Proteome analysis of soybean roots subjected to short-term drought stress

Drought is one of the most important constraints on the growth and productivity of many crops, including soybeans. However, as a primary sensing organ, the plant root response to drought has not been well documented at the proteomic level. In the present study, we carried out a proteome analysis in combination with physiological analyses of soybean roots subjected to severe but recoverable drought stress at the seedling stage. Drought stress resulted in the increased accumulation of reactive oxygen species and subsequent lipid peroxidation. The proline content increased in drought-stressed plants and then decreased during the period of recovery. The high-resolution proteome map demonstrated significant variations in about 45 protein spots detected on Comassie briliant blue-stained 2-DE gels. Of these, 28 proteins were identified by mass spectrometry; the levels of 5 protein spots were increased, 21 were decreased and 2 spots were newly detected under drought condition. When the stress was terminated by watering the plants for 4 days, in most cases, the protein levels tended towards the control level. The proteins identified in this study are involved in a variety of cellular functions, including carbohydrate and nitrogen metabolism, cell wall modification, signal transduction, cell defense and programmed cell death, and they contribute to the molecular mechanism of drought tolerance in soybean plants. Analysis of protein expression patterns revealed that proteins associated with osmotic adjustment, defense signaling and programmed cell death play important roles for soybean plant drought adaptation. The identification of these proteins provides new insight that may lead to a better understanding of the molecular basis of the drought stress responses.     

Acetaldehyde and ethanol biosynthesis in leaves of plants

Leaves of terrestrial plants are aerobic organs, and are not usually considered to possess the enzymes necessary for biosynthesis of ethanol, a product of anaerobic fermentation. We examined the ability of leaves of a number of plant species to produce acetaldehyde and ethanol anaerobically, by incubating detached leaves in N₂ and measuring headspace acetaldehyde and ethanol vapors. Greenhouse-grown maize and soybean leaves produced little or no acetaldehyde or ethanol, while leaves of several species of greenhouse-grown woody plants produced up to 241 nanograms per milliliter headspace ethanol in 24 hours, corresponding to a liquid-phase concentration of up to 3 milligrams per gram dry weight. When leaves of 50 plant species were collected in the field and incubated in N₂, all higher plants produced acetaldehyde and ethanol, with woody plants generally producing greater amounts (up to 1 microgram per milliliter headspace ethanol concentration). Maize and soybean leaves from the field produced both acetaldehyde and ethanol. Production of fermentation products was not due to phylloplane microbial activity: surface sterilized leaves produced as much acetaldehyde and ethanol as did unsterilized controls. There was no relationship between site flooding and foliar ethanol biosynthesis: silver maple and cottonwood from upland sites produced as much acetaldehyde and ethanol anaerobically as did plants from flooded bottomland sites. There was no relationship between flood tolerance of a species and ethanol biosynthesis rates: for example, the flood intolerant species Quercus rubra and the flood tolerant species Quercus palustris produced similar amounts of ethanol. Cottonwood leaves produced more ethanol than did roots, in both headspace and enzymatic assays. These results suggest a paradox: that the plant organ least likely to be exposed to anoxia or hypoxia is rich in the enzymes necessary for fermentation.     

Proteomic analysis of soybean root hairs after infection by Bradyrhizobium japonicum

Infection of soybean root hairs by Bradyrhizobium japonicum is the first of several complex events leading to nodulation. In the current proteomic study, soybean root hairs after inoculation with B. japonicum were separated from roots. Total proteins were analyzed by two-dimensional (2-D) polyacrylamide gel electrophoresis. In one experiment, 96 protein spots were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) to compare protein profiles between uninoculated roots and root hairs. Another 37 spots, derived from inoculated root hairs over different timepoints, were also analyzed by tandem MS (MS/MS). As expected, some proteins were differentially expressed in root hairs compared with roots (e.g., a chitinase and phosphoenolpyruvate carboxylase). Out of 37 spots analyzed by MS/MS, 27 candidate proteins were identified by database comparisons. These included several proteins known to respond to rhizobial inoculation (e.g., peroxidase and phenylalanine-ammonia lyase). However, novel proteins were also identified (e.g., phospholipase D and phosphoglucomutase). This research establishes an excellent system for the study of root-hair infection by rhizobia and, in a more general sense, the functional genomics of a single, plant cell type. The results obtained also indicate that proteomic studies with soybean, lacking a complete genome sequence, are practical.     

Functional Electron Microscopy in Studies of Plant response and adaptation to Anaerobic Stress

This article reviews the contribution made by functional electronmicroscopy towards identifying and understanding the reactionsof plant roots and shoots to anaerobic stress. Topics examinedinclude: (1) unexpected hypersensitivity, rather than hyper-resistance,to anoxia of root tips of flooding-tolerant plants; (2) protective,rather than damaging, effects of a stimulated energy metabolism(glycolysis and fermentation) under anaerobic conditions; (3)the concept of two main strategies of plant adaptation to anaerobicenvironments, namely avoidance of anaerobiosis on the wholeplant level, termed ‘apparent’ tolerance, and metabolicadaptation at the cellular and molecular levels, termed ‘true’tolerance; (4) the importance of protein synthesis during hypoxiaand anoxia for enhanced energy production and metabolic adaptation;(5) a general adaptive syndrome in plants to stress at the ultrastructurallevel and a possible molecular mechanism for its realizationunder anoxia; (6) the physiological role of anaerobically synthesizedlipids and nitrate as alternative electron acceptors in an oxygen-freemedium; and (7) the selection of cell lines derived from calluscultures that possess enhanced tolerance to anoxia and can regeneratewhole plants with improved tolerance of soil waterlogging.     

G proteins as regulators in ethylene-mediated hypoxia signaling

Waterlogging or flooding are frequently or constitutively encountered by many plant species. The resulting reduction in endogenous O₂ concentration poses a severe threat. Numerous adaptations at the anatomical, morphological and metabolic level help plants to either escape low oxygen conditions or to endure them. Formation of aerenchyma or rapid shoot elongation are escape responses, as is the formation of adventitious roots. The metabolic shift from aerobic respiration to anaerobic fermentation contributes to a basal energy supply at low oxygen conditions. Ethylene plays a central role in hypoxic stress signaling, and G proteins have been recognized as crucial signal transducers in various hypoxic signaling pathways. The programmed death of parenchyma cells that results in hypoxia-induced aerenchyma formation is an ethylene response. In maize, aerenchyma are induced in the absence of ethylene when G proteins are constitutively activated. Similarly, ethylene induced death of epidermal cells that cover adventitious roots at the stem node of rice is strictly dependent on heterotrimeric G protein activity. Knock down of the unique Gα gene RGA1 in rice prevents epidermal cell death. Finally, in Arabidopsis, induction of alcohol dehydrogenase with resulting increased plant survival relies on the balanced activities of a small Rop G protein and its deactivating protein RopGAP4. Identifying the general mechanisms of G protein signaling in hypoxia adaptation of plants is one of the tasks ahead.Key words: submergence, hypoxia, ethylene, G protein, reactive oxygen species, H₂O₂     

Effect of salinity and waterlogging on growth,anatomical and antioxidative responses in <Emphasis Type="Italic">Mentha aquatica</Emphasis> L.

Salinity and waterlogging are two stresses which in nature often occur simultaneously. In this work, effects of combined waterlogging and salinity stresses are studied on the anatomical alteration, changes of enzymatic antioxidant system and lipid peroxidation in Mentha aquatica L. plants. Seedlings were cultured in half-strength Hoagland medium 50 days after sowing, and were treated under combination of three waterlogging levels (well drained, moderately drained and waterlogging) and NaCl (0, 50, 100, 150 mM) for 30 days. Moderately drained and waterlogging conditions induced differently aerenchyma formation in roots of M. aquatica salt-treated and untreated plants. Moreover, stele diameter and endodermis layer were also affected by salt stress and waterlogging. Salt stress significantly decreased growth, relative water content (RWC), protein level, catalase (CAT) and polyphenol oxidase (PPO) activities, and increased proline content, MDA content, H₂O₂ level and activities of superoxide dismutase (SOD), peroxidase (POX), and ascorbate peroxidase (APX). Waterlogging in salt-untreated plants increased significantly growth parameters, RWC, protein content, antioxidant enzyme activity, and decreased proline content, H₂O₂ and MDA levels. In salt-treated plant, waterlogging caused strong induction of antioxidant enzymes activities especially at severe stress condition. These results suggest M. aquatica is a waterlogging tolerant plant due to significant increase of antioxidant activity, membrane stability and growth under water stress. High antioxidant capacity under waterlogging can be a protective strategy against oxidative damage, and help to salt stress alleviation.     

Identification of NaCl and NaHCO3 stress responsive proteins in tomato roots using iTRAQ-based analysis

Soil salinity and alkalinity are common constraints to crop productivity in low rainfall regions of the world. However, the physiological difference of plant response to these two stresses was short of deep investigation. This study has identified a set of differentially expressed proteins of tomato root exploring to NaCl and NaHCO₃ stress by iTRAQ (isobaric tags for relative and absolute quantitation) assay. A total of 313 proteins responsive to NaCl and NaHCO₃ were observed. Among these proteins, 70 and 114 proteins were up-regulated by salt and alkali stress, respectively. While down-regulated proteins were 80 in salt treatment and 83 in alkali treatment. Only 39 up-regulated proteins and 30 down-regulated proteins were shared by salt and alkali stresses. The majority of the down-regulated proteins accounted for metabolism and energy conversion, and the up-regulated proteins were involved in signaling or transport. Compared with salt stress, alkali stress down-regulated proteins related with the respiratory metabolism, fatty acid oxidative metabolism and nitrogenous metabolism of tomato roots, and up-regulated protein with the reactive oxygen species (ROS) scavenging and ion transport. This study provides a novel insight into tomato roots response to salt and alkali stress at a large translation level.     

外源亚精胺对淹涝胁迫下黄瓜根HSP70表达的影响

采用逆转录聚合酶链式反应(RT-PCR)及蛋白免疫印迹杂交(Western Blot)技术,研究0.5 mmol/L亚精胺浸种的黄瓜幼苗在淹水胁迫下,根热激蛋白70基因(HSP70)mRNA和蛋白质的表达量的变化。结果表明:淹水胁迫使黄瓜根HSP70的mRNA和蛋白的表达呈现先上升后下降的趋势,在淹水4 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理; 亚精胺浸种的黄瓜根HSP70的mRNA和蛋白的表达量在24 h内呈一直上升的趋势,在淹水24 h时,HSP70的mRNA和蛋白表达量均极显著高于未淹水处理。淹涝胁迫下,亚精胺浸种的黄瓜根HSP70的mRNA和蛋白表达量在淹水12 h和24 h时极显著高于蒸馏水浸种。外源亚精胺能诱导淹涝胁迫下黄瓜幼苗根HSP70 mRNA和蛋白质的表达量的增加,缓解淹涝胁迫对黄瓜造成的伤害。     

pH-dependent catabolic protein expression during anaerobic growth of Escherichia coli K-12

During aerobic growth of Escherichia coli, expression of catabolic enzymes and envelope and periplasmic proteins is regulated by pH. Additional modes of pH regulation were revealed under anaerobiosis. E. coli K-12 strain W3110 was cultured anaerobically in broth medium buffered at pH 5.5 or 8.5 for protein identification on proteomic two-dimensional gels. A total of 32 proteins from anaerobic cultures show pH-dependent expression, and only four of these proteins (DsbA, TnaA, GatY, and HdeA) showed pH regulation in aerated cultures. The levels of 19 proteins were elevated at the high pH; these proteins included metabolic enzymes (DhaKLM, GapA, TnaA, HisC, and HisD), periplasmic proteins (ProX, OppA, DegQ, MalB, and MglB), and stress proteins (DsbA, Tig, and UspA). High-pH induction of the glycolytic enzymes DhaKLM and GapA suggested that there was increased fermentation to acids, which helped neutralize alkalinity. Reporter lac fusion constructs showed base induction of sdaA encoding serine deaminase under anaerobiosis; in addition, the glutamate decarboxylase genes gadA and gadB were induced at the high pH anaerobically but not with aeration. This result is consistent with the hypothesis that there is a connection between the gad system and GabT metabolism of 4-aminobutanoate. On the other hand, 13 other proteins were induced by acid; these proteins included metabolic enzymes (GatY and AckA), periplasmic proteins (TolC, HdeA, and OmpA), and redox enzymes (GuaB, HmpA, and Lpd). The acid induction of NikA (nickel transporter) is of interest because E. coli requires nickel for anaerobic fermentation. The position of the NikA spot coincided with the position of a small unidentified spot whose induction in aerobic cultures was reported previously; thus, NikA appeared to be induced slightly by acid during aeration but showed stronger induction under anaerobic conditions. Overall, anaerobic growth revealed several more pH-regulated proteins; in particular, anaerobiosis enabled induction of several additional catabolic enzymes and sugar transporters at the high pH, at which production of fermentation acids may be advantageous for the cell.     

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.     

Antioxidative responses and morpho-anatomical adaptations to waterlogging in Sesbania virgata

Sesbania virgata (Leguminosae) is tolerant of long periods of soil inundation. However, its morphological adaptations to anoxia and its response to possible damage from oxidative stress are still unknown. Here, we provide new information that helps to explain the ability of S. virgata plants to grow in flooded environments. Plants containing six expanded leaves were placed in masonry tanks and were subjected to the following conditions: control (well watered), soil waterlogging (water to the setup level of 1 cm above the soil surface—roots and parts of the stems flooded), and complete submergence (whole plant flooded). Plants exposed to flooding (soil waterlogging and complete submergence) significantly increased their production of hydrogen peroxide (H₂O₂), indicating the extent of oxidative injury posed by stress conditions. We demonstrate that plants exposed to flooding develop an efficient scavenger of ROS (generated during stress) in the roots through the coordinated action of nonenzymatic ascorbic acid (Asc) and dehydroascorbate (DHA) as well as the enzymatic antioxidants superoxide dismutase (SOD), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), and glutathione reductase (GR) that are present in the tissues. Moreover, we observed the development of morpho-anatomical structures such as adventitious roots, lenticels, and cracks in the stem of plants under soil waterlogging. The secondary root of plants under soil waterlogging showed a thinner cortex and larger number of elements of small diameter vessels. Numerous aerenchymas were observed in the newly formed in the adventitious roots. We conclude that these antioxidative responses and morpho-anatomical adaptations in the roots are part of a suite of adaptations that allow S. virgata plants to survive long periods of flooding, notably under waterlogged conditions.     

Dinitro-o-cresol induces apoptosis-like cell death but not alternative oxidase expression in soybean cells

In plants, programmed cell death is thought to be activated during differentiation and in response to biotic and abiotic stresses. Although its mechanisms are far less clear, several morphological and biochemical features have been described in different experimental systems, including DNA laddering and cytosolic protease activation. Moreover, plant mitochondria have an alternative terminal oxidase (AOX), which is thought to be involved in protection against increased reactive oxygen species production, perhaps representing a mechanism to prevent programmed cell death. In this study, we analysed cell death induced by the herbicide dinitro-o-cresol (DNOC) in soybean (Glycine max) suspension cell cultures and evaluated biochemical and molecular events associated with programmed cell death. AOX capacity and expression were also determined. DNOC-treated cells showed fragmented nuclear DNA as assessed by an in situ assay that detects 3'-OH ends. In addition, specific colorimetric assays and immunoblot analysis revealed activation of caspase-3-like proteins and release of cytochrome c from mitochondria, respectively, confirming the apoptotic-like phenotype. Surprisingly, AOX capacity and protein levels decreased in DNOC-treated cells, suggesting no association between cell death and AOX under these experimental conditions. In conclusion, the results show that DNOC induces programmed cell death in soybean cells, suggesting that plants and animals might share similar pathways. Further, the role of AOX in cell death has not been confirmed, and may depend on the nature and intensity of stress conditions.     

淹水胁迫对青杨雌雄幼苗生理特性和生长的影响

为揭示青杨(Populus cathayana)雌雄幼苗对淹水胁迫的适应性, 在实验地内通过土培盆栽淹水方式从植株生理生态和生长发育方面探讨淹水胁迫对青杨扦插苗的影响。试验分为对照和淹水2个处理, 处理时间为40天。结果显示: (1)淹水胁迫导致青杨幼苗叶片中的丙二醛(MDA)含量和茎部淹水区的不定根数显著升高, 植株的净光合速率(P_n)、叶绿素含量、超氧化物歧化酶(SOD)活性、株高、基径、总叶面积、比叶面积(SLA)、根生物量、叶生物量、茎生物量、总生物量干重和根冠比(R/S)显著降低。(2)与雄株相比, 淹水胁迫显著增加了雌株幼苗的MDA含量, 降低了SOD活性、P_n、类胡萝卜素(Caro)含量、叶绿素a/b、SLA、根生物量和R/S, 并导致雄株在淹水胁迫下具有比雌株更高的气孔导度(G_s)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)、不定根数和株高。可见, 淹水胁迫对青杨雌雄幼苗的形态生长和生理过程均有严重的抑制作用, 但表现出显著的性别间差异。雄株可以通过维持更高的光合作用能力和增加不定根数量来维持植株的生长, 从而表现出比雌株更强的抗逆性。     

Morpho-anatomical and physiological responses to waterlogging stress in different barley (<Emphasis Type="Italic">Hordeum vulgare</Emphasis> L.) genotypes

Waterlogging is one of the major stresses limiting crop production worldwide. The understanding of the mechanisms of plant adaptations to waterlogging stress helps improve plant tolerance to stress. In this study, physiological responses and morpho-anatomical adaptations of seven different barley genotypes were investigated under waterlogging stress. The results showed that the waterlogging-tolerant varieties (TX9425, Yerong, TF58) showed less reduction in plant height, SPAD (soil–plant analyses development analyses) value, tillers, shoot and root biomasses than did the waterlogging-sensitive varieties (Franklin, Naso Nijo, TF57). Under waterlogging stress condition, the tolerant genotypes also showed a much larger number of adventitious roots than did the sensitive genotypes. More intercellular spaces and better integrated chloroplast membrane structures were observed in the leaves of the waterlogging-tolerant cultivars, which is likely due to increased ethylene content, decreased ABA content and less accumulation of O₂^.?. The ability to form new adventitious roots and intercellular spaces in shoots can also be used as selection criteria in breeding barley for waterlogging tolerance.