Fangping Gong

Protein extraction from plant tissues for 2DE and its application in proteomic analysis

Plant tissues contain large amounts of secondary compounds that significantly interfere with protein extraction and 2DE analysis. Thus, sample preparation is a crucial step prior to 2DE in plant proteomics. This tutorial highlights the guidelines that need to be followed to perform an adequate total protein extraction before 2DE in plant proteomics. We briefly describe the history, development, and feature of major sample preparation methods for the 2DE analysis of plant tissues, that is, trichloroacetic acid/acetone precipitation and phenol extraction. We introduce the interfering compounds in plant tissues and the general guidelines for tissue disruption, protein precipitation and resolubilization. We describe in details the advantages, limitations, and application of the trichloroacetic acid/acetone precipitation and phenol extraction methods to enable the readers to select the appropriate method for a specific species, tissue, or cell type. The current applications of the sample preparation methods in plant proteomics in the literature are analyzed. A comparative proteomic analysis between male and female plants of Pistacia chinensis is used as an example to represent the sample preparation methodology in 2DE‐based proteomics. Finally, the current limitations and future development of these sample preparation methods are discussed. This Tutorial is part of the International Proteomics Tutorial Programme (IPTP17).

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.

Proteomic analysis of crop plants under abiotic stress conditions: where to focus our research?

Approximately 80% of human food is composed of crops, which are dominated by cereals that collectively make up 50% of global food production (Langridge and Fleury, 2011). Among cereal crops, rice, wheat, and maize provide approximately half of the calories consumed worldwide. Nevertheless, crop production is seriously hampered by influential abiotic stresses like drought, climate fluctuations, and salinity. It is estimated that up to 50–70% decline in crop productivity is attributed to abiotic stress (Mittler, 2006). Therefore, to ensure the security of global food production, it is essential to produce sustainable crop varieties that can adapt to climate variability, and to develop a broad spectrum of abiotic stress tolerant crops (Tester and Langridge, 2010). This has driven much research into the study of crop responses to abiotic stresses. Proteomics has been successfully used to study abiotic stress responses in a wide range of crops (Abreu et al., 2013; Barkla et al., 2013; Ngara and Ndimba, 2014), especially rice (Kim et al., 2014), wheat (Komatsu et al., 2014), and maize (Benesova et al., 2012; Gong et al., 2014). It is generally envisioned that at this stage, proteomic-based discoveries in rice are likely to be translated into improving other crop plants against ever-changing environmental factors (Kim et al., 2014). Despite the potential role of proteomics to advance the study of stress tolerance in crops, thus far little useful information has been made available for crop improvement and breeding, even with the numerous proteomics studies undertaken in recent years. In our opinion, crop stress proteomics should be better focused on the following aspects: dissecting cell specific stress response (especially initial stress responses), identification of stress proteins, and the analysis of post translational modifications (PTMs) of proteins (Figure ​(Figure11). Open in a separate window Figure 1 A graphic summary on current research and future research in proteomic analysis of crop plants under abiotic stress conditions.

Advances in crop proteomics: PTMs of proteins under abiotic stress.

Under natural conditions, crop plants are frequently subjected to various abiotic environmental stresses such as drought and heat wave, which may become more prevalent in the coming decades. Plant acclimation and tolerance to an abiotic stress are always associated with significant changes in PTMs of specific proteins. PTMs are important for regulating protein function, subcellular localization and protein activity and stability. Studies of plant responses to abiotic stress at the PTMs level are essential to the process of plant phenotyping for crop improvement. The ability to identify and quantify PTMs on a large‐scale will contribute to a detailed protein functional characterization that will improve our understanding of the processes of crop plant stress acclimation and stress tolerance acquisition. Hundreds of PTMs have been reported, but it is impossible to review all of the possible protein modifications. In this review, we briefly summarize several main types of PTMs regarding their characteristics and detection methods, review the advances in PTMs research of crop proteomics, and highlight the importance of specific PTMs in crop response to abiotic stress.

Protein sHSP26 improves chloroplast performance under heat stress by interacting with specific chloroplast proteins in maize (Zea mays).

UNLABELLED We recently demonstrated that chloroplast small HSP26 (sHSP26) is abundant in maize leaves under heat stress and potentially involved in maize heat tolerance. However, it largely remains unclear how sHSP26 functions in maize under heat stress. Here, 2-DE-based proteomics, RNA interference (RNAi), co-immunoprecipitation (Co-IP) and yeast two-hybrid (Y2H) were used to reveal chloroplast proteins interacting with sHSP26 and how sHSP26 functions under heat stress. After the silencing of sHSP26, a total of 45 protein spots from isolated protoplasts were greatly changed in abundance, of which 33 spots are chloroplastic. Co-IP revealed that nine proteins possibly associated with sHSP26. Y2H demonstrated that six chloroplast proteins interact with sHSP26 under heat stress. In particular, four proteins, including ATP synthase subunit β, chlorophyll a-b binding protein, oxygen-evolving enhancer protein 1 and photosystem I reaction center subunit IV, strongly interacted with sHSP26 and their abundance greatly declined after RNAi of sHSP26 under heat stress. In addition, H2O2 accumulation in the chloroplasts significantly increased the expression of sHSP26, and the suppression of sHSP26 expression significantly reduced the O2 evolution rate of photosystem II under heat stress. Overall, these findings demonstrate the relevance of sHSP26 in protecting maize chloroplasts under heat stress. BIOLOGICAL SIGNIFICANCE Maize is one of the most important crops worldwide. Frequent heat stress reduces significantly the yield and quality of maize. Our results demonstrated that sHSP26 improved maize chloroplast performance under heat stress by interacting with specific proteins. These findings are useful for understanding the mechanism of heat stress response and heat-tolerant molecular breeding in maize.

Making better maize plants for sustainable grain production in a changing climate

Achieving grain supply security with limited arable land is a major challenge in the twenty-first century, owing to the changing climate and increasing global population. Maize plays an increasingly vital role in global grain production. As a C4 plant, maize has a high yield potential. Maize is predicted to become the number one cereal in the world by 2020. However, maize production has plateaued in many countries, and hybrid and production technologies have been fully exploited. Thus, there is an urgent need to shape maize traits and architectures for increased stress tolerance and higher yield in a changing climate. Recent achievements in genomics, proteomics, and metabolomics have provided an unprecedented opportunity to make better maize. In this paper, we discuss the current challenges and potential of maize production, particularly in China. We also highlight the need for enhancing maize tolerance to drought and heat waves, summarize the elite shoot and root traits and phenotypes, and propose an ideotype for sustainable maize production in a changing climate. This will facilitate targeted maize improvement through a conventional breeding program combined with molecular techniques.

Diversity and function of maize pollen coat proteins: from biochemistry to proteomics

Maize (Zea mays L.) is globally cultivated as one of the most important grain crops. As a wind-pollinated species, maize produces a large quantity of pollen grains that heavier and larger compared to Arabidopsis. Maize is an important model plant in pollen biology of monocots. The pollen coat, the outermost layer of pollen, plays a vital role in pollen–stigma interactions and successful fertilization. Pollen coat proteins (PCPs), which confer species specificity, are required for pollen adhesion, recognition, hydration, and germination on the stigma. Thus, PCPs have attracted intensive research efforts in plant science for decades. However, only a few PCPs in maize have been characterized to date, whereas the functions of most maize PCPs remain unclear. In this review, we summarize the current knowledge of maize PCPs with regard to protein constituents, synthesis and transport, and functions by comparison with the model plant Arabidopsis thaliana and Brassica plants. An understanding of the comprehensive knowledge of maize PCPs will help to illuminate the mechanism by which PCPs are involved in pollen–stigma interactions in maize and other crop plants.

Functional assignment to maize group 1 LEA protein EMB564 within the cell nucleus using computational analysis.

Maize protein EMB564 is a member of group 1 LEA (late embryogenesis abundant) proteins. Currently, the molecular functions of group 1 LEA proteins remain largely unclear. We here report on the functional assignment to EMB564 by computational analysis. EMB564 is predicted as nuclear localization by five different predictors including CELLO, Plant-mPLoc, WoLF PSORT, Predotar and TargetP. EMB564 is found to be remote homologous with DNA/RNA helicases and single-stranded DNA-binding proteins, and their sequences contains similar DNA/RNA binding sites. Furthermore, the three-dimensional (3D) model of EMB564 structurally resembles a variety of nuclear and DNA/RNA-binding proteins, especially those involving in the regulation of cell division, chromosomal replication and DNA unwinding or repairing. Our results reveal that EMB564 protein is most likely to function within the cell nucleus.

Sequential Extraction Results in Improved Proteome Profiling of Medicinal Plant Pinellia ternata Tubers, Which Contain Large Amounts of High-Abundance Proteins

Pinellia ternata tuber is one of the well-known Chinese traditional medicines. In order to understand the pharmacological properties of tuber proteins, it is necessary to perform proteome analysis of P. ternata tubers. However, a few high-abundance proteins (HAPs), mainly mannose-binding lectin (agglutinin), exist in aggregates of various sizes in the tubers and seriously interfere with proteome profiling by two-dimensional electrophoresis (2-DE). Therefore, selective depletion of these HAPs is a prerequisite for enhanced proteome analysis of P. ternata tubers. Based on differential protein solubility, we developed a novel protocol involving two sequential extractions for depletion of some HAPs and prefractionation of tuber proteins prior to 2-DE. The first extraction using 10% acetic acid selectively extracted acid-soluble HAPs and the second extraction using the SDS-containing buffer extracted remaining acid-insoluble proteins. After application of the protocol, 2-DE profiles of P. ternata tuber proteins were greatly improved and more protein spots were detected, especially low-abundance proteins. Moreover, the subunit composition of P. ternata lectin was analyzed by electrophoresis. Native lectin consists of two hydrogen-bonded subunits (11 kDa and 25 kDa) and the 11 kDa subunit was a glycoprotein. Subsequently, major HAPs in the tubers were analyzed by mass spectrometry, with nine protein spots being identified as lectin isoforms. The methodology was easy to perform and required no specialized apparatus. It would be useful for proteome analysis of other tuber plants of Araceae.

Proteome Profiling of Maize Pollen Coats Reveals Novel Protein Components

The pollen coat, which covers the exine wall of pollen, is essential for initial sexual contact and thus successful fertilization in flowering plants. Pollen coat proteins (PCPs) not only mediate species specificity but are also needed for pollen–stigma recognition and pollen germination on the stigma. Maize (Zea mays L.) is one of the most common cereal crops in the world. To date, only a few PCPs from maize have been identified and characterized. In the present study, we extracted the pollen coat fraction from maize inbred line B73 with chloroform and purified the PCPs via a phenol-based protocol prior to two-dimensional gel electrophoresis (2-DE). By proteomic analysis, 26 protein spots were successfully identified and classified into 12 unique proteins. The protein composition of the maize pollen coat is distinctly different from those of the dicot plants Arabidopsis and rapeseed. Of the proteins identified in this study, eight (including profilins, caleosin, Zea m 2, β-expansin-10, exopolygalacturonase, Rho GDP-dissociation inhibitor 1, Ras-related protein Rab-2-A, and putative subtilase) had not previously been observed in the maize pollen coat. Bioinformatic analysis showed that nine of the PCPs were secreted proteins and that most of them were extracellular. These PCPs are potentially involved in pollen germination and tube growth. The current study extracted and identified the pollen coat proteins of maize, one of the most important crops throughout the world. Proteome profiling of the maize pollen coat revealed many novel protein components potentially involved in pollen–stigma interactions and pollen germination. Our results provide basic knowledge and further the functional characterization of PCPs in wind-pollinated species such as maize.