Soybean production statistics (FAOSTAT 2010)
Soil is a dynamic environment due to fluctuations in climatic conditions that affect pH, temperature, water and nutrient availability. These factors, along with agricultural management practices, affect the soil micro-flora health and the capacity for effective plant-microbe interactions. Despite these constant changes, soil constitutes one of the most productive of earth’s ecospheres and is a hub for evolutionary and other adaptive activities.
1.1. Biological nitrogen fixation
Biological nitrogen fixation (BNF) is one of the most important phenomena occurring in nature, only exceeded by photosynthesis [1,2]. One of the most common limiting factors in plant growth is the availability of nitrogen . Although 4/5ths of earth’s atmosphere is comprised of nitrogen, the ability to utilize atmospheric nitrogen is restricted to a few groups of prokaryotes that are able to covert atmospheric nitrogen to ammonia and, in the case of the legume symbiosis, make some of this available to plants. Predominantly, members of the plant family Leguminosae have evolved with nitrogen fixing bacteria from the family Rhizobiaceae. In summary, the plants excrete specific chemical signals to attract the nitrogen fixing bacteria towards their roots. They also give the bacteria access to their roots, allowing them to colonize and reside in the root nodules, where the modified bacteria (bacteroids) can perform nitrogen fixation [1,4,5]. This process is of great interest to scientists in general, and agriculture specifically, since this highly complex recognition and elicitation is co-ordinated through gene expression and cellular differentiation, followed by plant growth and development; it has the potential to minimize the use of artificial nitrogen fertilizers and pesticides in crop management. This biological nitrogen fixation process is complex, but has been best examined in some detail in the context of soybean-
1.2. Soybean – The plant
Soybean originated in China, where it has been under cultivation for more than 5000 years . The annual wild soybean (
Soybean is a well-known nitrogen fixer and has been a model plant for the study of BNF. Its importance in BNF led to the genome sequencing of soybean; details of the soybean genome are available at soybase.org (
The efficiency of BNF depends on climatic factors such as temperature and photoperiod ; the effectiveness of a given soybean cultivar in fixing atmospheric nitrogen depends on the interaction between the cultivar’s genome and conditions such as soil moisture and soil nutrient availability [17,18]; and the competitiveness of the bacterial strains available, relative to indigenous and less effective strains, plus the amount and type of inoculants applied, and interactions with other, possibly antagonistic, agrochemicals that are used in crop protection . The most important criteria, however, is the selection of an appropriate strain of
Initially attached to the root-hair tips of soybean plants, rhizobia colonize within the roots and are eventually localized within symbiosomes, surrounded by plant membrane. This symbiotic relationship provides a safe niche and a constant carbon source for the bacteria while the plant derives the benefits of bacterial nitrogen fixation, which allows for the use of readily available nitrogen for plant growth. Inoculation of soybean with
1.4. Lipo-chitooligosaccharide (LCO) from
As mentioned earlier in this review, the process of nodulation in legumes begins with a complex signal exchange between host plants and rhizobia. The first step in rhizobial establishment in plant roots is production of isoflavonoids as plant-to-bacterial signals; the most common in the soybean-
Nodulation and subsequent nitrogen fixation are affected by environmental factors. It has been observed that, under sub-optimal root zone temperatures (for soybean 15-17 ºC), pH stress and in the presence of nitrogen, isoflavanoid signal levels are reduced; while high temperature (39 ºC) increases non-specific isoflavanoid production and reduces
LCOs also positively and directly affect plant growth and development in legumes and non-legumes. The potential role of LCOs in plant growth regulation was first reported by Denarie and Cullimore ). Nod genes A and B from
Since the protein quality of soybean plays an important role in overall agricultural and in nutraceuticals production, it is imperative that we study the proteomics of soybean and its symbiont
1.5. Proteomics as a part of integrative systems biology
The “omics” approach to knowledge gain in biology has advanced considerably in the recent years. The triangulation approach of integrating transcriptomics, proteomics and metabolomics is being used currently to study interconnectivity of molecular level responses of crop plants to various conditions of stress tolerance and adaptation of plants, thus improving systems level understanding of plant biology [60, 61].
While transcriptomics is an important tool for studying gene expression, proteomics actually portrays the functionality of the genes expressed. Several techniques are available for studying differential expression of protein profiles, and can be broadly classified as gel-based and MS (mass spectrometry)-based quantification methods. The gel based approach uses conventional, two-dimensional (2-D) gel electrophoresis, and 2-D fluorescence difference gel electrophoresis (2D-DIGE), both based on separation of proteins according to isoelectric point, followed by separation by molecular mass. The separated protein spots are then isolated and subjected to MS analysis for identification. Major drawbacks of these techniques are laborious sample preparation and inability to identify low abundance, hydrophobic and basic proteins.
The MS based approach can be a label-based quantitation, where the plants or cells are grown in media containing 15N metabolite label or using 15N as the nitrogen source. Label-free quantitation, however, is easier and allows analysis of multiple and unlimited samples. This technique, also referred to as MudPIT (multidimensional protein identification technology), is a method used to study proteins from whole-cell lysate and/or a purified complex of proteins [62,63]. The total set of proteins or proteins from designated target sites are isolated and subjected to standard protease digestions (eg. such as tryptic digestion). In brief, flash frozen leaf samples are ground in liquid nitrogen and polyphenols; tannins and other interfering substances such as chlorophyll are removed. The processed tissue is resuspended in a chaotropic reagent to extract proteins in the upper phase, and the plant debris is discarded [64-70]. The total protein set, in the resulting solution, is further quantified using the Lowry method . The protein samples (2 µg of total protein each), once digested with trypsin, can then be loaded onto a microcapillary column packed with reverse phase and strong cation exchange resins. The peptides get separated in the column, based on their charge and hydrophobicity. The columns are connected to a quarternary high-performance liquid chromatography pump and coupled with an ion trap mass spectrometer, to ionize the samples within the column and spray them directly into a tandem mass spectrometer. This allows for a very effective and high level of peptide separation within the mixture, and detects the eluting peptides to produce a mass spectrum. The detected peptide ions, at measured mass-to-charge (m/z) ratios with sufficient intensity, are selected for collision-induced dissociation (CID). This procedure allows for the fragmenting of the peptides to produce a product ion spectrum, the MS/MS spectrum. In addition, the fragmentation occurs preferentially at the amide bonds, to generate N-terminal fragments (b ions) and C-terminal fragments (y ions) at specific m/z ratios, providing structural information about the amino acid sequence and sites of modification. The b ion and y ion patterns are matched to a peptide sequence in a translated genomic database to help identify the proteins present in the sample [72-75]. A variety of database searching and compiling algorithms are used to interpret the data obtained for structure and function of the identified proteins.
2. Analyses of soybean proteomics
2.1. Physiological and biological changes in the soybean proteome
2.1.1. Whole plant organs
The various tissues of soybean have specific groups of associated proteins at each developmental stage. While leaves at various developmental stages showed 26 differentially expressed proteins, the first trifoliate stage manifested the greatest increase in protein types of the outer/inner envelope of choloroplast membrane and also of the protein transport machineries. Young leaves showed abundant chaperonin-60, while HSP 70 and TP-synthase b were present in all the tissues analyzed. Age dependent correlation was observed in net photosynthesis rate, chlorophyll content and carbon assimilation. During the flowering stage, flower tissue expressed 29 proteins that were exclusively involved in protein transport and assembly of mitochondria, secondary metabolism and pollen tube growth (Ahsan and Komatsu., 2009 . Soybean peroxisomal adenine nucleotide carrier (GmPNC1) is associated with the peroxisomal membrane and facilitates ATP and ADP importing activities. The proteins At PNC1 and At PNC2 are arabidopsis orthologs of Gm PNC1. Under constant darkness, Gm PNC1 increased in cotyledons up to 5 days post germination and the levels were rapidly reduced when the seedlings were exposed to light. RNA interference studies on arabidopsis At PNC1 and At PNC2 suggests that PNC1 assists with transport of ATP/ADP in the peroxisomal fatty acid-b oxidation pathway post germination (Arai et al., 2008 . This probably helps the seedling establish vigour for future growth.
In order to establish if xylem proteins and the apoplast conduit are involved in long distance signalling in autoregulation of nodulation (AON) in the soybean-
Proteomic studies on chasmogamous (CH) CH cv. Toyosuzu and cleistogamous (CL) CL cv. Karafuto-1 flowerbuds using 2D gel revealed differential protein levels of β-galactosidase and protein disulfide isomerase. Cleistogamy occurs in plants under diverse stress conditions, such as drought and cold, and can also vary with temperature and light . Soybean cv Maverick was used to study proteomics during seed filling stages, at 2, 3, 4, 5 and 6 weeks after flowering, using 2D and MALDI-TOF-MS. Storage proteins, proteins involved in metabolism and metabolite transport and defense related proteins were the most abundant, along with cysteine and methionine biosynthesis proteins, lipoxygenases and 14-3-3-like proteins [80,81].
Based on these findings, it is clear that the plant partitions its proteomics based on ontogeny and this specificity probably plays a crucial role in organ maturation and transition from one stage to another in the plants life cycle. Understanding this is of fundamental importance in agriculture, global food production, biofuel production and issues such as plant responses to climate change.
Both 2D gel and peptide mass fingerprinting techniques (MALDI-TOF-MS) were used to study the proteins of mature and dry soybean (cv. Jefferson) seeds. Sucrose binding proteins, alcohol dehydrogenase and seed maturation proteins were some of the key proteins identified (Mooney and Thelen 2004 . A comparison of four methods for protein isolation and purification from soybean seed was one of the first reports on soybean proteomics; thiourea/urea and TCA protocols were found to be the best. Proteins extracted with these two methods and further characterized by MALDI-TOF-MS and LC-MS helped identify proteins such as β-conglycinin, glycinin, Kunitz trypsin inhibitor, alcohol dehydrogenase, Gm Bd 28K allergen and sugar binding proteins in seeds . The two major soybean storage proteins are α-conglycinin and glycinin. While the α-conglycinin subunits separated well in the pH range 3.0-10.0, glycinin polypeptides could be separated in pH ranges 4.0-7.0 and 6.0-11.0. Apart from these major storage proteins, this combined proteomic approach (2D-PAGE and immobilized pH gradient strips) also identified 44 storage proteins in wild soybean (
Synthesis of soybean glycinin and conglycinin, was suppressed by RNA interference. The storage protein knockdown (SP2) seeds were very similar to the wild type during development and at maturity. Proteomic analysis of the SP2 soybean genotypes and next-generation transcript sequencing (RNA-Seq) suggested that the seeds could rebalance their transcriptome and metabolome in the face of at least some alterations. GFP quantification for glycinin allele mimics further revealed that glycinin was not involved in proteome rebalance and that seeds are capable of compensating through increases in other storage proteins, to maintain normal protein content, even if the major storage proteins were not available .
Transgenic soybean seeds have higher amounts of malondialdehyde, ascorbate peroxidase, glutathione reductase, and catalase (29.8, 30.6, 71.4, and 35.3%, respectively) than non-transgenic seeds. Precursors of glycinin, allergen Gly m Bd 28k, actin and sucrose binding proteins were the other proteins identified [87,88]. High protein accessions of soybean (with 45 % or more protein in seeds) were compared with soybean cultivar Williams 82. 2-DE-MALDI-TOF-MS followed by Delta2D image analysis showed huge differences in 11S storage globulins amongst the accessions. In addition, the trait for high protein from PI407788A was moved to experimental line LG99-469 and was stable upon transformation [89,90].
2.1.3. Roots, root hairs and nodules
Since the root apical meristem (RAM) is responsible for the growth of the plant root system and root architecture plays and important role in determining the performance of crop plants, a proteome reference map of the soybean root apex and the differentiated root zone was established. The root apex samples comprised of 1 mm of the root apex, encasing the RAM, the quiescent center and the root cap. The predominant proteins in the root belonged to those of stress response, glycolysis, redox homeostasis and protein processing machinery. The root apex contained key proteins, such as those involved in redox homeostasis and flavonoid biosynthesis, but was underrepresented in glycolysis, stress response and TCA cycle related proteins . Analysis of the proteome of isolated soybean root hair cells using 2-D gel and shotgun proteomics approaches identified proteins involved in basic cell metabolism, those whose functions are specific to root hair cell activities, including water and nutrient uptake, vesicle trafficking, and hormone and secondary metabolism [92, 93]. Proteomic studies of soybean roots and root hairs after
2.2. Soybean proteomics under stress conditions
Like all plants, soybean also encounters various stressors during its life cycle. Work related to flooding, drought, salt, heat, biotic stressors, metal toxicity, ozone, phosphorous deficiency and seed protein allergens are reviewed here.
2.2.1. Flooding stress
Plasma membrane proteins from the root and hypocotyl of soybean seedlings were purified and subjected to 2-D gel electrophoresis, followed by MS and protein sequencing, and also using nanoliquid chromatography followed by nano-LC-MS/MS based proteomics. The two techniques were used to compare the proteins present, and this indicated that during flooding stress proteins typically found in the cell wall were up-regulated in the plasma membrane. Also, the anti-oxidative proteins were up-regulated to protect the cells from oxidative damage, heat shock proteins to protect protein degradation and signaling proteins to regulate ion homeostasis . MS based proteomics applied to root tips of two-day-old seedlings flooded for 1 day showed increased levels of proteins involved in energy production. Proteins involved in cell structure maintenance and protein folding were negatively affected, as was their phosphorylation status .
Two-day-old germinated soybean seeds were subjected to water logging for 12 h and total RNA and proteins were analyzed from the root and hypocotyl. At the transcriptional level, the expression of genes for alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell wall loosening were all significantly up-regulated, while scavengers and chaperons of reactive oxygen species were seen to change only at the translational level. Transcriptional and translational level changes were observed for hemoglobin, acid phosphatase, and Kunitz trypsin protease inhibitors. This adaptive strategy might be for both hypoxia and more direct damage of cells by excessive water ). Proteins from 2-day-old soybean seedlings flooded for 12 h were analyzed using 2-D gel MS, 2-D fluorescence difference gel electrophoresis, and nanoliquid chromatography. Early responses to flooding involved proteins related to glycolysis and fermentation, and inducers of heat shock proteins. Glucose degradation and sucrose accumulation increased due to activation of glycolysis and down-regulation of sucrose degrading enzymes, in addition the methylglyoxal pathway, a detoxification system linked to glycolysis, was up-regulated. 2-D gel based phosphoproteomic analysis showed that proteins involved in protein synthesis and folding were dephosphorylated under flooding conditions . Water logging stress imposed on very early soybean seedlings (V2 stage) resulted in a gradual increase of lipid peroxidation and
Soybean seeds germinated for 48 h were subjected to water logging stress for 6-48 h. In addition to general stress responses due to increases in reactive oxygen species scavengers, several glycolytic enzymes were up-regulated, suggesting changes in energy generation .
2.2.2. Water stress – Drought
Soybean root activities are affected during water stress. The root can be partitioned into zones 1 (apical 4 mm zone) and 2 (4-8 mm zone), based on maximum elongation during well watered conditions. Soluble proteins from these regions, studied under both well-watered and water deficit stress conditions, revealed region-specific regulation of the phenylpropanoid pathway. Zone 1 of roots manifested increases in isoflavanoid biosynthesis related enzymes and proteins that contribute to growth and maintenance of the roots under water stress conditions. However, zone 2 of water stressed roots manifested up-regulation of caffeoyl-CoA
2.2.3. High temperature stress
Tissue specific proteomics under high temperature stress revealed 54, 35 and 61 differentially expressed proteins in the leaves, stems and roots, respectively. Heat shock proteins and those involved in antioxidant defense were up-regulated while proteins for photosynthesis, amino acid and protein synthesis and secondary metabolism were down- regulated. HSP70 and other low molecular weight HSPs were seen in all the tissues analyzed. ChsHSP and CPN-60 were tissue specific and the sHSPs were found only in tissues under heat stress, and were not induced by other stresses such as cold or hydrogen peroxide exposure .
2.2.4. Salt stress
Salt stress is also an important abiotic stressor that affects crop growth and productivity. Of the 20% of agricultural land available globally, 50% of the cropland is estimated by the United Nations Environment Program (The UNEP) to be salt-stressed . As the plant grows under salt stresses conditions, depending on the severity of the stress, the plants can experience reduced photosynthesis, protein and energy production, and changes in lipid metabolism [109,110]. As soil salinity increase, the effects on seed germination and germinating seedlings are profound. Responses to salinity and drought stress are similar; they affect the osmotic activity of the root system, thereby affecting the movement of water and nutrients into the plants. In Canadian soils, salinity varies between spring and fall and the most saline conditions are seen at the soil surface just after spring thaw. In the Canadian prairies, the dominant salts of saline seeps include calcium (Ca), magnesium (Mg) and sodium (Na) cations, and sulphate (SO4-) anions . Soybean is very sensitive to Cl-, but not greatly affected by Na+, because of its ability to restrict movement of Na+ to leaves .
This first report regarding soybean seedling proteomic responses to salt stress evaluated length and fresh weight of the hypocotyl and roots of soybean exposed to a series of NaCl concentrations. At 200 mM NaCl, the length and fresh weight of hypocotyl and roots were greatly reduced, with a simultaneous increase in proline content, suggesting activation of mechanisms for coping with salt stress. In addition, hypocotyl and root samples from 100 mM NaCl treated seedlings up-regulated seven key proteins, such as late embryogenesis-abundant protein, b-conglycinin, elicitor peptide three precursor, and basic/helix-loop-helix protein. The same treatment caused down-regulation of protease inhibitor, lectin, and stem 31-kDa glycoprotein precursor. This combination of up- and down-regulated proteins indicates a metabolic shift and could represent a strategy used by soybean seedlings to enhance tolerance of, or adapt to, salt stress .
Sobhanian et al. [110,114] found that treatment of soybean seedlings with 80 mM NaCl arrests the growth and development of both hypocotyl and roots. This study assessed effects on leaf, hypocotyl and root proteomics of salt treated soybean seedlings and found that reduction of glyceraldehyde-3-phospahte dehydrogenase was indicative of reduction in ATP production, and down-regulation of calreticulin was associated with disruption in the calcium signalling pathway, both of which are associated with decreased plant growth. The levels of other proteins, such as kinesin motor protein, trypsin inhibitor, alcohol dehydrogenase and annexin, were also found to change, suggesting that these proteins might play different roles in soybean salt tolerance and adaptation [110,114].
Soybean cultivars Lee68 and N2899 are salt-tolerant and salt-sensitive, respectively. The percentage germination was not affected when exposed to 100 mmol L-1 NaCl, however, the mean germination time for Lee68 (0.3 days) and N2899 (1.0 day) was delayed, compared with control plants. Hormonal responses to salt stress differed between these cultivars. Both cultivars, increased abscisic acid levels and decreased giberrelic acid (GA 1, 3) and isopentyladenosine concentrations; auxin (IAA) increased in Lee68, but remained unchanged in N2899. 2-D gel electrophoresis, followed by MALDI-TOF-MS analysis, of the proteins from germinated seeds suggested increases in ferritin and the 20S proteasome subunit β-6 in both the cultivars. Glyceraldehyde 3-phosphate dehydrogenase, glutathione
2.2.5. Biotic stress
Soybean mosaic virus (SBMV) causes one of the most serious viral infections of soybean; leaves of infected plants were studied at a series of time points using 2-D gel electrophoresis, followed by MALDI-TOF-MS and tandem TOF/TOF-MS. Proteins expressed in the inoculated leaves were identified and were seen to be involved in protein degradation, defense signalling, coping with changes in the levels of reactive oxygen species, cell wall reinforcement, and energy and metabolism regulation. Quantitative real time PCR was used to focus on gene expression related to some of these proteins. Photosynthesis and metabolism related genes were down-regulated at all the time points, while most of the energy related genes (respiration in this case) were up-regulated for at least five of the six time points studied . At the time of this writing, this report is the only one addressing the proteomic approach to molecular understanding of soybean-SBMV interaction.
2.2.6. Other miscellaneous stress related reports
Aluminium toxicity is often observed in acidic soils and Baxi 10 (BX10) is an Al-resistant cultivar. One-week-old soybean seedlings treated with 50 mM AlCl3 for 24, 48 and 72 h were studied for characterization of root proteins in response to Al; and 2-D gel electrophoresis followed by MS revealed 39 proteins expressed differentially following Al treatment. Of these 21 were up-regulated (such as heat shock proteins, glutathione S-transferase, chalcone related synthetase, GTP-binding protein, ABC transporters and ATP binding proteins). Five proteins were also down-regulated and 15 newly induced proteins were present following AL treatment .
Label free proteomics, coupled with multiple reaction monitoring (MRM) with synthetic isotope labelled peptides, was used to study 10 allergens from 20 non-genetically modified commercial varieties of soybean. The concentration of these allergens varied between 0.5-5.7
The responses of soybean plants exposed to 116 ppb O3 involved significant changes to carbon metabolism, photosynthesis, amino acid, flavanoid and isoprenoid biosynthesis, signaling, homeostasis, anti-oxidant and redox pathways , as indicated by shifts in expression of the relevant proteins.
More information regarding soybean functional genomics and proteomics is available at the publicly accessible Soybean Knowledgebase (SoyKB) http://soykb.org/ .
Bradyrhizobium japonicumand its proteomics/exoproteomics
Since competitiveness plays an important role in this symbiotic relationship, 2-D gel electrophoresis, image and data analysis, and in-gel digestion proteomic studies, were conducted on
A study of 2-D gel electrophoresis combined with MALDI-TOF MS for the identification of
4. Other dimensions to soybean-rhizobacteria interactions
Bacteriocins are grouped into four distinct classes based on the peptide characteristics such as post translational modifications, side chains, heat stability, N-terminal sequence homology and molecular weight .
Proteomic profiling of both these bacteria are underway in our laboratory and we hope to acquire some indications of plant proteomic shifts related to biological nitrogen fixation through these experiments over the next few months.
5. Conclusions and future perspectives
Soybean is an important protein and oil seed crop and BNF is an important source of nitrogen for the crop. Considerable work has been conducted regarding soybean proteomics, facilitated by recent advancements in technology, but a more systematic approach to this method is required in order to understand the intricacies of plant growth and development in the face of interactions with various symbionts. There is wide variation in the ability of