Xiaojian Yin

Phosphoproteomics reveals the effect of ethylene in soybean root under flooding stress.

Flooding has severe negative effects on soybean growth. To explore the flooding-responsive mechanisms in early-stage soybean, a phosphoproteomic approach was used. Two-day-old soybean plants were treated without or with flooding for 3, 6, 12, and 24 h, and root tip proteins were then extracted and analyzed at each time point. After 3 h of flooding exposure, the fresh weight of soybeans increased, whereas the ATP content of soybean root tips decreased. Using a gel-free proteomic technique, a total of 114 phosphoproteins were identified in the root tip samples, and 34 of the phosphoproteins were significantly changed with respect to phosphorylation status after 3 h of flooding stress. Among these phosphoproteins, eukaryotic translation initiation factors were dephosphorylated, whereas several protein synthesis-related proteins were phosphorylated. The mRNA expression levels of sucrose phosphate synthase 1F and eukaryotic translation initiation factor 4 G were down-regulated, whereas UDP-glucose 6-dehydrogenase mRNA expression was up-regulated during growth but down-regulated under flooding stress. Furthermore, bioinformatic protein interaction analysis of flooding-responsive proteins based on temporal phosphorylation patterns indicated that eukaryotic translation initiation factor 4 G was located in the center of the network during flooding. Soybean eukaryotic translation initiation factor 4 G has homology to programmed cell death 4 protein and is implicated in ethylene signaling. The weight of soybeans was increased with treatment by an ethylene-releasing agent under flooding condition, but it was decreased when plants were exposed to an ethylene receptor antagonist. These results suggest that the ethylene signaling pathway plays an important role, via the protein phosphorylation, in mechanisms of plant tolerance to the initial stages of flooding stress in soybean root tips.

Quantitative proteomics of nuclear phosphoproteins in the root tip of soybean during the initial stages of flooding stress.

UNLABELLED Soybean is sensitive to flooding stress, which has affected many proteins in roots. To identify the upstream events controlling the regulation of flooding-responsive proteins, nuclear phosphoproteomics of soybean-root tip was performed. Nuclei were isolated from the root tip of 2-day-old soybeans treated with flooding for 3h. The purity of nuclear fractions was confirmed by Western blotting and enzyme-activity assays for subcellular-specific enzymes. Phosphopeptides in the fractions were enriched and analyzed using gel-free proteomic technique. Fourteen phosphoproteins significantly changed in root tip in response to flooding stress. Of these phosphoproteins, 10 proteins including 5 protein synthesis-related proteins were predicted to be localized in the nucleus. In particular, zinc finger/BTB domain-containing protein 47, glycine-rich protein, and rRNA processing protein Rrp5, which are related to abscisic acid (ABA) response, were clearly phosphorylated in response to flooding stress. The mRNA expression levels of these nuclear phosphoproteins were down-regulated in root tip exposed to flooding stress with ABA. In addition, the fresh weight of soybean decreased under flooding stress with ABA, although the fresh weight of plant increased during the initial stage of flooding stress. These results suggest that ABA may affect the flooding response of early-stage soybean through the regulation of nuclear-localized phosphoproteins. BIOLOGICAL SIGNIFICANCE This study reported nuclear phosphoprotein analysis of root tip under initial flooding stress using gel-free quantitative proteomics. The main findings of this study are as follows: (i) Fourteen nuclear phosphoproteins in soybean root tip cells were significantly changed in the initial stages of flooding stress; (ii) Zinc finger protein, glycine-rich protein, and Rrp5 were phosphorylated in the nuclei of root tip in response to flooding; and (iii) The mRNA expression levels of these genes were down-regulated by ABA under flooding conditions. These results suggest that ABA may be involved in the initial responses of early-stage soybean to flooding stress by altering the phosphorylation of nuclear-localized phosphoproteins. This study provides not only the nuclear phosphoproteomic analysis but also the molecular mechanism underlying the initial flooding responsive nuclear phosphoproteins functions in the root tip of soybean.

Analysis of initial changes in the proteins of soybean root tip under flooding stress using gel-free and gel-based proteomic techniques.

UNLABELLED Flooding has a severe negative effect on soybean cultivation in the early stages of growth. To obtain a better understanding of the response mechanisms of soybean to flooding stress, initial changes in root tip proteins under flooding were analyzed using two proteomic techniques. Two-day-old soybeans were treated with flooding for 3, 6, 12, and 24h. The weight of soybeans increased during the first 3h of flooding, but root elongation was not observed. Using gel-based and gel-free proteomic techniques, 115 proteins were identified in root tips, of which 9 proteins were commonly detected by both methods. The 71 proteins identified by the gel-free proteomics were analyzed by a hierarchical clustering method based on induction levels during the flooding, and the proteins were divided into 5 clusters. Additional interaction analysis of the proteins revealed that ten proteins belonging to cluster I formed the center of a protein interaction network. mRNA expression analysis of these ten proteins showed that citrate lyase and heat shock protein 70 were down-regulated, whereas calreticulin was up-regulated in initial phase of flooding. These results suggest that flooding stress to soybean induces calcium-related signal transduction, which might play important roles in the early responses to flooding. BIOLOGICAL SIGNIFICANCE Flooding has a severe negative effect on soybean cultivation, particularly in the early stages of growth. To better understand the response mechanisms of soybean to the early stages of flooding stress, two proteomic techniques were used. Two-day-old soybeans were treated without or with flooding for 3, 6, 12, and 24h. The fresh weight of soybeans increased during the first 3h of flooding stress, but the growth then slowed and no root elongation was observed. Using gel-based and gel-free proteomic techniques, 115 proteins were identified in root tips, of which 9 proteins were commonly detected by both methods. The 71 proteins identified by the gel-free proteomics were analyzed by a hierarchical clustering method based on induction levels during the flooding stress, and 5 protein clusters were recognized. Protein interaction analysis revealed that ten proteins belonging to cluster I formed the center of a protein interaction network. mRNA expression analysis of these ten proteins showed that citrate lyase and heat shock protein 70 were down-regulated in response to flooding stress, whereas calreticulin was up-regulated. These results suggest that flooding stress to soybean induces calcium-related signal transduction, which might play important roles in the early responses to flooding.

Quantitative Proteomics Reveals the Flooding-Tolerance Mechanism in Mutant and Abscisic Acid-Treated Soybean.

Flooding negatively affects the growth of soybean, and several flooding-specific stress responses have been identified; however, the mechanisms underlying flooding tolerance in soybean remain unclear. To explore the initial flooding tolerance mechanisms in soybean, flooding-tolerant mutant and abscisic acid (ABA)-treated plants were analyzed. In the mutant and ABA-treated soybeans, 146 proteins were commonly changed at the initial flooding stress. Among the identified proteins, protein synthesis-related proteins, including nascent polypeptide-associated complex and chaperonin 20, and RNA regulation-related proteins were increased in abundance both at protein and mRNA expression. However, these proteins identified at the initial flooding stress were not significantly changed during survival stages under continuous flooding. Cluster analysis indicated that glycolysis- and cell wall-related proteins, such as enolase and polygalacturonase inhibiting protein, were increased in abundance during survival stages. Furthermore, lignification of root tissue was improved even under flooding stress. Taken together, these results suggest that protein synthesis- and RNA regulation-related proteins play a key role in triggering tolerance to the initial flooding stress in soybean. Furthermore, the integrity of cell wall and balance of glycolysis might be important factors for promoting tolerance of soybean root to flooding stress during survival stages.

Nuclear Proteomics Reveals the Role of Protein Synthesis and Chromatin Structure in Root Tip of Soybean during the Initial Stage of Flooding Stress

To identify the upstream events controlling the regulation of flooding-responsive proteins in soybean, proteomic analysis of nuclear proteins in root tip was performed. By using nuclear fractions, which were highly enriched, a total of 365 nuclear proteins were changed in soybean root tip at initial stage of flooding stress. Four exon-junction complex-related proteins and NOP1/NOP56, which function in upstream of 60S preribosome biogenesis, were decreased in flooded soybean. Furthermore, proteomic analysis of crude protein extract revealed that the protein translation was suppressed by continuous flooding stress. Seventeen chromatin structure-related nuclear proteins were decreased in response to flooding stress. Out of them, histone H3 was clearly decreased with protein abundance and mRNA expression levels at the initial flooding stress. Additionally, a number of protein synthesis-, RNA-, and DNA-related nuclear proteins were decreased in a time-dependent manner. mRNA expressions of genes encoding the significantly changed flooding-responsive nuclear proteins were inhibited by the transcriptional inhibitor, actinomycin D. These results suggest that protein translation is suppressed through inhibition of preribosome biogenesis- and mRNA processing-related proteins in nuclei of soybean root tip at initial flooding stress. In addition, flooding stress may regulate histone variants with gene expression in root tip.

Transcriptomic analysis reveals the flooding tolerant mechanism in flooding tolerant line and abscisic acid treated soybean

Soybean is highly sensitive to flooding stress and exhibits markedly reduced plant growth and grain yield under flooding conditions. To explore the mechanisms underlying initial flooding tolerance in soybean, RNA sequencing-based transcriptomic analysis was performed using a flooding-tolerant line and ABA-treated soybean. A total of 31 genes included 12 genes that exhibited similar temporal patterns were commonly changed in these plant groups in response to flooding and they were mainly involved in RNA regulation and protein metabolism. The mRNA expression of matrix metalloproteinase, glucose-6-phosphate isomerase, ATPase family AAA domain-containing protein 1, and cytochrome P450 77A1 was up-regulated in wild-type soybean under flooding conditions; however, no changes were detected in the flooding-tolerant line or ABA-treated soybean. The mRNA expression of cytochrome P450 77A1 was specifically up-regulated in root tips by flooding stress, but returned to the level found in control plants following treatment with the P450 inhibitor uniconazole. The survival ratio and root fresh weight of plants were markedly improved by 3-h uniconazole treatment under flooding stress. Taken together, these results suggest that cytochrome P450 77A1 is suppressed by uniconazole treatment and that this inhibition may enhance soybean tolerance to flooding stress.

Proteomic Analysis of Phosphoproteins in the Rice Nucleus During the Early Stage of Seed Germination

The early stage of seed germination is the first step in the plant life cycle without visible morphological change. To investigate the mechanism controlling the early stage of rice seed germination, we performed gel-and label-free nuclear phosphoproteomics. A total of 3467 phosphopeptides belonging to 102 nuclear phosphoproteins from rice embryos were identified. Protein-synthesis-related proteins were mainly phosphorylated. During the first 24 h following imbibition, 115 nuclear phosphoproteins were identified, and significant changes in the phosphorylation level over time were observed in 29 phosphoproteins. Cluster analysis indicated that nucleotide-binding proteins and zinc finger CCCH- and BED-type proteins increased in abundance during the first 12 h of imbibition and then decreased. The in silico protein-protein interactions for 29 nuclear phosphoproteins indicated that the Sas10/Utp3 protein, which functions in snoRNA binding and gene silencing, was the center of the phosphoprotein network in nuclei. The germination rate of seeds was significantly slowed with phosphatase inhibitor treatment. The mRNA expression of the zinc finger CCCH-type protein did not change, and the zinc finger BED-type protein was upregulated in rice embryos during the early stage of germination with phosphatase inhibitor treatment. These results suggest that the phosphorylation and dephosphorylation of nuclear proteins are involved in rice seed germination. Furthermore, transcription factors such as zinc finger CCCH- and BED-type proteins might play a key role through nuclear phosphoproteins, and Sas10/Utp3 protein might interact with nuclear phosphoproteins in rice embryos to mediate the early stage of seed germination.

Plant nuclear proteomics for unraveling physiological function

The nucleus is the subcellular organelle that functions as the regulatory hub of the cell and is responsible for regulating several critical cellular functions, including cell proliferation, gene expression, and cell survival. Nuclear proteomics is a useful approach for investigating the mechanisms underlying plant responses to abiotic stresses, including protein-protein interactions, enzyme activities, and post-translational modifications. Among abiotic stresses, flooding is a major limiting factor for plant growth and yields, particularly for soybean. In this review, plant nuclei purification methods, modifications of plant nuclear proteins, and recent contributions to the field of plant nuclear proteomics are summarized. In addition, to reveal the upstream regulating mechanisms controlling soybean responses to flooding stress, the functions of flooding-responsive nuclear proteins are reviewed based on the results of nuclear proteomic analysis of soybean in the early stages of flooding stress.

Comprehensive analysis of response and tolerant mechanisms in early-stage soybean at initial-flooding stress

Soybean is one of the most cultivated crops in the world; however, it is very sensitive to flooding stress, which markedly reduces its growth and yield. Morphological and biochemical changes such as an increase of fresh weight and a decrease of ATP content happen in early-stage soybean at initial-flooding stress, indicating that soybean responses to flooding stress are keys for its survival and seedling growth. Phosphoproteomics and nuclear proteomics are useful tools to detect protein-phosphorylation status and to identify transcriptional factors. In the review, the effect of flooding on soybean response to initial flooding stress is discussed based on recent results of proteomic, phosphoproteomic, nuclear proteomic, and nuclear phosphoproteomic studies. In addition, soybean survival under flooding stress, which is defined as tolerance mechanism, is discussed with the results of comprehensive analysis in flooding-tolerant mutant line and abscisic acid-treated soybean. BIOLOGICAL SIGNIFICANCE Soybean is one of the most cultivated crops in the world; however, it is very sensitive to flooding stress, especially soybean responses to initial flooding stress is key for its survival and seedling growth. Recently, proteomic techniques are applied to investigate the response and tolerant mechanisms of soybean at initial flooding condition. In this review, the progress in proteomic, phosphoproteomic, nuclear proteomic, and nuclear phosphoproteomic studies about the initial-flooding response mechanism in early-stage soybean is presented. In addition, the tolerant mechanism in soybean is discussed with the results of comprehensive analysis in flooding-tolerant mutant line and abscisic acid-treated soybean. Through this review, the key proteins and genes involved in initial flooding response and tolerance at early stage soybean are summarized and they contribute greatly to uncover response and tolerance mechanism at early stage under stressful environmental conditions in soybean.

Phosphoproteomics: Protein Phosphorylation in Regulation of Seed Germination and Plant Growth

Seed germination and plant growth are key stages in the plant life cycle and they are affected by abiotic stresses. Protein phosphorylation is commonly involved in key regulatory process, which mediates plant growth/development and stress responses. In plant, phosphoproteins play roles in stress responses and provide basic backbone in complex signaling networks. The mechanism of plant responses to abiotic stresses has been uncovered through study of protein phosphorylation. Recently, new methods for enrichment of phosphopeptides and phosphoproteins have been developed. Phosphoproteomic techniques are widely applied to studies of seed germination and plant growth. The signaling networks of phytohormones have been explored using the phosphoproteomic approach. In this review, the enrichment methods of phosphopeptides and phosphoproteins are summarized for phosphoproteomics. The roles of protein kinases and phosphatases in plant are described. The significant achievements of phosphoproteomics in the areas of seed germination and plant growth under abiotic stresses are reviewed. Applications of phosphoproteomics are valuable resources to uncover functional proteins regulating plant growth and stress responses.