Analysis of proteomic changes in roots of soybean seedlings during recovery after flooding

A proteomic approach was used to identify proteins involved in post-flooding recovery in soybean roots. Two-day-old soybean seedlings were flooded with water for up to 3 days. After the flooding treatment, seedlings were grown until 7 days after sowing and root proteins were then extracted and separated using two-dimensional polyacrylamide gel electrophoresis (2-DE). Comparative analysis of 2-D gels of control and 3 day flooding-experienced soybean root samples revealed 70 differentially expressed protein spots, from which 80 proteins were identified. Many of the differentially expressed proteins are involved in protein destination/storage and metabolic processes. Clustering analysis based on the expression profiles of the 70 differentially expressed protein spots revealed that 3 days of flooding causes significant changes in protein expression, even during post-flooding recovery. Three days of flooding resulted in downregulation of ion transport-related proteins and upregulation of proteins involved in cytoskeletal reorganization, cell expansion, and programmed cell death. Furthermore, 7 proteins involved in cell wall modification and S-adenosylmethionine synthesis were identified in roots from seedlings recovering from 1 day of flooding. These results suggest that alteration of cell structure through changes in cell wall metabolism and cytoskeletal organization may be involved in post-flooding recovery processes in soybean seedlings.     

Comparative proteomics analysis of differentially expressed proteins in soybean cell wall during flooding stress

Flooding is a major problem for soybean crop as it reduces the growth and grain yield. To investigate the function of the soybean cell wall in the response to flooding stress, cell wall proteins were analyzed. Cell wall proteins from roots and hypocotyls of soybeans, which were germinated for 2 days and subjected to 2 days of flooding, were purified, separated by two-dimensional polyacrylamide gel electrophoresis and stained with Coomassie brilliant blue. Sixteen out of 204 cell wall proteins showed responses to flooding stress. Of these, two lipoxygenases, four germin-like protein precursors, three stem 28/31 kDa glycoprotein precursors, and one superoxide dismutase [Cu–Zn] were downregulated. A copper amine oxidase was found to have shifted from the basic to acidic zone following flooding stress. Based on these results, it was confirmed by the lignin staining that the lignification was suppressed in the root of soybean under the flooding stress. These results suggest that the roots and hypocotyls of soybean caused the suppression of lignification through decrease of these proteins by downregulation of reactive oxygen species and jasmonate biosynthesis under flooding stress.     

Proteins involved in biophoton emission and flooding-stress responses in soybean under light and dark conditions

To know the molecular systems basically flooding conditions in soybean, biophoton emission measurements and proteomic analyses were carried out for flooding-stressed roots under light and dark conditions. Photon emission was analyzed using a photon counter. Gel-free quantitative proteomics were performed to identify significant changes proteins using the nano LC–MS along with SIEVE software. Biophoton emissions were significantly increased in both light and dark conditions after flooding stress, but gradually decreased with continued flooding exposure compared to the control plants. Among the 120 significantly identified proteins in the roots of soybean plants, 73 and 19 proteins were decreased and increased in the light condition, respectively, and 4 and 24 proteins were increased and decreased, respectively, in the dark condition. The proteins were mainly functionally grouped into cell organization, protein degradation/synthesis, and glycolysis. The highly abundant lactate/malate dehydrogenase proteins were decreased in flooding-stressed roots exposed to light, whereas the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme was increased in both light and dark conditions. Notably, however, specific enzyme assays revealed that the activities of these enzymes and biophoton emission were sharply increased after 3 days of flooding stress. This finding suggests that the source of biophoton emission in roots might involve the chemical excitation of electron or proton through enzymatic or non-enzymatic oxidation and reduction reactions. Moreover, the lysine ketoglutarate reductase/saccharopine dehydrogenase bifunctional enzyme may play important roles in responses in flooding stress of soybean under the light condition and as a contributing factor to biophoton emission.     

Analysis of flooding-responsive proteins localized in the nucleus of soybean root tips

Flooding stress has negative impact on soybean cultivation as it severely impairs plant growth and development. To examine whether nuclear function is affected in soybean under flooding stress, abundance of nuclear proteins and their mRNA expression were analyzed. Two-day-old soybean seedlings were treated with flooding for 2 days, and nuclear proteins were purified from root tips. Gel-free proteomics analysis identified a total of 39 flooding-responsive proteins, of which abundance of 8 and 31 was increased and decreased, respectively, in soybean root tips. Among these differentially regulated proteins, the mRNA expression levels of five nuclear-localized proteins were further analyzed. The mRNA levels of four proteins, which are splicing factor PWI domain-containing protein, epsilon2-COP, beta-catenin, and clathrin heavy chain decreased under flooding stress, were also down-regulated. In addition, mRNA level of a receptor for activated protein kinase C1(RACK1) was down-regulated, though its protein was accumulated in the soybean nucleus in response to flooding stress. These results suggest that several nuclear-related proteins are decreased at both the protein and mRNA level in the root tips of soybean under flooding stress. Furthermore, RACK1 might have an important role with accumulation in the soybean nucleus under flooding-stress conditions.     

Morphological and photosynthetic responses of riparian plant Distylium chinense seedlings to simulated Autumn and Winter flooding in Three Gorges Reservoir Region of the Yangtze River, China

To evaluate the tolerance of riparian plant Distylium chinense in Three Gorges Reservoir Region to anti-season flooding, a simulation flooding experiment was conducted during Autumn and Winter, and morphology and photosynthesis of D. chinense seedlings and their recovery growth after soil drainage were analyzed in different duration of flooding and flooding depth. The seedlings were submitted to four treatments: (1) 40 seedlings unflooded and watered daily as control (Unflooded, CK); (2) 120 seedlings flooded at 1 cm above the ground level (F-1 cm); (3) 120 seedlings flooded at 12 cm above the ground level (F-12 cm) and (4) 120 seedlings completely submerged with 2 m water depth (F-2 m, top of plants at 2 m below water surface). The flooding survival, plant height, stem diameter, adventitious roots, stem lenticels, epicormic shoots, chlorophyll content and photosynthesis parameters were determined at 0, 15, 30, 90 days in flooding stress and 15, 60 days after soil drainage. The results showed that the survival of the seedlings subjected to flooding was 100% for all repeated measurements in all treatments. Adventitious roots, hypertrophied lenticels and stem hypertrophy were observed in the seedlings flooded for more than 15 d, and increased with the prolonged flooding duration, while disappeared after the soil was drained. Flooding duration and flooding depth showed significant individual and interactive effects on leaf chlorophyll a (Chl a), chlorophyll b (Chl b), and their ratio, chlorophyll (a + b), the net photosynthesis rate (P_n), transpiration rate (T_r), stomatal conductance (C_s), and inter-cellular CO₂ concentration (C_i) of D. chinense seedlings (P < 0.01). After 15 days of flooding, there was no significant decrease in P_n of the flooded seedlings as compared with that of the control seedlings. P_n of the flooded seedlings was significantly lower than that of the control seedlings after 30 days of flooding (P < 0.05), whereas P_n showed no significant difference among seedlings from three flooding depths. After 90 days of flooding, P_n of the F-2 m flooded seedlings was significantly lower than that of the controls, F-1 cm and F-12 cm flooded seedlings (P < 0.05), but still maintained high photosynthetic capacity. P_n of the F-1 cm and F-12 cm flooded seedlings rose gradually after soil drainage, while, it was significantly lower than that of the control seedlings after 15 days of recovery (P < 0.05). After 60 days of recovery, P_n of all seedlings flooded with different depths showed no significant difference as compared with that of the control seedlings and new leaves grew out in the F-2 m flooded seedlings. The effect of all flooding treatments on Gs, T_r, Chl a, Chl b, Chl a/Chl b and chl (a + b) was basically the same as their effect on P_n, while the effect of all flooding treatments on C_i was quite the contrary. Correlation analysis showed that P_n was positively relative with Gs, T_r, Chl a, Chl b and chl (a + b) (P < 0.05) and significantly negative with C_i (P < 0.05). Therefore, the present study demonstrates that D. chinense has high survival and good recovery growth after long-term flooding in anti-season flooding and could be taken as an excellent candidate species in the re-vegetation of water-level-fluctuation areas in Three Gorges Reservoir Region.     

Morphological and photosynthetic responses of riparian plant Distylium chinense seedlings to simulated Autumn and Winter flooding in Three Gorges Reservoir Region of the Yangtze River, China

To evaluate the tolerance of riparian plant Distylium chinense in Three Gorges Reservoir Region to anti-season flooding, a simulation flooding experiment was conducted during Autumn and Winter, and morphology and photosynthesis of D. chinense seedlings and their recovery growth after soil drainage were analyzed in different duration of flooding and flooding depth. The seedlings were submitted to four treatments: (1) 40 seedlings unflooded and watered daily as control (Unflooded, CK); (2) 120 seedlings flooded at 1 cm above the ground level (F-1 cm); (3) 120 seedlings flooded at 12 cm above the ground level (F-12 cm) and (4) 120 seedlings completely submerged with 2 m water depth (F-2 m, top of plants at 2 m below water surface). The flooding survival, plant height, stem diameter, adventitious roots, stem lenticels, epicormic shoots, chlorophyll content and photosynthesis parameters were determined at 0, 15, 30, 90 days in flooding stress and 15, 60 days after soil drainage. The results showed that the survival of the seedlings subjected to flooding was 100% for all repeated measurements in all treatments. Adventitious roots, hypertrophied lenticels and stem hypertrophy were observed in the seedlings flooded for more than 15 d, and increased with the prolonged flooding duration, while disappeared after the soil was drained. Flooding duration and flooding depth showed significant individual and interactive effects on leaf chlorophyll a (Chl a), chlorophyll b (Chl b), and their ratio, chlorophyll (a + b), the net photosynthesis rate (P_n), transpiration rate (T_r), stomatal conductance (C_s), and inter-cellular CO₂ concentration (C_i) of D. chinense seedlings (P < 0.01). After 15 days of flooding, there was no significant decrease in P_n of the flooded seedlings as compared with that of the control seedlings. P_n of the flooded seedlings was significantly lower than that of the control seedlings after 30 days of flooding (P < 0.05), whereas P_n showed no significant difference among seedlings from three flooding depths. After 90 days of flooding, P_n of the F-2 m flooded seedlings was significantly lower than that of the controls, F-1 cm and F-12 cm flooded seedlings (P < 0.05), but still maintained high photosynthetic capacity. P_n of the F-1 cm and F-12 cm flooded seedlings rose gradually after soil drainage, while, it was significantly lower than that of the control seedlings after 15 days of recovery (P < 0.05). After 60 days of recovery, P_n of all seedlings flooded with different depths showed no significant difference as compared with that of the control seedlings and new leaves grew out in the F-2 m flooded seedlings. The effect of all flooding treatments on Gs, T_r, Chl a, Chl b, Chl a/Chl b and chl (a + b) was basically the same as their effect on P_n, while the effect of all flooding treatments on C_i was quite the contrary. Correlation analysis showed that P_n was positively relative with Gs, T_r, Chl a, Chl b and chl (a + b) (P < 0.05) and significantly negative with C_i (P < 0.05). Therefore, the present study demonstrates that D. chinense has high survival and good recovery growth after long-term flooding in anti-season flooding and could be taken as an excellent candidate species in the re-vegetation of water-level-fluctuation areas in Three Gorges Reservoir Region.     

Characterization of S-adenosylmethionine synthetases in soybean under flooding and drought stresses

Soybean is stress-sensitive crop that exhibits markedly reduced growth under flooding and drought conditions. Three S-adenosylmethionine synthetases (SAMs) proteins were identified as flooding and drought responsive proteins in soybean using a proteomic technique. To better understand the role of these SAMs proteins in soybean under flooding and drought stresses, temporal, organ, and stress specificities were examined at mRNA and enzyme activity levels. The activity of SAMs decreased in response to the flooding, however, it was not significantly changed by NaCl, cold, gibberellic acid, and calcium in soybean roots. The activity of SAMs was induced in roots and hypocotyls under drought. The mRNA expression of the S-adenosylmethionine synthetase (SAMs) family was down-regulated in root tips and roots under the flooding and the drought, and SAMs 1 and SAMs 2 were down-regulated in roots under both stresses. A gene 1-aminocyclopropane-1-carboxylate synthase was up-regulated in root tips, roots, and hypocotyls under drought, however, it was not changed in root tips and roots under the flooding. In addition, 1-aminocyclopropane-1-carboxylate oxidase was induced in root tips under flooding and drought. These results suggest that SAMs was involved in the response to the flooding and drought and it might affect ethylene biosynthesis in soybean.     

淹水对玉米不定根形态结构和ATP酶活性的影响

淹水２天后,玉米苗基节内即有不定根原基一进于正常植株。淹水１６天后,从基节部长出的不定根数多于正常植株,但淹水导致根系生长和干物质积累大幅度下降。淹水幼苗不定根伸长区内有发达的通气组织形成,使根内部组织孔隙度大幅提高。电镜细胞化学研究表明,经１５天淹一,不定根根尖细胞内ＡＴＰ酶的分布与正常功苗基本相同,酶活性尽管有一定的下降,但仍保持较高水平。根据实验结果,本文重点讨论了不定根的发生及其内部通气组     

Formation and function of secondary aerenchyma in hypocotyl, roots and nodules of soybean (Glycine max) under flooded conditions

Flooding is a major problem in many areas of the world and soybean is susceptible to the stress. Understanding the morphological mechanisms of flooding tolerance is important for developing flood-tolerant genotypes. We investigated secondary aerenchyma formation and function in soybean (Glycine max) seedlings grown under flooded conditions. Secondary aerenchyma, a white and spongy tissue, was formed in the hypocotyl, tap root, adventitious roots and root nodules after 3 weeks of flooding. Under irrigated conditions aerenchyma development was either absent or rare and phellem was formed in the hypocotyl, tap root, adventitious roots and root nodules. Secondary meristem partially appeared at the outer parts of the interfascicular cambium and girdled the stele, and then cells differentiated to construct secondary aerenchyma in the flooded hypocotyl. These morphological changes proceeded for 4 days after the initiation of the flooding. After 14 days of treatment, porosity exceeded 30% in flooded hypocotyl with well-developed secondary aerenchyma, while it was below 10% in hypocotyl of irrigated plants that had no aerenchyma. When Vaseline was applied to the hypocotyl of plants from a flooded treatment to prevent the entry of atmospheric oxygen into secondary aerenchyma, plant growth, especially that of roots, was sharply inhibited. Thus secondary aerenchyma might be an adaptive response to flooding.     

The major Nod factor of Bradyrhizobium japonicum promotes early growth of soybean and corn

Greenhouse experiments were conducted to evaluate the effect of Nod factor Nod Bj-V (C18:1, MeFuc) of Badyrhizobium japonicum on the growth of soybean and corn. Three-day-old seedlings of soybean and corn were grown in hydroponic solutions containing four concentrations (0, 10(-7), 10(-9) or 10(-11) M) of Nod factor. After 7 d of treatment, Nod factor enhanced soybean and corn biomass. Nod factor elicited profound effects on root growth resulting in 34-44% longer roots in soybean. More detailed analyses of the roots, using a scanner based image analysis system, revealed that Nod factor increased the total length, projected area and surface area of the roots and decreased the diameter of soybean roots, while it increased the total length of corn roots. Stem injection of soybean plants with 10(-7) M Nod factor resulted in increased dry matter accumulation. These results suggest that Nod factor, besides mediating early stages of nodulation, has more general plant growth-promoting effects.     

Responses of Melaleuca quinquenervia seedlings to flooding

Abstract Studies were conducted on effects of flooding for 15, 30, 60, and 90 days on morphological changes, stomatal aperture, water potential, and growth of seedlings of Melaleuca quinquenervia, a species often planted for reclamation of swamps. Flooding rapidly induced formation of many hair-like adventitious roots as well as a few thick adventitious roots that originated on the original root system. Some adventitious roots also formed on submerged portions of the stem. Melaleuca seedlings were very tolerant of flooding as shown by only slight reduction in dry weight increment of shoots after 30 days of flooding in stagnant water. Although flooding for 60 or 90 days significantly reduced dry weight increment of leaves, dry weight increment of roots was not inhibited by any flooding treatment, reflecting both degeneration of some of the original roots and compensatory growth of adventitious roots. On certain days flooding induced stomatal closure on both adaxial and abaxial leaf surfaces. Extensive production of adventitious roots and some stomatal reopening after a critical period of flooding appeared to be important factors in the flooding tolerance of Melaleuca and are consistent with its aggressiveness and vigorous growth on wet sites.     

Mass spectrometry-based analysis of proteomic changes in the root tips of flooded soybean seedlings

Flooding injury is a major problem in soybean cultivation. A proteomics approach was used to clarify the occurrence of changes in protein expression level and phosphorylation in soybeans under flooding stress. Two-day-old seedlings were flooded for 1 day, proteins were extracted from root tips of the seedlings and digested with trypsin, and their expression levels and phosphorylation states were compared to those of untreated controls using mass spectrometry-based proteomics techniques. Phosphoproteins were enriched using a phosphoprotein purification column prior to digestion and mass spectrometry. The expression of proteins involved in energy production increased as a result of flooding, while expression of proteins involved in protein folding and cell structure maintenance decreased. Flooding induced changes of phosphorylation status of proteins involved in energy generation, protein synthesis and cell structure maintenance. The response to flooding stress may be regulated by both modulation of protein expression and phosphorylation state. Energy-demanding and production-related metabolic pathways may be particularly subject to regulation by changes in protein phosphorylation during flooding.     

Microarray Analysis of Gene Expression in Soybean Roots Susceptible to the Soybean Cyst Nematode Two Days Post Invasion

Soybean root cells undergo dramatic morphological and biochemical changes during the establishment of a feeding site in a compatible interaction with the soybean cyst nematode (SCN). We constructed a cDNA microarray with approximately 1,300 cDNA inserts targeted to identify differentially expressed genes during the compatible interaction of SCN with soybean roots 2 days after infection. Three independent biological replicates were grown and inoculated with SCN, and 2 days later RNA was extracted for hybridization to microarrays and compared to noninoculated controls. Statistical analysis indicated that approximately 8% of the genes monitored were induced and more than 50% of these were genes of unknown function. Notable genes that were more highly expressed 2 days after inoculation with SCN as compared to noninoculated roots included the repetitive proline-rich glycoprotein, the stress-induced gene SAM22, ß-1,3-endoglucanase, peroxidase, and those involved in carbohydrate metabolism, plant defense, and signaling.     

Ecophysiological adaptations of black spruce (<Emphasis Type="Italic"> Picea mariana</Emphasis>) and tamarack (<Emphasis Type="Italic"> Larix laricina</Emphasis>) seedlings to flooding

Black spruce [ Picea mariana (Mill.) B.S.P.] and tamarack [ Larix laricina (Du Roi) K. Koch] are the predominant tree species in boreal peatlands. The effects of 34 days of flooding on morphological and physiological responses were investigated in the greenhouse for black spruce and tamarack seedlings in their second growing season (18 months old). Flooding resulted in reduced root hydraulic conductance, net assimilation rate and stomatal conductance and increased needle electrolyte leakage in both species. Flooded tamarack seedlings maintained a higher net assimilation rate and stomatal conductance compared to flooded black spruce. Flooded tamarack seedlings were also able to maintain higher root hydraulic conductance compared to flooded black spruce seedlings at a comparable time period of flooding. Root respiration declined in both species under flooding. Sugar concentration increased in shoots while decreasing in roots in both species under flooding. Needles of flooded black spruce appeared necrotic and electrolyte leakage increased over time with flooding and remained significantly higher than in flooded tamarack seedlings. No visible damage symptoms were observed in flooded tamarack seedlings. Flooded tamarack seedlings developed adventitious roots beginning 16 days after the start of flooding treatment. Adventitious roots exhibited significantly higher root hydraulic conductivity than similarly sized flooded tamarack roots. Flooded black spruce lacked any such morphological adaptation. These results suggest that tamarack is better able to adjust both morphologically and physiologically to prolonged soil flooding than black spruce seedlings.     

The effect of Ca2+-stress on fluxes of Ca2+ and K+ in wheat and cucumber

Betula papyrifera Marsh, seedlings adapted very poorly to flooding for up to 60 days. Responses to flooding included increased ethylene production; stomatal closure; leaf senescence; drastic inhibition of shoot growth, cambial growth, and root growth; decay of roots, and death of many seedlings. Flooding inhibited growth of leaves that formed prior to flooding, inhibited formation of new leaves, and induced abscission of old leaves. As a result of extensive leaf abscission, fewer leaves were present after flooding than before flooding was initiated. The drastic reduction in leaf area was associated with greatly decreased growth of the lower stem and roots. No evidence was found of adaptive morphological changes to flooding. The data indicate that intolerance of B. papyrifera seedlings to flooding is an important barrier to regeneration of the species on sites subject to periodic inundation.     

Identification of flooding stress responsible cascades in root and hypocotyl of soybean using proteome analysis

Flooding inducible proteins were analyzed using a proteomic technique to understand the mechanism of soybean response to immersion in water. Soybeans were germinated for 2 days, and then subjected to flooding for 2 days. Proteins were extracted from root and hypocotyl, separated by two-dimensional polyacrylamide gel electrophoresis, stained by Coomassie brilliant blue, and analyzed by protein sequencing and mass spectrometry. Out of 803 proteins, 21 proteins were significantly up-regulated, and seven proteins were down-regulated by flooding stress. Of the total, 11 up-regulated proteins were classified as related to protein destination/storage and three proteins to energy, while four down-regulated proteins were related to protein destination/storage and three proteins to disease/defense. The expression of 22 proteins significantly changed within 1 day after flooding stress. The effects of flooding, nitrogen substitution without flooding, or flooding with aeration were analyzed for 1–4 days. The expression of alcohol dehydrogenase increased remarkably by nitrogen substitution compared to flooding. The expression of many proteins that changed due to flooding showed the same tendencies observed for nitrogen substitution; however, the expression of proteins classified into protein destination/storage did not.