Cu-II-directed self-assembly of fullerenols to ameliorate copper stress in maize seedlings.

Widespread use of copper-based agrochemical may cause copper excessive accumulation in agricultural soil to seriously threaten crop production. Recently, fullerenols are playing important roles in helping crops build resistance to abiotic stresses by giving ingenious and successful resolutions. However, there is a lack of knowledge on their beneficial effects in crops under stresses induced by heavy metals. Herein, the visual observation of Cu2+-mediated assembly of fullerenols via electrostatic and coordination actions was carried out in vitro, showing that water-soluble nanocomplexes and water-insoluble cross-linking nanohybrids were selectively fabricated by precisely adjusting feeding ratios of fullerenols and CuSO4. Furthermore, maize simultaneous exposure of fullerenols and CuSO4 solutions was tested to investigate the comparative effects of seed germination and seedling growth relative to exposure of CuSO4 alone. Under moderate Cu2+ stresses (40 and 80 µM), fullerenols significantly mitigated the detrimental effects of seedlings, including phenotype, root and shoot elongation, biomass accumulation, antioxidant capacity, and Cu2+ uptake and copper transporter-related gene expressions in roots. Under 160 µM of Cu2+ as a stressor, fullerenols also accelerated germination of Cu2+-stressed seeds eventually up to the level of the control. Summarily, fullerenols can enhance tolerance of Cu2+-stressed maize mainly due to direct detoxification through fullerenol-Cu2+ interactions restraining the Cu2+ intake into roots and reducing free Cu2+ content in vivo, as well as fullerenol-maize interactions to enhance resistance by maintaining balance of reactive oxygen species and optimizing the excretion and transport of Cu2+. This will unveil valuable insights into the beneficial roles of fullerenols and its mechanism mode in alleviating heavy metal stress on crop plants.

Directed assembly of fullerenols via electrostatic and coordination interactions to fabricate diverse and water-soluble metal cation-fullerene nanocluster complexes.

Metal ion-nanocluster coordination complexes can produce a variety of functional engineered nanomaterials with promising characteristics to enable widespread applications. Herein, the visualization observation of the interactions of metal ions and fullerene derivatives, particularly anionic fullerenols (Fol), were carried out in aqueous solutions. The alkali metal salts only resulted in salting out of Fol to gain re-soluble sediments, whereas multivalent metal cations (Mn+, n = 2, 3) modulated further assembly of Fol to produce insoluble hybrids. These provide crucial insights into the directed assembly of Fol that two major forces involved in actuation are electrostatic and coordination effects. Through the precise modulation of feed ratios of Fol to Mn+, a variety of water-soluble Mn+@Fol coordination complexes were facilely prepared and subsequently characterized by various measurements. Among them, X-ray photoelectron spectra validated the coordination effects through the metal cation and oxygen binding feature. Transmission electron microscopy delivered valuable information about diverse morphologies and locally-ordered microstructures at the nanoscale. This study opens a new opportunity for developing a preparation strategy to fabricate water-soluble metal cation-fullerenol coordination complexes with various merits for potential application in biomedical fields.

Calcineurin B-like protein ZmCBL8-1 promotes salt stress resistance in Arabidopsis.

MAIN CONCLUSION: ZmCBL8-1 enhances salt stress tolerance in maize by improving the antioxidant system to neutralize ROS homeostasis and inducing Na+/H+ antiporter gene expressions of leaves. Calcineurin B-like proteins (CBLs) as plant-specific calcium sensors have been explored for their roles in the regulation of abiotic stress tolerance. Further, the functional variations in ZmCBL8, encoding a component of the salt overly sensitive pathway, conferred the salt stress tolerance in maize. ZmCBL8-1 is a transcript of ZmCBL8 found in maize, but its function in the salt stress response is still unclear. The present study aimed to characterize the protein ZmCBL8-1 that was determined to be composed of 194 amino acids (aa) with three conserved EF hands responsible for binding Ca2+. However, a 20-aa fragment was found to be missing from its C-terminus relative to another transcript of ZmCBL8. Results indicated that it harbored a dual-lipid modification motif MGCXXS at its N-terminus and was located on the cell membrane. The accumulation of ZmCBL8-1 transcripts was high in the roots but relatively lower in the leaves of maize under normal condition. In contrast, its expression was significantly decreased in the roots, while increased in the leaves under NaCl treatment. The overexpression of ZmCBL8-1 resulted in higher salt stress resistance of transgenic Arabidopsis in a Ca2+-dependent manner relative to that of the wild type (WT). In ZmCBL8-1-overexpressing plants exposed to NaCl, the contents of malondialdehyde and hydrogen peroxide were decreased in comparison with those in the WT, and the expression of key genes involved in the antioxidant defense system and Na+/H+ antiporter were upregulated. These results suggested that ZmCBL8-1 played a positive role in the response of leaves to salt stress by inducing the expression of Na+/H+ antiporter genes and enhancing the antioxidant system to neutralize the accumulation of reactive oxygen species. These observations further indicate that ZmCBL8-1 confers salt stress tolerance, suggesting that transcriptional regulation of the ZmCBL8 gene is important for salt tolerance.

Cadmium tolerance and hyperaccumulation in plants - A proteomic perspective of phytoremediation.

Cadmium (Cd) is a major environmental pollutant and poses a risk of transfer into the food chain through contaminated plants. Mechanisms underlying Cd tolerance and hyperaccumulation in plants are not fully understood. Proteomics-based approaches facilitate an in-depth understanding of plant responses to Cd stress at the systemic level by identifying Cd-inducible differentially abundant proteins (DAPs). In this review, we summarize studies related to proteomic changes associated with Cd-tolerance mechanisms in Cd-tolerant crops and Cd-hyperaccumulating plants, especially the similarities and differences across plant species. The enhanced DAPs identified through proteomic studies can be potential targets for developing Cd-hyperaccumulators to remediate Cd-contaminated environments and Cd-tolerant crops with low Cd content in the edible organs. This is of great significance for ensuring the food security of an exponentially growing global population. Finally, we discuss the methodological drawbacks in current proteomic studies and propose that better protocols and advanced techniques should be utilized to further strengthen the reliability and applicability of future Cd-stress-related studies in plants. This review provides insights into the improvement of phytoremediation efficiency and an in-depth study of the molecular mechanisms of Cd enrichment in plants.

Modifying the Expression of Cysteine Protease Gene PCP Affects Pollen Development, Germination and Plant Drought Tolerance in Maize.

Cysteine proteases (CPs) are vital proteolytic enzymes that play critical roles in various plant processes. However, the particular functions of CPs in maize remain largely unknown. We recently identified a pollen-specific CP (named PCP), which highly accumulated on the surface of maize pollen. Here, we reported that PCP played an important role in pollen germination and drought response in maize. Overexpression of PCP inhibited pollen germination, while mutation of PCP promoted pollen germination to some extent. Furthermore, we observed that germinal apertures of pollen grains in the PCP-overexpression transgenic lines were excessively covered, whereas this phenomenon was not observed in the wild type (WT), suggesting that PCP regulated pollen germination by affecting the germinal aperture structure. In addition, overexpression of PCP enhanced drought tolerance in maize plants, along with the increased activities of the antioxidant enzymes and the decreased numbers of the root cortical cells. Conversely, mutation of PCP significantly impaired drought tolerance. These results may aid in clarifying the precise functions of CPs in maize and contribute to the development of drought-tolerant maize materials.

ZmCIPK32 positively regulates germination of stressed seeds via gibberellin signal.

Calcineurin B-like proteins (CBLs) as specific calcium sensors that interact with CBL-interacting protein kinases (CIPKs) play a key role in the regulation of plant development and abiotic stress tolerance. In this study, we isolated and characterized the CIPK32 gene from Zea mays. ZmCIPK32 showed that it comprised 440 amino acids and a conserved NAF motif responsible for the interaction with CBLs localized in the cytoplasm and cell membrane. The interaction of ZmCIPK32 with ZmCBL1 and ZmCBL9 demonstrated using yeast two-hybrid system and bimolecular fluorescence complementation assay required the presence of the NAF domain. Overexpression of ZmCIPK32 promoted early germination in transgenic Arabidopsis seeds relative to that observed in wild-type (WT) plants under mannitol treatment. In addition, ZmCIPK32-overexpressing plants were insensitive to treatments with exogenous abscisic acid and paclobutrazol (PBZ) at seed germination and early seedling stages. Expression levels of the key genes GA20ox and GA3ox involved in the synthesis of gibberellin (GA) were increased, whereas expression levels of genes involved in the conversion of active GA to inactive forms and GA signaling were reduced in ZmCIPK32-overexpressing plants relative to those in WT plants under mannitol and PBZ treatments. Furthermore, overexpression of ZmCIPK32 increased GA level but decreased abscisic acid level in transgenic lines compared to the respective levels in WT plants under PBZ or mannitol treatments. Our results suggest that ZmCIPK32 positively regulates seed germination under stressed conditions by modulating GA signals.

Quaternary ammonium iminofullerenes improve root growth of oxidative-stress maize through ASA-GSH cycle modulating redox homeostasis of roots and ROS-mediated root-hair elongation.

BACKGROUND: Various environmental factors are capable of oxidative stress to result in limiting plant development and agricultural production. Fullerene-based carbon nanomaterials can enable radical scavenging and positively regulate plant growth. Even so, to date, our knowledge about the mechanism of fullerene-based carbon nanomaterials on plant growth and response to oxidative stress is still unclear. RESULTS: 20 or 50 mg/L quaternary ammonium iminofullerenes (IFQA) rescued the reduction in root lengths and root-hair densities and lengths of Arabidopsis and maize induced by accumulation of endogenous hydrogen peroxide (H2O2) under 3-amino-1,2,4-triazole or exogenous H2O2 treatment, as well as the root active absorption area and root activity under exogenous H2O2 treatment. Meanwhile, the downregulated contents of ascorbate acid (ASA) and glutathione (GSH) and the upregulated contents of dehydroascorbic acid (DHA), oxidized glutathione (GSSG), malondialdehyde (MDA), and H2O2 indicated that the exogenous H2O2 treatment induced oxidative stress of maize. Nonetheless, application of IFQA can increase the ratios of ASA/DHA and GSH/GSSG, as well as the activities of glutathione reductase, and ascorbate peroxidase, and decrease the contents of H2O2 and MDA. Moreover, the root lengths were inhibited by buthionine sulfoximine, a specific inhibitor of GSH biosynthesis, and subsequently rescued after addition of IFQA. The results suggested that IFQA could alleviate exogenous-H2O2-induced oxidative stress on maize by regulating the ASA-GSH cycle. Furthermore, IFQA reduced the excess accumulation of ROS in root hairs, as well as the NADPH oxidase activity under H2O2 treatment. The transcript levels of genes affecting ROS-mediated root-hair development, such as RBOH B, RBOH C, PFT1, and PRX59, were significantly induced by H2O2 treatment and then decreased after addition of IFQA. CONCLUSION: The positive effect of fullerene-based carbon nanomaterials on maize-root-hair growth under the induced oxidative stress was discovered. Application IFQA can ameliorate oxidative stress to promote maize-root growth through decreasing NADPH-oxidase activity, improving the scavenging of ROS by ASA-GSH cycle, and regulating the expressions of genes affecting maize-root-hair development. It will enrich more understanding the actual mechanism of fullerene-based nanoelicitors responsible for plant growth promotion and protection from oxidative stress.

Genome-Wide Identification and Comparison of Cysteine Proteases in the Pollen Coat and Other Tissues in Maize.

Cysteine proteases, belonging to the C1-papain family, play a major role in plant growth and development, senescence, and immunity. There is evidence to suggest that pollen cysteine protease (CP) (ZmCP03) is involved in regulating the anther development and pollen formation in maize. However, there is no report on the genome-wide identification and comparison of CPs in the pollen coat and other tissues in maize. In this study, a total of 38 homologous genes of ZmCP03 in maize were identified. Subsequently, protein motifs, conserved domains, gene structures, and duplication patterns of 39 CPs are analyzed to explore their evolutionary relationship and potential functions. The cis-elements were identified in the upstream sequence of 39 CPs, especially those that are related to regulating growth and development and responding to environmental stresses and hormones. The expression patterns of these genes displayed remarked difference at a tissue or organ level in maize based on the available transcriptome data in the public database. Quantitative reverse transcription PCR (RT-qPCR) analysis showed that ZmCP03 was preferably expressed at a high level in maize pollen. Analyses by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot, immunofluorescence and immunogold electron microscopy all validated the cellular localization of ZmCP03 in both the pollen coat and pollen cytoplasm. In addition, 142 CP genes from Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa) and cotton (Gossypium hirsutum), together with 39 maize CPs, were retrieved to analyze their evolution by comparing with orthologous genes. The results suggested that ZmCP03 was relatively conservative and stable during evolution. This study may provide a referential evidence on the function of ZmCP03 in pollen development and germination in maize.

Quaternary ammonium iminofullerenes promote root growth and osmotic-stress tolerance in maize via ROS neutralization and improved energy status.

In the present study, the role of quaternary ammonium iminofullerenes (IFQA) on the root growth of plant seedlings was investigated. The root elongation of Arabidopsis and maize exposed to 20 and 50 mg/L of IFQA was promoted under normal and osmotic stress conditions, respectively. In the meantime, the root active absorption area and adenosine triphosphate content in roots of maize seedlings were enhanced by IFQA treatment, however, the contents of hydrogen peroxide (H2O2) and malondialdehyde in roots were down-regulated. IFQA application improved glutathione transferase and glutathione reductase activities and the ratios of glutathione/oxidized glutathione and ascorbic acid/dehydroascorbic acid, and restored the inhibition of root elongation caused by the excess accumulation of H2O2 in roots of maize seedlings under osmotic stress. Furthermore, the expression of 14 proteins involved in cell growth, energy metabolism, and stress response in maize roots was upregulated by two-dimensional electrophoresis combined with mass spectrometry. This analysis revealed that IFQA stimulated the redox pathway to maintain balance levels of reactive oxygen species to ensure normal cell metabolism, promote energy production for root growth, and enhance osmotic-stress tolerance. It provided crucial information to elucidate the mechanism of the root growth of crop seedlings enhanced by water-soluble fullerene-based nanomaterials.

A new adenylyl cyclase, putative disease-resistance RPP13-like protein 3, participates in abscisic acid-mediated resistance to heat stress in maize.

In plants, 3´,5´-cyclic adenosine monophosphate (cAMP) is an important second messenger with varied functions; however, only a few adenylyl cyclases (ACs) that synthesize cAMP have been identified. Moreover, the biological roles of ACs/cAMP in response to stress remain largely unclear. In this study, we used quantitative proteomics techniques to identify a maize heat-induced putative disease-resistance RPP13-like protein 3 (ZmRPP13-LK3), which has three conserved catalytic AC centres. The AC activity of ZmRPP13-LK3 was confirmed by in vitro enzyme activity analysis, in vivo RNAi experiments, and functional complementation in the E. coli cyaA mutant. ZmRPP13-LK3 is located in the mitochondria. The results of in vitro and in vivo experiments indicated that ZmRPP13-LK3 interacts with ZmABC2, a possible cAMP exporter. Under heat stress, the concentrations of ZmRPP13-LK3 and cAMP in the ABA-deficient mutant vp5 were significantly less than those in the wild-type, and treatment with ABA and an ABA inhibitor affected ZmRPP13-LK3 expression in the wild-type. Application of 8-Br-cAMP, a cAMP analogue, increased heat-induced expression of heat-shock proteins in wild-type plants and alleviated heat-activated oxidative stress. Taken together, our results indicate that ZmRPP13-LK3, a new AC, can catalyse ATP for the production of cAMP and may be involved in ABA-regulated heat resistance.

Quaternary Ammonium Salts of Iminofullerenes: Fabrication and Effect on Seed Germination.

In this study, novel water-soluble quaternary ammonium salts of iminofullerenes (IFQA) were synthesized by nitrene chemistry in combination with quaternization and identified as [C60(NCH2CH2NH3+·CF3COO-)4·10H2O]n by various spectroscopies. Maize and Arabidopsis seeds were used to test the bioactivity of IFQA in seed germination. Compared with the control, maize seed exposure to 50 mg/L IFQA (normal: 73.1% vs 58.7%; drought: 66.7% vs 50.0% at the second day) and Arabidopsis seed exposure to 20 mg/L IFQA (normal: 77.5% vs 58.8%; drought: 63.3% vs 36.7% at the second day) had higher germination rates and quicker germination. The results of two-dimensional gel electrophoresis combined with mass spectroscopy showed that the abundance of 21 proteins in embryo proteome of maize seeds was significantly changed (>1.5 fold). The downregulated six storage proteins and upregulated four proteins induced by IFQA for energy production and sugar metabolism indicated a faster metabolic activity of maize seed germination. The upregulated eight stress-related proteins and antioxidant enzymes suggested that the role of IFQA was to activate the metabolic processes in seed germination and also increase seed stress response. The results provide important information to understand the mechanism of seed germination enhancement by carbon nanomaterials.

Subcellular locations of potential cell wall proteins in plants: predictors, databases and cross-referencing.

The cell wall is the most striking feature that distinguishes plant cells from animal cells. It plays an essential role in cell shape, stability, growth and protection. Despite being present in small amounts, cell wall proteins (CWPs) are crucial components of the cell wall. The cell wall proteome generally consists of sensu stricto CWPs, apoplast proteins and extracellular secreted proteins. Currently, there is a need for the bioinformatics analysis of a tremendous number of protein sequences that have been generated from genomic, transcriptomic and proteomics research. Compared with intracellular proteins, the location prediction of CWPs is challenging because many aspects of these proteins have not been experimentally characterized, and there are no CWP-trained, specific predictors available. By introducing the biological relevance (particularly molecular aspects) of the cell wall and CWPs, we critically evaluated the accuracy of 16 state-of-the-art predictors and classical predictors for the prediction of CWPs using an independent database of Arabidopsis and rice proteins. All experimentally verified CWPs and non-CWPs were retrieved from the UniProt Knowledgebase. Based on the evaluation, we currently recommend the predictors mGOASVM, HybridGO-Loc and FUEL-mLoc for CWPs. Furthermore, we outlined the public databases that can be used to cross-reference the subcellular location of CWPs. We illustrate a flowchart of the subcellular location prediction and a cross-reference of possible CWPs. Finally, we discuss challenges and perspectives in the bioinformatics analysis of CWPs. It is hoped that this article will provide practical guidance regarding CWPs for nonspecialists and provide insight for bioinformatics experts to develop computational tools for CWPs.

The Difference of Physiological and Proteomic Changes in Maize Leaves Adaptation to Drought, Heat, and Combined Both Stresses.

At the eight-leaf stage, maize is highly sensitive to stresses such as drought, heat, and their combination, which greatly affect its yield. At present, few studies have analyzed maize response to combined drought and heat stress at the eight-leaf stage. In this study, we measured certain physical parameters of maize at the eight-leaf stage when it was exposed to drought, heat, and their combination. The results showed an increase in the content of H2O2 and malondialdehyde (MDA), and in the enzyme activities of superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR), but a decrease in the quantum efficiency of photosystem II (ΦPSII). The most obvious increase or decrease in physical parameters was found under the combined stress condition. Moreover, to identify proteins differentially regulated by the three stress conditions at the eight-leaf stage, total proteins from the maize leaves were identified and quantified using multiplex iTRAQ-based quantitative proteomic and LC-MS/MS methods. In summary, the expression levels of 135, 65, and 201 proteins were significantly changed under the heat, drought and combined stress conditions, respectively. Of the 135, 65, and 201 differentially expressed proteins, 61, 28, and 16 responded exclusively to drought stress, heat stress, and combined stress, respectively. Bioinformatics analysis implied that chaperone proteins and proteases play important roles in the adaptive response of maize to heat stress and combined stress, and that the leaf senescence promoted by ethylene-responsive protein and ripening-related protein may play active roles in maize tolerance to combined drought and heat stress. The signaling pathways related to differentially expressed proteins were obviously different under all three stress conditions. Thus, the functional characterization of these differentially expressed proteins will be helpful for discovering new targets to enhance maize tolerance to stress.

Chloroform-assisted phenol extraction improving proteome profiling of maize embryos through selective depletion of high-abundance storage proteins.

The presence of abundant storage proteins in plant embryos greatly impedes seed proteomics analysis. Vicilin (or globulin-1) is the most abundant storage protein in maize embryo. There is a need to deplete the vicilins from maize embryo extracts for enhanced proteomics analysis. We here reported a chloroform-assisted phenol extraction (CAPE) method for vicilin depletion. By CAPE, maize embryo proteins were first extracted in an aqueous buffer, denatured by chloroform and then subjected to phenol extraction. We found that CAPE can effectively deplete the vicilins from maize embryo extract, allowing the detection of low-abundance proteins that were masked by vicilins in 2-DE gel. The novelty of CAPE is that it selectively depletes abundant storage proteins from embryo extracts of both monocot (maize) and dicot (soybean and pea) seeds, whereas other embryo proteins were not depleted. CAPE can significantly improve proteome profiling of embryos and extends the application of chloroform and phenol extraction in plant proteomics. In addition, the rationale behind CAPE depletion of abundant storage proteins was explored.

"Omics" of maize stress response for sustainable food production: opportunities and challenges.

Maize originated in the highlands of Mexico approximately 8700 years ago and is one of the most commonly grown cereal crops worldwide, followed by wheat and rice. Abiotic stresses (primarily drought, salinity, and high and low temperatures), together with biotic stresses (primarily fungi, viruses, and pests), negatively affect maize growth, development, and eventually production. To understand the response of maize to abiotic and biotic stresses and its mechanism of stress tolerance, high-throughput omics approaches have been used in maize stress studies. Integrated omics approaches are crucial for dissecting the temporal and spatial system-level changes that occur in maize under various stresses. In this comprehensive analysis, we review the primary types of stresses that threaten sustainable maize production; underscore the recent advances in maize stress omics, especially proteomics; and discuss the opportunities, challenges, and future directions of maize stress omics, with a view to sustainable food production. The knowledge gained from studying maize stress omics is instrumental for improving maize to cope with various stresses and to meet the food demands of the exponentially growing global population. Omics systems science offers actionable potential solutions for sustainable food production, and we present maize as a notable case study.

Proteomic analysis reveals differential accumulation of small heat shock proteins and late embryogenesis abundant proteins between ABA-deficient mutant vp5 seeds and wild-type Vp5 seeds in maize.

ABA is a major plant hormone that plays important roles during many phases of plant life cycle, including seed development, maturity and dormancy, and especially the acquisition of desiccation tolerance. Understanding of the molecular basis of ABA-mediated plant response to stress is of interest not only in basic research on plant adaptation but also in applied research on plant productivity. Maize mutant viviparous-5 (vp5), deficient in ABA biosynthesis in seeds, is a useful material for studying ABA-mediated response in maize. Due to carotenoid deficiency, vp5 endosperm is white, compared to yellow Vp5 endosperm. However, the background difference at proteome level between vp5 and Vp5 seeds is unclear. This study aimed to characterize proteome alterations of maize vp5 seeds and to identify ABA-dependent proteins during seed maturation. We compared the embryo and endosperm proteomes of vp5 and Vp5 seeds by gel-based proteomics. Up to 46 protein spots, most in embryos, were found to be differentially accumulated between vp5 and Vp5. The identified proteins included small heat shock proteins (sHSPs), late embryogenesis abundant (LEA) proteins, stress proteins, storage proteins and enzymes among others. However, EMB564, the most abundant LEA protein in maize embryo, accumulated in comparable levels between vp5 and Vp5 embryos, which contrasted to previously characterized, greatly lowered expression of emb564 mRNA in vp5 embryos. Moreover, LEA proteins and sHSPs displayed differential accumulations in vp5 embryos: six out of eight identified LEA proteins decreased while nine sHSPs increased in abundance. Finally, we discussed the possible causes of global proteome alterations, especially the observed differential accumulation of identified LEA proteins and sHSPs in vp5 embryos. The data derived from this study provides new insight into ABA-dependent proteins and ABA-mediated response during maize seed maturation.

Improving gel-based proteome analysis of soluble protein extracts by heat prefractionation.

The presence of high-abundance proteins in complex protein mixtures often masks low-abundance proteins and causes loss of resolution of 2DE. Protein fractionation steps conducted prior to 2DE can enhance the detection of low-abundance proteins and improve the resolution of 2DE. Here, we report a method to prefractionate soluble protein extracts based on protein thermal denaturation. Soluble proteins were extracted from maize embryos and leaves and Escherichia coli cells. Through heating at 95°C for 5 min, soluble protein extracts were prefractionated as heat stable protein fraction (the supernatant) and heat labile protein fraction (the precipitate). Our results showed that heat prefractionation enhanced the separation of proteins in both fractions by 2DE, thereby increasing the chance of detecting low-abundance proteins, many of which were nonvisible in unfractionated extract. In maize embryo, 330 spots were detected in soluble protein extract, while 577 spots were detected after prefractionation. Furthermore, this prefractionation method facilitated the enrichment, detection, and identification of de novo synthesized stress proteins. Because of its simplicity, the one-step heat prefractionation minimizes protein loss. Finally, heat prefractionation requires no expensive special hardware or reagents, and provides an alternative prefractionation for increasing the resolving power of 2DE.

Two cotton Cys2/His2-type zinc-finger proteins, GhDi19-1 and GhDi19-2, are involved in plant response to salt/drought stress and abscisic acid signaling.

Cotton (Gossypium hirsutum) often encounters abiotic stress such as drought and high salinity during its development, and its productivity is significantly limited by those adverse factors. To investigate the molecular adaptation mechanisms of this plant species to abiotic stress, we identified two genes encoding Di19-like Cys2/His2 zinc-finger proteins in cotton. GFP fluorescence assay demonstrated that GhDi19-1 and GhDi19-2 are two nuclear-localized proteins. Quantitative RT-PCR and Northern blot analyses revealed that mRNA accumulation of both GhDi19-1 and GhDi19-2 was significantly promoted by salinity and drought. Expression of GUS gene driven by the GhDi19-1 and GhDi19-2 promoters, respectively, was intensively induced in cotyledons under NaCl and mannitol stresses. Overexpression of GhDi19-1 and GhDi19-2 in Arabidopsis resulted in the seedlings displaying hypersensitivity to high salinity and abscisic acid (ABA). Seed germination and seedling growth of the transgenic Arabidopsis were dramatically inhibited by salinity and ABA, compared with wild type. In addition, expression levels of the ABA-responsive genes ABF3, ABF4, ABI5 and KIN1 were also remarkably altered in the transgenic plants under ABA treatment. Collectively, our results suggested that both GhDi19-1 and GhDi19-2 may be involved in response to salt/drought stress and ABA signaling during early stages of plant development.

A cotton gene encodes a tonoplast aquaporin that is involved in cell tolerance to cold stress.

To enhance the survival probability in cold stress, plant cells often increase their cold- and freezing-tolerance in response to low, nonfreezing temperatures by expressing some cold-related genes. In present study, a cotton gene encoding tonoplast intrinsic protein (TIP) was isolated from a cotton seedling cDNA library, and designated as GhTIP1;1. GFP fluorescent microscopy indicated that GhTIP1;1 protein was localized to the vacuolar membrane. Assay on GhTIP1;1 expression in Xenopus laevis oocytes demonstrated that GhTIP1;1 protein displayed water channel activity and facilitated water transport to the cells. At normal conditions, GhTIP1;1 transcripts were predominantly accumulated in roots and hypocotyls, but less abundance in other tissues of cotton. The GhTIP1;1 expression was dramatically up-regulated in cotyledons, but down-regulated in roots within a few hours after cotton seedlings were cold-treated. Overexpression of GhTIP1;1 in yeast (Schizosaccharomyces pombe) significantly enhanced the cell survival probability, suggesting that the GhTIP1;1 protein is involved in cell freezing-tolerance.

Optimizing protein extraction from plant tissues for enhanced proteomics analysis.

Plant tissues usually contain high levels of proteases and secondary metabolites that severely interfere with protein extraction, separation, and identification. Preparation of high-quality protein samples from plant tissues for proteomic analysis represents a great challenge. This article briefly describes the critical points in protein separation, especially secondary metabolites in plant tissues, and removal strategy. It provides an updated overview of three total protein extraction methods and their applications in proteomic analysis of various recalcitrant tissues.