Pingfang Yang

In-Depth Proteome Analysis of Arabidopsis Leaf Peroxisomes Combined with in Vivo Subcellular Targeting Verification Indicates Novel Metabolic and Regulatory Functions of Peroxisomes

Peroxisomes are metabolically diverse organelles with essential roles in plant development. The major protein constituents of plant peroxisomes are well characterized, whereas only a few low-abundance and regulatory proteins have been reported to date. We performed an in-depth proteome analysis of Arabidopsis (Arabidopsis thaliana) leaf peroxisomes using one-dimensional gel electrophoresis followed by liquid chromatography and tandem mass spectrometry. We detected 65 established plant peroxisomal proteins, 30 proteins whose association with Arabidopsis peroxisomes had been previously demonstrated only by proteomic data, and 55 putative novel proteins of peroxisomes. We subsequently tested the subcellular targeting of yellow fluorescent protein fusions for selected proteins and confirmed the peroxisomal localization for 12 proteins containing predicted peroxisome targeting signals type 1 or 2 (PTS1/2), three proteins carrying PTS-related peptides, and four proteins that lack conventional targeting signals. We thereby established the tripeptides SLM> and SKV> (where > indicates the stop codon) as new PTS1s and the nonapeptide RVx5HF as a putative new PTS2. The 19 peroxisomal proteins conclusively identified from this study potentially carry out novel metabolic and regulatory functions of peroxisomes. Thus, this study represents an important step toward defining the complete plant peroxisomal proteome.

Constructing the metabolic and regulatory pathways in germinating rice seeds through proteomic approach

Construction of metabolic and regulatory pathways from proteomic data can contextualize the large‐scale data within the overall physiological scheme of an organism. It is an efficient way to predict metabolic phenotype or regulatory style. We did protein profiling in the germinating rice seeds through 1‐DE via LC MS/MS proteomic shotgun strategy. In total, 673 proteins were identified, and could be sorted into 14 functional groups. The largest group was metabolism related. The metabolic proteins were integrated into different metabolic pathways to show the style of reserves mobilization and precursor preparation during the germination. Analysis of the regulatory proteins indicated that regulation of redox homeostasis and gene expression also play important roles for the rice seed germination. Although transcription is unnecessary for the germination, it could ensure the rapidity and uniformity of germination. On the contrary, translation with the stored mRNA is required for the germination. This study will help us to further understand the metabolic style, regulation of redox homeostasis, and gene expression during rice seed germination.

Exploring the Mechanism of Physcomitrella patens Desiccation Tolerance through a Proteomic Strategy

The moss Physcomitrella patens has been shown to tolerate abiotic stresses, including salinity, cold, and desiccation. To better understand this plants mechanism of desiccation tolerance, we have applied cellular and proteomic analyses. Gametophores were desiccated over 1 month to 10% of their original fresh weight. We report that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling, and microtubule depolymerization. Despite the severe desiccation, the membrane system maintains integrity. Through two-dimensional gel electrophoresis and image analysis, we identified 71 proteins as desiccation responsive. Following identification and functional categorization, we found that a majority of the desiccation-responsive proteins were involved in metabolism, cytoskeleton, defense, and signaling. Degradation of cytoskeletal proteins might result in cytoskeletal disassembly and consequent changes in the cell structure. Late embryogenesis abundant proteins and reactive oxygen species-scavenging enzymes are both prominently induced, and they might help to diminish the damage brought by desiccation.Bryophytes as the first land plants are believed to have colonized the land from a fresh water origin, requiring adaptive mechanisms that survival of dehydration. Physcomitrella patens is such a non-vascular bryophyte and shows rare desiccation tolerance in its vegetative tissues. Previous studies showed that during the course of dehydration, several related processes are set in motion: plasmolysis, chloroplast remodeling and microtubule depolymerization. And proteomic alteration supported the cellular structural changes in respond to desiccation stress. In this addendum, we report that Golgi bodies are absent and adaptor protein complex AP-1 large subunit is downregulated during the course of dehydration. Those phenomena may be adverse in protein posttranslational modification, protein sorting and cell walls synthesis under the desiccation condition.

Manipulation of arginase expression modulates abiotic stress tolerance in Arabidopsis: effect on arginine metabolism and ROS accumulation

Arginine is an important medium for the transport and storage of nitrogen, and arginase (also known as arginine amidohydrolase, ARGAH) is responsible for catalyse of arginine into ornithine and urea in plants. In this study, the impact of AtARGAHs on abiotic stress response was investigated by manipulating AtARGAHs expression. In the knockout mutants of AtARGAHs, enhanced tolerances were observed to multiple abiotic stresses including water deficit, salt, and freezing stresses, while AtARGAH1- and AtARGAH2-overexpressing lines exhibited reduced abiotic stress tolerances compared to the wild type. Consistently, the enhanced tolerances were confirmed by the changes of physiological parameters including electrolyte leakage, water loss rate, stomatal aperture, and survival rate. Interestingly, the direct downstream products of arginine catabolism including polyamines and nitric oxide (NO) concentrations significantly increased in the AtARGAHs-knockout lines, but decreased in overexpressing lines under control conditions. Additionally, the AtARGAHs-overexpressing and -knockout lines displayed significantly reduced relative arginine (% of total free amino acids) relative to the wild type. Similarly, reactive oxygen species accumulation was remarkably regulated by AtARGAHs under abiotic stress conditions, as shown from hydrogen peroxide (H2O2), superoxide radical () concentrations, and antioxidant enzyme activities. Taken together, this is the first report, as far as is known, to provide evidence that AtARGAHs negatively regulate many abiotic stress tolerances, at least partially, attribute to their roles in modulating arginine metabolism and reactive oxygen species accumulation. Biotechnological strategy based on manipulation of AtARGAHs expression will be valuable for future crop breeding.

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.

Proteomic analysis of rice seedlings infected by Sinorhizobium meliloti 1021

Rhizobial endophytes infect and colonize not only leguminous plants, but several non‐leguminous species as well. Using green fluorescent protein tagging technique, it has been shown that Rhizobia infect different varieties of rice species and migrate from plant roots to aerial tissues such as leaf sheaths and leaves. The interaction between them was found to promote the growth of rice. The growth promotion is the cumulative result of enhanced photosynthesis and stress resistance. In addition, indole‐3‐acetic acid also contributes to the promotion. Gel‐based comparative proteomic approaches were applied to analyze the protein profiles of three different tissues (root, leaf sheath and leaf) of Sinorhizobium meliloti 1021 inoculated rice in order to get an understanding about the molecular mechanism. Upon the inoculation of rhizobia, proteins involved in nine different functional categories were either up‐regulated or down‐regulated. Photosynthesis related proteins were up‐regulated only in leaf sheath and leaf, while the up‐regulated proteins in root were exclusively defense related. The results implied that there might have been an increase in the import and transport of proteins involved in light and dark reactions to the chloroplast as well as more efficient distribution of nutrients, hence enhanced photosynthesis. Although the initiation of defensive reactions mainly occurred in roots, some different defense mechanisms were also evoked in the aerial tissues.

Responses of Jatropha curcas seedlings to cold stress: photosynthesis related proteins and chlorophyll fluorescence characteristics

Photosynthesis-related proteins and PSII functions of Jatropha curcas seedlings under cold stress were studied using proteomic and chlorophyll fluorescence approaches. The results of chlorophyll fluorescence measurement indicated that electron transport flux per reaction center (ET(o)/RC) and performance index (PI(ABS)) were relatively sensitive to low temperature, especially at early stage of cold stress. The increase in O-J phase and decrease in J-I phase of chlorophyll fluorescence transient indicated a protection mechanism of J. curcas to photoinhibition at early stage of cold stress. Eight photosynthesis-related proteins significantly changed during cold stress were identified using liquid chromatography MS/MS. Results of correlation analyses between photosynthesis-related proteins and chlorophyll fluorescence parameters indicated that (1) ATP synthase and Rieske FeS protein were significantly correlated with electron transport of reaction center in PSII; (2) precursor for 33-kDa protein was positively correlated with fluorescence quenching of the O-J and J-I phases and PI(ABS) during cold stress, which implies that it might be related to multiple process in PSII; (3) contrary correlations were found between F(J) - F(o) and two enzymes in the Calvin cycle, and the relations between these proteins and PSII function were unclear. The combined study using proteomic approaches and chlorophyll fluorescence measurements indicated that the early-stage (0-12 h) acclimation of PSII and the late-stage (after 24 h) H(2)O(2) scavenging might be involved in the cold response mechanisms of J. curcas seedlings.

The Cysteine2/Histidine2-Type Transcription Factor ZINC FINGER OF ARABIDOPSIS THALIANA6 Modulates Biotic and Abiotic Stress Responses by Activating Salicylic Acid-Related Genes and C-REPEAT-BINDING FACTOR Genes in Arabidopsis.

An Arabidopsis C2H2-type zinc finger protein increases both biotic and abiotic stress resistances through gene activation, including salicylic acid-related genes. The cysteine2/histidine2-type zinc finger proteins are a large family of transcription regulators, and some of them play essential roles in plant responses to biotic and abiotic stress. In this study, we found that expression of C2H2-type ZINC FINGER OF ARABIDOPSIS THALIANA6 (AtZAT6) was transcriptionally induced by salt, dehydration, cold stress treatments, and pathogen infection, and AtZAT6 was predominantly located in the nucleus. AtZAT6-overexpressing plants exhibited improved resistance to pathogen infection, salt, drought, and freezing stresses, while AtZAT6 knockdown plants showed decreased stress resistance. AtZAT6 positively modulates expression levels of stress-related genes by directly binding to the TACAAT motifs in the promoter region of pathogen-related genes (ENHANCED DISEASE SUSCEPTIBILITY1, PHYTOALEXIN DEFICIENT4, PATHOGENESIS-RELATED GENE1 [PR1], PR2, and PR5) and abiotic stress-responsive genes (C-REPEAT-BINDING FACTOR1 [CBF1], CBF2, and CBF3). Moreover, overexpression of AtZAT6 exhibited pleiotrophic phenotypes with curly leaves and small-sized plant at vegetative stage and reduced size of floral organs and siliques at the reproductive stage. Modulation of AtZAT6 also positively regulates the accumulation of salicylic acid and reactive oxygen species (hydrogen peroxide and superoxide radical). Taken together, our findings indicated that AtZAT6 plays important roles in plant development and positively modulates biotic and abiotic stress resistance by activating the expression levels of salicylic acid-related genes and CBF genes.

Proteomic analysis of the cold stress response in the moss, Physcomitrella patens.

Cold stress has adverse effects on plant growth and development. Plants respond and acclimate to cold stress through various biochemical and physiological processes, thereby acquiring stress tolerance. To better understand the basis for tolerance, we carried out a proteomic study in the model moss, Physcomitrella patens, characterizing gametophore proteins with 2‐DE and mass spectroscopy. Following exposure to 0°C for up to 3 days, out of the more than 1000 protein spots reproducibly resolved, only 45 changed in abundance by at least 1.5‐fold. Of these, 35 were identified by tryptic digestion and mass spectroscopy. Photosynthetic proteins decreased, whereas many catabolic proteins increased. In addition, cold stress up‐regulated a variety of signaling, cytoskeleton, and defense proteins and few proteins in these classes were down‐regulated. Up‐regulated proteins include the 14‐3‐3‐like protein, actin, HSP70s, lipoxygenases, and cytochrome P450 proteins. These results point to pathways that are important for the mechanism of cold stress response in P. patens and by extension to the entire plant kingdom.

Proteomics of rice seed germination

Seed is a condensed form of plant. Under suitable environmental conditions, it can resume the metabolic activity from physiological quiescent status, and mobilize the reserves, biosynthesize new proteins, regenerate organelles, and cell membrane, eventually protrude the radicle and enter into seedling establishment. So far, how these activities are regulated in a coordinated and sequential manner is largely unknown. With the availability of more and more genome sequence information and the development of mass spectrometry (MS) technology, proteomics has been widely applied in analyzing the mechanisms of different biological processes, and proved to be very powerful. Regulation of rice seed germination is critical for rice cultivation. In recent years, a lot of proteomic studies have been conducted in exploring the gene expression regulation, reserves mobilization and metabolisms reactivation, which brings us new insights on the mechanisms of metabolism regulation during this process. Nevertheless, it also invokes a lot of questions. In this mini-review, we summarized the progress in the proteomic studies of rice seed germination. The current challenges and future perspectives were also discussed, which might be helpful for the following studies.