromatography followed by mass spectrometry (nanoLC-MS/MS)-based proteomics. The number of hydrophobic proteins identified by nanoLC-MS/MS-based proteomics was compared with those identified by 2-DE MS/sequencer-based proteomics. These techniques were applied to identify the proteins in soybean that are responsive to flooding stress. Results indicate insights of plasma membrane into the response of soybean to flooding stress: (i) the proteins located in the cell wall are up-regulated in plasma membrane; (ii) the proteins related to antioxidative system play a crucial role in protecting cells from oxidative damage; (iii) the heat shock cognate protein plays a role in protecting proteins from denaturation and degradation during flooding stress; and (iv) the signaling related proteins might regulate ion homeostasis.

First Report of Soybean Stem Canker Caused by Diaporthe phaseolorum var. caulivora in Argentina.

A soybean stem canker (SSC) outbreak caused by Diaporthe phaseolorum (Cooke & Ellis) Sacc. var. meridionalis Fernández was reported in Santa Fe, Argentina, in 1997 (3). In 1999 an isolate, which was morphologically distinct from D. phaseolorum var. meridionalis, was obtained from stems of field-grown soybean plants exhibiting SSC symptoms, at Oliveros, Santa Fe, Argentina (Lat. 32° 33'S, Lon. 60° 51'W). Disease incidence was 76% in the field where samples were collected. The pathogen was isolated in darkness at 25°C on potatoglucose agar acidified with 0.2% lactic acid (3). The isolate produced white colonies with compact and tufted mycelium that changed to yellow and light tan with age. Stromata and pycnidia were not produced. After 35 days in culture, clustered perithecia were frequently observed on stem segments. Fifty asci, five from each of 10 perithecia, and bicellular, biguttulated ascospores were measured. Ascus mean length was 26.9 ± 2.5 μm and width was 5.3 ± 0.5 μm; ascospore mean length was 8.3 ± 0.6 μm and width was 2.6 ± 0.1μm. Based on these features, the new isolate was classified as D. phaseolorum var. caulivora Athou & Caldwell (1). To further compare the new isolate with previous identified ones, a principal component analysis (PCA, SAS Systems) was performed using seven isolates of D. phaseolorum var. meridionalis, three isolates of D. phaseolorum var. sojae, and two isolates of Phomopsis longicolla. Seventeen morphological characters, all related with the color and texture of the colonies, the presence and shape of the pycnidia and conidia, the presence and type of stromata and perithecia, and the length of the asci, were compared. According to the PCA analysis, the principal characters that discriminated SSC producing isolates (D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora) from non-SSC producing ones (D. phaseolorum var. sojae and P. longicolla) were the development of perithecia (r = 0.98) and low frequency stromata (r = 0.98) in D. phaseolorum var. meridionalis and D. phaseolorum var. caulivora isolates. The principal components that discriminated SSC producing isolates were the more compact and tufted aspect of the mycelia (r = 0.95) and the shorter length of the asci (r = 0.83) in D. phaseolorum var. caulivora compared with D. phaseolorum var. meridionalis. Pathogenicity trials were performed under greenhouse conditions by inoculating D. phaseolorum var. caulivora mycelia in hypocotyls of soybean seedlings by the toothpick method (2). Typical SSC symptoms were observed on susceptible plants and the pathogen was re-isolated and identified from stem portions of the first internode above the inoculation point. Pathogenicity trials were repeated twice with similar results. This is the first report of D. phaseolorum var. caulivora in Argentina and, as far as we know, in all of South America.

TRANSCRIPT ACCUMULATION OF POLYGALACTURONASE INHIBITING PROTEIN (PGIP) FOLLOWING PATHOGEN INFECTIONS IN SOYBEAN

SUMMARY Proteins inhibiting fungal endo-polygalacturonase (PGIP) are constitutively expressed and localized on cell walls of most plant species. Induction of pgip transcripts following pathogen infection would demonstrate a role of PGIP in active plant defence mechanisms. We investigated pgip expression in hypocotyls of soybean seedlings (cvs ‘Sapporo’ and ‘Kure’) infected with the fungal pathogens Diaporthe phaseolorum var. caulivora, Sclerotinia sclerotiorum and an avirulent (race 1) and virulent (race 20) races of Phytophthora sojae. Two pgip transcripts were transiently expressed in all types of interaction with similar timing and maximum accumulation at 16-24 h after infection. However, soybean seedlings of both cvs infected with the virulent race 20 of P. sojae showed higher levels of pgip expression than seedlings infected with the avirulent race 1. A delay of pgip accumulation was observed when plants were inoculated with zoospores in place of mycelium of P. sojae. The presence of endo-polygalacturonase (endo-PG) in infected tissue was monitored both by immunological detection and by determining PG activity. PG was detected only in soybean seedlings infected with S. sclerotiorum and not in those infected with D. phaseolorum var. caulivora or P. sojae. The apparent inability in vivo of the virulent and avirulent races of P. sojae to produce PG suggests that PG-PGIP interaction is not required for the resistance response of soybean against this pathogen.

Isoflavonoids in root and hypocotyl of soybean seedlings (Glycine max, Fabaceae)

Isoflavonoids, some of which are highly fluorescent, are produced by soybean [Glycine max (L.) Merr.] and serve as chemical signals for certain aspects of nitrogen fixation and microbial resistance. This study was conducted to determine whether soybean mutants with nonfluorescent roots contained abnormal concentrations of isoflavonoids. Isoflavonoids were extracted from the root and hypocotyl of 4‐d‐old wild‐type soybean seedlings (cv. Hark) having fluorescent roots and from four nonallelic mutant, near isogenic lines of Hark having nonfluorescent roots. In addition, isoflavonoids were extracted from the root and hypocotyl of 4‐d‐old seedlings of near isogenic lines of Hark harboring two pairs of the mutant alleles for nonfluorescent roots. Malonyl daidzin, daidzin, malonyl genistin, and genistein were the most abundant isoflavonoids extracted from either the root or hypocotyl of seedlings with either fluorescent or nonfluorescent roots. Extracted malonyl daidzin, malonyl genistin, and malonyl glycitin decomposed readily, yielding daidzin, genistin, and glycitin, respectively. The concentrations of malonyl genistin and genistein, two highly fluorescent compounds, were similar in both fluorescent and nonfluorescent roots. Thus, root fluorescence was not correlated with abundance per se of fluorescent isoflavonoids in roots. In addition, the abundance of isoflavonoids extractable from the hypocotyl did not correlate with root fluorescence.

Endogenous Inhibitors of Growth of Soybean Seedlings Associated with the Inhibition of Growth Induced by Red Light

Summary In order to correlate the role of endogenous inhibitors of growth of soybean plants with the inhibition of such growth by red light (R), a search for specific growth inhibitors was undertaken in the neutral and acidic fractions of acetone extracts from R- and dark-grown hypocotyls of soybean seedlings ( Glycine max L.). Two neutral and two acidic inhibitors were isolated. They were named N-I and N-II (neutral inhibitors) and A-I and A-II (acidic inhibitors), respectively, based on the order of their increasing Rf values during chromatography. Variations in the activity of the inhibitors in the hypocotyls of dark-grown seedlings after the onset of R irradiation were determined in terms of «cress units» which were defined as the activity that causes 50 % inhibition in the cress-root bioassay. The inhibitory activity of N-I and N-II tripled after exposure of plants to R for 24 h, whereas the activity of A-I and A-II remained almost unchanged. These results suggest that R-induced inhibition of growth of soybean hypocotyls may be controlled by variations in the levels of the neutral inhibitors, N-I and/or N-II, in the hypocotyls rather than by the acidic inhibitors.

Relative Elemental Rates of Elongation and the Protoxylem-Metaxylem Transition in Hypocotyls of Soybean Seedlings

The transition from helical to reticulate wall patterns in the conducting elements of the xylem was located in etiolated soybean hypocotyls in conjunction with the distribution of elongation in the axis. The most acropetal reticulate elements are interpreted as the most acropetal metaxylem. Evidence is given that these elements experience strain as they mature. Predicted strain correlated significantly with maximum relative elemental rate of elongation (RELEL), which ranged from 0.08 to 0.12-hr-1. RELEL varied from 0.03 to 0.06hr-1 at the point of transition from helical to reticulate xylem element wall patterns. Estimates of strain to be experienced in the reticulate elements varied from 0.11 to 0.13 in untransplanted plants and from 0.04 to 0.11 in plants transplanted to the same, fully watered medium. Transplanting suppressed elongation in the region of maximum RELEL for ca. 12 hr and caused an upward migration of the region of depressed rates on the outside of the axis that accounts for hook-opening. Attention is called to the fact that this region of depressed growth corresponds with the onset of transverse enlargement of the hypocotyl axis.

A mechanism for ethylene-induced disease resistance in soybean: enhanced synthesis of an elicitor-releasing factor, β-1,3-endoglucanase

Exogenous ethylene increased the level of β-1,3-endoglucanase (EC 3.2.1.39) activity and the level ofβ-1,3-endoglucanase protein 2- to 4-fold higher than constitutive levels in hypocotyls of soybean seedlings. Ethylene-treated soybean hypocotyls showed partial resistance upon infection with a compatible race of Phytophthora megasperma f. sp. glycinea, accompanied with higher levels of the phytoalexin, glyceollin. Purified soybean β-1,3-endoglucanase had no direct fungitoxic activity to the fungus. The purified glucanase, however, released soluble elicitors from cell walls of the fungus. Higher levels of glyceollin accumulated in hypocotyls of ethylene-treated soybean seedlings than in control hypocotyls when they were treated with native fungal cell walls. Similar levels of glyceollin accumulated when ethylene-treated and control hypocotyls were treated with soluble elicitor released by the glucanase. These results indicate that more efficient elicitor release occurred in the ethylene-treated soybean tissue. In addition, co-treatment of hypocotyls with the purified glucanase and the native cell walls, but not the glucanase-released elicitor, resulted in greater glyceollin accumulation. Exogenous application of the purified glucanase to inoculation sites also increased resistance of soybean hypocotyls against attack by the fungus. In contrast, resistance of soybean hypocotyls to an incompatible race of P. megasperma f. sp. glycinea was partially negated when the seedlings were absorbed with laminarin, which had been shown to inhibit the reaction of elicitor release from fungus cell walls. These results support the idea that the increase in resistance in ethylene-treated soybean tissue may be due, in part, to enhanced β-1, 3-endoglucanase activity which may release elicitor-active molecules from fungal cell walls. The results also suggest that the process of elicitor release mediated by host β-1,3-endoglucanase is an important process leading to active disease resistance in soybean.

Requirement of Ca2+ for the gelatinization of pectin solution by a proteinous cell wall-bound factor and solubilization of the gel by acid treatment

The cell wall-bound pectin-gelatinizing factor, whose existence was reported in a previous paper (Yamaoka and Sato, 1981), was found to be composed of a proteinous factor (PGF) and calcium ion. Magnesium ion could not replace Ca2+ whereas Sr2+ could replace Ca2+ and Ba2+ could cause gelation without the action of PGF. In the hypocotyl of soybean seedlings, the basal region contained more PGF than did the apical region. The gel of apple pectin formed by the action of PGF and Ca2+ was solubilized by acid treatment for some time after the gelation. It was also found that the gel became more and more resistant to acidity with time. The hardened gel was almost completely dissolved by boiling in 0.2 M NaCl solution. The idea that the gelation of apple pectinin vitro may simulate the gelation of pectin in the primary cell wall was discussed in view of the results and the phenomenon of acid growth.

A study on a cell wall-bound factor which causes the gelation of pectin solution

By extracting the cell wall fraction obtained from hypocotyls of soybean seedlings with 1 M NaCl solution, a factor which caused the gelation of the pectin solution was released. Mixed with the extract, the pectin solution increased in viscosity and eventually solidified. The pectin-gelatinizing factor (PGF) was of protein nature and seemed to modify pectin molecules to make them coagulate. The physiological significance of the PGF action was discussed. PGF seemed to be widely distributed among seed plants and to be cell wall-boundin situ.

Host-Pathogen Interactions

Resistance of soybean (Glycine max L.) seedlings to Phytophthora megasperma var. sojae (Pms) is in part due to the accumulation in infected tissue of a compound which is toxic to Pms. The accumulation of this compound, a phytoalexin called glyceollin, is triggered by infection, but it can also be triggered by molecules, "elicitors," present in cultures of Pms. The ability of the Pms elicitor to stimulate phytoalexin accumulation in soybean tissues has been used as the basis for biological assays of elicitor activity. Two bioassays were developed and characterized in this study of the Pms elicitor. These bioassays use the cotyledons and the hypocotyls of soybean seedlings. The cotyledon assay was used to characterize the extracellular Pms elicitor. This elicitor was isolated from Pms cultures and purified by ion exchange and molecular sieving chromatography. The extracellular Pms elicitor was determined to be a predominantly 3-linked glucan, which is similar in composition and structure to a polysaccharide component of Pms mycelial walls.