Yohei Nanjo

Proteome analysis of soybean leaves, hypocotyls and roots under salt stress.

BackgroundSalinity is one of the most widespread agricultural problems in arid and semi-arid regions that makes fields unproductive, and soil salinization is a serious problem in the entire world. To determine the effects of salt stress on soybean seedlings, a proteomic technique was used.ResultsSoybean plants were exposed to 0, 20, 40, or 80 mM NaCl for one week. The effect of treatment at 20 mM NaCl on plant growth was not severe, at 80 mM NaCl was lethal, and at 40 mM NaCl was significant but not lethal. Based on these results, proteins were extracted from the leaves, hypocotyls and roots of soybean treated with 40 mM NaCl. Nineteen, 22 and 14 proteins out of 340, 330 and 235 proteins in the leaves, hypocotyls and roots, respectively, were up- and down-regulated by NaCl treatment. In leaves, hypocotyls and roots, metabolism related proteins were mainly down-regulated with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was down-regulated in the leaf/hypocotyls, and fructokinase 2 was down-regulated in the hypocotyls/root with NaCl treatment. Stem 31 kDa glycoprotein precursor was up-regulated in all three organs with NaCl treatment. Glyceraldehyde-3-phosphate dehydrogenase was specifically down-regulated at the RNA and protein levels by salt stress.ConclusionThese results suggest that metabolism related proteins play a role in each organ in the adaptation to saline conditions.

Rice Plastidial N-Glycosylated Nucleotide Pyrophosphatase/Phosphodiesterase Is Transported from the ER-Golgi to the Chloroplast through the Secretory Pathway

A nucleotide pyrophosphatase/phosphodiesterase (NPP) activity that catalyzes the hydrolytic breakdown of ADP-glucose (ADPG) has been shown to occur in the plastidial compartment of both mono- and dicotyledonous plants. To learn more about this enzyme, we purified two NPPs from rice (Oryza sativa) and barley (Hordeum vulgare) seedlings. Both enzymes are glycosylated, since they bind to concanavalin A, stain with periodic acid–Schiff reagent, and are digested by Endo-H. A complete rice NPP cDNA, designated as NPP1, was isolated, characterized, and overexpressed in transgenic plants displaying high ADPG hydrolytic activity. Databank searches revealed that NPP1 belongs to a functionally divergent group of plant nucleotide hydrolases. NPP1 contains numerous N-glycosylation sites and a cleavable hydrophobic signal sequence that does not match with the N-terminal part of the mature protein. Both immunocytochemical analyses and confocal fluorescence microscopy of rice cells expressing NPP1 fused with green fluorescent protein (GFP) revealed that NPP1-GFP occurs in the plastidial compartment. Brefeldin A treatment of NPP1-GFP–expressing cells prevented NPP1-GFP accumulation in the chloroplasts. Endo-H digestibility studies revealed that both NPP1 and NPP1-GFP in the chloroplast are glycosylated. Collectively, these data demonstrate the trafficking of glycosylated proteins from the endoplasmic reticulum–Golgi system to the chloroplast in higher plants.

The Rice α-Amylase Glycoprotein Is Targeted from the Golgi Apparatus through the Secretory Pathway to the Plastids

The well-characterized secretory glycoprotein, rice (Oryza sativa) α-amylase isoform I-1 (AmyI-1), was localized within the plastids and proved to be involved in the degradation of starch granules in the organelles of rice cells. In addition, a large portion of transiently expressed AmyI-1 fused to green fluorescent protein (AmyI-1-GFP) colocalized with a simultaneously expressed fluorescent plastid marker in onion (Allium cepa) epidermal cells. The plastid targeting of AmyI-1 was inhibited by both dominant-negative and constitutively active mutants of Arabidopsis thaliana ARF1 and Arabidopsis SAR1, which arrest endoplasmic reticulum-to-Golgi traffic. In cells expressing fluorescent trans-Golgi and plastid markers, these fluorescent markers frequently colocalized when coexpressed with AmyI-1. Three-dimensional time-lapse imaging and electron microscopy of high-pressure frozen/freeze-substituted cells demonstrated that contact of the Golgi-derived membrane vesicles with cargo and subsequent absorption into plastids occur within the cells. The transient expression of a series of C-terminal-truncated AmyI-1-GFP fusion proteins in the onion cell system showed that the region from Trp-301 to Gln-369 is necessary for plastid targeting of AmyI-1. Furthermore, the results obtained by site-directed mutations of Trp-302 and Gly-354, located on the surface and on opposite sides of the AmyI-1 protein, suggest that multiple surface regions are necessary for plastid targeting. Thus, Golgi-to-plastid traffic appears to be involved in the transport of glycoproteins to plastids and plastid targeting seems to be accomplished in a sorting signal–dependent manner.

A Comprehensive Analysis of the Soybean Genes and Proteins Expressed under Flooding Stress using Transcriptome and Proteome Techniques

The inducible genes and proteins were analyzed using transcriptome and proteome techniques to explore the mechanisms underlying soybean response to flooding stress. Soybean seedlings were germinated for 2 days and subjected to flooding for 12 h, and the total RNAs and proteins were extracted from the root and hypocotyl. High-coverage gene expression profiling analysis as transcriptome technique was performed. Ninety-seven out of the 29,388 peaks observed demonstrated a greater than 25-fold change following 12 h of flood-induced stress. Furthermore, 34 proteins out of 799 proteins were changed by 12 h stress. Genes associated with alcohol fermentation, ethylene biosynthesis, pathogen defense, and cell wall loosening were significantly up-regulated. Hemoglobin, acid phosphatase, and Kunitz trypsin protease inhibitor were altered at both transcriptional and translational levels. Reactive oxygen species scavengers and chaperons were changed only at the translational level. It is suggested that the early response of soybean under flooding might be important stress adaptation to ensure survival against not only hypoxia but also the direct damage of cell by water.

Comprehensive Analysis of Mitochondria in Roots and Hypocotyls of Soybean under Flooding Stress using Proteomics and Metabolomics Techniques

Flooding is a serious problem for soybeans because it reduces growth and grain yield. Proteomic and metabolomic techniques were used to examine whether mitochondrial function is altered in soybeans by flooding stress. Mitochondrial fractions were purified from the roots and hypocotyls of 4-day-old soybean seedlings that had been flooded for 2 days. Mitochondrial matrix and membrane proteins were separated by two-dimensional polyacrylamide gel electrophoresis and blue-native polyacrylamide gel electrophoresis, respectively. Differentially expressed proteins and metabolites were identified using mass spectrometry. Proteins and metabolites related to the tricarboxylic acid cycle and γ-amino butyrate shunt were up-regulated by flooding stress, while inner membrane carrier proteins and proteins related to complexes III, IV, and V of the electron transport chains were down-regulated. The amounts of NADH and NAD were increased; however, ATP was significantly decreased by flooding stress. These results suggest that flooding directly impairs electron transport chains, although NADH production increases in the mitochondria through the tricarboxylic acid cycle.

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.

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.

Transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings

To understand the transcriptional responses to flooding stress in roots including hypocotyl of soybean seedlings, genome-wide changes in gene expression were analyzed using a soybean microarray chip containing 42,034 60-mer oligonucleotide probes. More than 6,000 of flooding-responsive genes in the roots including hypocotyl of soybean seedlings were identified. The transcriptional analysis showed that genes related to photosynthesis, glycolysis, Ser-Gly-Cys group amino acid synthesis, regulation of transcription, ubiquitin-mediated protein degradation and cell death were significantly up-regulated by flooding. Meanwhile, genes related to cell wall synthesis, secondary metabolism, metabolite transport, cell organization, chromatin structure synthesis, and degradation of aspartate family amino acid were significantly down-regulated. Comparison of the responses with other plants showed that genes encoding pyrophosphate dependent phosphofructokinase were down-regulated in flooded soybean seedlings, however, those in tolerant plants were up-regulated. Additionally, genes related to RNA processing and initiation of protein synthesis were not up-regulated in soybean, however, those in tolerant plants were up-regulated. Furthermore, we found that flooding-specific up-regulation of genes encoding small proteins which might have roles in acclimation to flooding. These results suggest that functional disorder of acclimative responses to flooding through transcriptional and post-transcriptional regulations is involved in occurring flooding injury to soybean seedlings.

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.

Label-Free Quantitative Proteomic Analysis of Abscisic Acid Effect in Early-Stage Soybean under Flooding

Flooding is a serious problem for soybean cultivation because it markedly reduces growth. To investigate the role of phytohormones in soybean under flooding stress, gel-free proteomic technique was used. When 2-day-old soybeans were flooded, the content of abscisic acid (ABA) did not decrease in the root, though its content decreased in untreated plant. When ABA was added during flooding treatment, survival ratio was improved compared with that of soybeans flooded without ABA. When 2-day-old soybeans were flooded with ABA, the abundance of proteins related to cell organization, vesicle transport and glycolysis decreased compared with those in root of soybeans flooded without ABA. Furthermore, the nuclear proteins were analyzed to identify the transcriptional regulation. The abundance of 34 nuclear proteins such as histone deacetylase and U2 small nuclear ribonucleoprotein increased by ABA supplementation under flooding; however, 35 nuclear proteins such as importin alpha, chromatin remodeling factor, zinc finger protein, transducin, and cell division 5 protein decreased. Of them, the mRNA expression levels of cell division cycle 5 protein, C2H2 zinc finger protein SERRATE, CCCH type zinc finger family protein, and transducin were significantly down-regulated under the ABA treatment. These results suggest that ABA might be involved in the enhancement of flooding tolerance of soybean through the control of energy conservation via glycolytic system and the regulation on zinc finger proteins, cell division cycle 5 protein and transducin.