Theodore W. Thannhauser

Analysis for disulfide bonds in peptides and proteins

Publisher Summary This chapter discusses the analysis for disulfide bonds in peptides and proteins. Disulfide bonds make major contributions to the stability of the native conformations of proteins. The determination of the number of disulfide bonds per molecule is therefore crucial to structural studies of proteins. Many methods for the determination of disulfide bond concentration are proposed. Recently, a new method involving the use the reagent 2-nitro-5-thiosulfobenzoate (NTSB) is introduced. This method has significant advantages, the two most important being that it is both sensitive and quantitative. The reaction of NTSB with thiols and disulfides can be carried out in the presence of both dissolved oxygen and the reducing agent, sodium sulfite, which eliminates the inaccuracy and inconvenience associated with the necessity to work under an oxygen-free atmosphere as well as the need to remove the reducing agent. The NTSB assay is actually composed of two sequential reactions. The first reaction is the cleavage of a disulfide bond with sodium sulfite. The second reaction involves the nucleophilic attack of the thiolate produced in first reaction on NTSB to yield 1 mol each of a thiosulfonate and 2-nitro-5-thiobenzoate (NTB).

Proteome changes induced by aluminium stress in tomato roots

Growth inhibition in acid soils due to Al stress affects crop production worldwide. To understand mechanisms in sensitive crops that are affected by Al stress, a proteomic analysis of primary tomato root tissue, grown in Al-amended and non-amended liquid cultures, was performed. DIGE-SDS-MALDI-TOF-TOF analysis of these tissues resulted in the identification of 49 proteins that were differentially accumulated. Dehydroascorbate reductase, glutathione reductase, and catalase enzymes associated with antioxidant activities were induced in Al-treated roots. Induced enzyme proteins associated with detoxification were mitochondrial aldehyde dehydrogenase, catechol oxidase, quinone reductase, and lactoylglutathione lyase. The germin-like (oxalate oxidase) proteins, the malate dehydrogenase, wali7 and heavy-metal associated domain-containing proteins were suppressed. VHA-ATP that encodes for the catalytic subunit A of the vacuolar ATP synthase was induced and two ATPase subunit 1 isoforms were suppressed. Several proteins in the active methyl cycle, including SAMS, quercetin 3-O-methyltransferase and AdoHcyase, were induced by Al stress. Other induced proteins were isovaleryl-CoA dehydrogenase and the GDSL-motif lipase hydrolase family protein. NADPH-dependent flavin reductase and beta-hydroxyacyl-ACP dehydratase were suppressed.

Arabidopsis OR proteins are the major posttranscriptional regulators of phytoene synthase in controlling carotenoid biosynthesis

Significance Carotenoids are indispensable to plants and humans. Despite significant achievements in carotenoid research, we still lack the fundamental knowledge of the regulatory mechanisms underlying carotenogenesis in plants. Phytoene synthase (PSY) and ORANGE (OR) are the two key proteins for carotenoid biosynthesis and accumulation in plastids. This study shows that OR family proteins interact directly with PSY and function as the major regulators of active PSY protein abundance in mediating carotenoid biosynthesis. The findings establish posttranscriptional regulation of PSY as a novel way to control carotenoid biosynthesis in plants and provide strategies for crop nutritional quality improvement. Carotenoids are indispensable natural pigments to plants and humans. Phytoene synthase (PSY), the rate-limiting enzyme in the carotenoid biosynthetic pathway, and ORANGE (OR), a regulator of chromoplast differentiation and enhancer of carotenoid biosynthesis, represent two key proteins that control carotenoid biosynthesis and accumulation in plants. However, little is known about the mechanisms underlying their posttranscriptional regulation. Here we report that PSY and OR family proteins [Arabidopsis thaliana OR (AtOR) and AtOR-like] physically interacted with each other in plastids. We found that alteration of OR expression in Arabidopsis exerted minimal effect on PSY transcript abundance. However, overexpression of AtOR significantly increased the amount of enzymatically active PSY, whereas an ator ator-like double mutant exhibited a dramatically reduced PSY level. The results indicate that the OR proteins serve as the major posttranscriptional regulators of PSY. The ator or ator-like single mutant had little effect on PSY protein levels, which involves a compensatory mechanism and suggests partial functional redundancy. In addition, modification of PSY expression resulted in altered AtOR protein levels, corroborating a mutual regulation of PSY and OR. Carotenoid content showed a correlated change with OR-mediated PSY level, demonstrating the function of OR in controlling carotenoid biosynthesis by regulating PSY. Our findings reveal a novel mechanism by which carotenoid biosynthesis is controlled via posttranscriptional regulation of PSY in plants.

Mining the surface proteome of tomato (Solanum lycopersicum) fruit for proteins associated with cuticle biogenesis

The aerial organs of plants are covered by the cuticle, a polyester matrix of cutin and organic solvent-soluble waxes that is contiguous with the polysaccharide cell wall of the epidermis. The cuticle is an important surface barrier between a plant and its environment, providing protection against desiccation, disease, and pests. However, many aspects of the mechanisms of cuticle biosynthesis, assembly, and restructuring are entirely unknown. To identify candidate proteins with a role in cuticle biogenesis, a surface protein extract was obtained from tomato (Solanum lycopersicum) fruits by dipping in an organic solvent and the constituent proteins were identified by several complementary fractionation strategies and two mass spectrometry techniques. Of the ∼200 proteins that were identified, a subset is potentially involved in the transport, deposition, or modification of the cuticle, such as those with predicted lipid-associated protein domains. These include several lipid-transfer proteins, GDSL-motif lipase/hydrolase family proteins, and an MD-2-related lipid recognition domain-containing protein. The epidermal-specific transcript accumulation of several of these candidates was confirmed by laser-capture microdissection and quantitative reverse transcription-PCR (qRT-PCR), together with their expression during various stages of fruit development. This indicated a complex pattern of cuticle deposition, and models for cuticle biogenesis and restructuring are discussed.

The Or Gene Enhances Carotenoid Accumulation and Stability During Post-Harvest Storage of Potato Tubers

Provitamin A carotenoids in staple crops are not very stable during storage and their loss compromises nutritional quality. To elucidate the fundamental mechanisms underlying carotenoid accumulation and stability, we investigated transgenic potato tubers that expressed the cauliflower Orange (Or) gene. We found that the Or transgene not only promoted retention of β-carotene level, but also continuously stimulated its accumulation during 5 months of cold storage. In contrast, no increased levels of carotenoids were observed in the tubers of vector-only controls or a yellow-flesh variety during the same period of storage. The increased carotenoid accumulation was found to be associated with the formation of lipoprotein-carotenoid sequestering structures, as well as with the enhanced abundance of phytoene synthase, a key enzyme in the carotenoid biosynthetic pathway. Furthermore, the provitamin A carotenoids stored were shown to be stable during simulated digestion and accessible for uptake by human intestinal absorptive cells. Proteomic analysis identified three major functional groups of proteins (i.e. heat shock proteins, glutathione-S-transferases, and carbohydrate metabolic proteins) that are potentially important in the Or-regulated carotenoid accumulation. Our results show that regulation of carotenoid sequestration capacity is an important mechanism by which carotenoid stability is regulated. Our findings suggest that induction of a proper sink structure formation in staple crops may provide the crops with a unique ability to promote and/or stabilize provitamin A accumulation during plant growth and post-harvest storage.

Evaluation of Different Multidimensional LC–MS/MS Pipelines for Isobaric Tags for Relative and Absolute Quantitation (iTRAQ)-Based Proteomic Analysis of Potato Tubers in Response to Cold Storage

Cold-induced sweetening in potato tubers is a costly problem for the food industry. To systematically identify the proteins associated with this process, we employed a comparative proteomics approach using isobaric, stable isotope coded labels to compare the proteomes of potato tubers after 0 and 5 months of storage at 5 °C. We evaluated both high pH reverse phase (hpRP) liquid chromatography (LC) and off-gel electrophoresis (OGE) as first dimension fractionation methods followed by nanoLC-MS/MS, using two high performance mass spectrometry platforms (Q-TOF and Orbitrap). We found that hpRP-LC consistently offered better resolution, reduced expression ratio compression, and a more MS-compatible workflow than OGE and consistently yielded more unique peptide/protein identifications and higher sequence coverage with better quantification. In this study, a total of 4463 potato proteins were identified, of which 46 showed differential expressions during potato tuber cold storage. Several key proteins important in controlling starch-sugar conversion, which leads to cold-induced sweetening, as well as other proteins that are potentially involved in this process, were identified. Our results suggest that the hpRP-RP shotgun approach is a feasible and practical workflow for discovering potential protein candidates in plant proteomic analysis.

Coupling Genetics and Proteomics To Identify Aphid Proteins Associated with Vector-Specific Transmission of Polerovirus (Luteoviridae)

ABSTRACT Cereal yellow dwarf virus-RPV (CYDV-RPV) is transmitted specifically by the aphids Rhopalosiphum padi and Schizaphis graminum in a circulative nonpropagative manner. The high level of vector specificity results from the vector aphids having the functional components of the receptor-mediated endocytotic pathways to allow virus to transverse the gut and salivary tissues. Studies of F2 progeny from crosses of vector and nonvector genotypes of S. graminum showed that virus transmission efficiency is a heritable trait regulated by multiple genes acting in an additive fashion and that gut- and salivary gland-associated factors are not genetically linked. Utilizing two-dimensional difference gel electrophoresis to compare the proteomes of vector and nonvector parental and F2 genotypes, four aphid proteins (S4, S8, S29, and S405) were specifically associated with the ability of S. graminum to transmit CYDV-RPV. The four proteins were coimmunoprecipitated with purified RPV, indicating that the aphid proteins are capable of binding to virus. Analysis by mass spectrometry identified S4 as a luciferase and S29 as a cyclophilin, both of which have been implicated in macromolecular transport. Proteins S8 and S405 were not identified from available databases. Study of this unique genetic system coupled with proteomic analysis indicated that these four virus-binding aphid proteins were specifically inherited and conserved in different generations of vector genotypes and suggests that they play a major role in regulating polerovirus transmission.

An evaluation of the basis and consequences of a stay-green mutation in the navel negra citrus mutant using transcriptomic and proteomic profiling and metabolite analysis.

A Citrus sinensis spontaneous mutant, navel negra (nan), produces fruit with an abnormal brown-colored flavedo during ripening. Analysis of pigment composition in the wild-type and nan flavedo suggested that typical ripening-related chlorophyll (Chl) degradation, but not carotenoid biosynthesis, was impaired in the mutant, identifying nan as a type C stay-green mutant. nan exhibited normal expression of Chl biosynthetic and catabolic genes and chlorophyllase activity but no accumulation of dephytylated Chl compounds during ripening, suggesting that the mutation is not related to a lesion in any of the principal enzymatic steps in Chl catabolism. Transcript profiling using a citrus microarray indicated that a citrus ortholog of a number of SGR (for STAY-GREEN) genes was expressed at substantially lower levels in nan, both prior to and during ripening. However, the pattern of catabolite accumulation and SGR sequence analysis suggested that the nan mutation is distinct from those in previously described stay-green mutants and is associated with an upstream regulatory step, rather than directly influencing a specific component of Chl catabolism. Transcriptomic and comparative proteomic profiling further indicated that the nan mutation resulted in the suppressed expression of numerous photosynthesis-related genes and in the induction of genes that are associated with oxidative stress. These data, along with metabolite analyses, suggest that nan fruit employ a number of molecular mechanisms to compensate for the elevated Chl levels and associated photooxidative stress.

Enabling proteomic studies with RNA-Seq: The proteome of tomato pollen as a test case.

Effective proteome profiling is generally considered to depend heavily on the availability of a high‐quality DNA reference database. As such, proteomics has long been taxonomically restricted, with limited inroads being made into the proteomes of “non‐model” organisms. However, next generation sequencing (NGS), and particularly RNA‐Seq, now allows deep coverage detection of expressed genes at low cost, which in turn potentially facilitates the matching of peptide mass spectra with cognate gene sequence. To test this, we performed a quantitative analysis of the proteomes of pollen from domesticated tomato (Solanum lycopersicum) and two wild relatives that exhibit differences in mating systems and in interspecific reproductive barriers. Using a custom tomato RNA‐Seq database created through 454 pyrosequencing, more than 1200 proteins were identified, with subsets showing expression differences between genotypes or in the accumulation of the corresponding transcripts. Importantly, no major qualitative or quantitative differences were observed in the characterized proteomes when mass spectra were used to interrogate either a highly curated community database of tomato sequences generated through traditional sequencing technologies, or the RNA‐Seq database. We conclude that RNA‐Seq provides a cost‐effective and robust platform for protein identification and will be increasingly valuable to the field of proteomics.

Proteomic analysis of chromoplasts from six crop species reveals insights into chromoplast function and development

Chromoplasts are unique plastids that accumulate massive amounts of carotenoids. To gain a general and comparative characterization of chromoplast proteins, this study performed proteomic analysis of chromoplasts from six carotenoid-rich crops: watermelon, tomato, carrot, orange cauliflower, red papaya, and red bell pepper. Stromal and membrane proteins of chromoplasts were separated by 1D gel electrophoresis and analysed using nLC-MS/MS. A total of 953–2262 proteins from chromoplasts of different crop species were identified. Approximately 60% of the identified proteins were predicted to be plastid localized. Functional classification using MapMan bins revealed large numbers of proteins involved in protein metabolism, transport, amino acid metabolism, lipid metabolism, and redox in chromoplasts from all six species. Seventeen core carotenoid metabolic enzymes were identified. Phytoene synthase, phytoene desaturase, ζ-carotene desaturase, 9-cis-epoxycarotenoid dioxygenase, and carotenoid cleavage dioxygenase 1 were found in almost all crops, suggesting relative abundance of them among the carotenoid pathway enzymes. Chromoplasts from different crops contained abundant amounts of ATP synthase and adenine nucleotide translocator, which indicates an important role of ATP production and transport in chromoplast development. Distinctive abundant proteins were observed in chromoplast from different crops, including capsanthin/capsorubin synthase and fibrillins in pepper, superoxide dismutase in watermelon, carrot, and cauliflower, and glutathione-S-transferease in papaya. The comparative analysis of chromoplast proteins among six crop species offers new insights into the general metabolism and function of chromoplasts as well as the uniqueness of chromoplasts in specific crop species. This work provides reference datasets for future experimental study of chromoplast biogenesis, development, and regulation in plants.