Cynthia M. B. Damasceno

Sample extraction techniques for enhanced proteomic analysis of plant tissues

Major improvements in proteomic techniques in recent years have led to an increase in their application in all biological fields, including plant sciences. For all proteomic approaches, protein extraction and sample preparation are of utmost importance for optimal results; however, extraction of proteins from plant tissues represents a great challenge. Plant tissues usually contain relatively low amounts of proteins and high concentrations of proteases and compounds that potentially can limit tissue disintegration and interfere with subsequent protein separation and identification. An effective protein extraction protocol must also be adaptable to the great variation in the sets of secondary metabolites and potentially contaminating compounds that occurs between tissues (e.g., leaves, roots, fruit, seeds and stems) and between species. Here we present two basic protein extraction protocols that have successfully been used with diverse plant tissues, including recalcitrant tissues. The first method is based on phenol extraction coupled with ammonium acetate precipitation, and the second is based on trichloroacetic acid (TCA) precipitation. Both extraction protocols can be completed within 2 d.

Systems Biology of Tomato Fruit Development: Combined Transcript, Protein, and Metabolite Analysis of Tomato Transcription Factor (nor, rin) and Ethylene Receptor (Nr) Mutants Reveals Novel Regulatory Interactions

Tomato (Solanum lycopersicum) is an established model to study fleshy fruit development and ripening. Tomato ripening is regulated independently and cooperatively by ethylene and transcription factors, including nonripening (NOR) and ripening-inhibitor (RIN). Mutations of NOR, RIN, and the ethylene receptor Never-ripe (Nr), which block ethylene perception and inhibit ripening, have proven to be great tools for advancing our understanding of the developmental programs regulating ripening. In this study, we present systems analysis of nor, rin, and Nr at the transcriptomic, proteomic, and metabolomic levels during development and ripening. Metabolic profiling marked shifts in the abundance of metabolites of primary metabolism, which lead to decreases in metabolic activity during ripening. When combined with transcriptomic and proteomic data, several aspects of the regulation of metabolism during ripening were revealed. First, correlations between the expression levels of a transcript and the abundance of its corresponding protein were infrequently observed during early ripening, suggesting that posttranscriptional regulatory mechanisms play an important role in these stages; however, this correlation was much greater in later stages. Second, we observed very strong correlation between ripening-associated transcripts and specific metabolite groups, such as organic acids, sugars, and cell wall-related metabolites, underlining the importance of these metabolic pathways during fruit ripening. These results further revealed multiple ethylene-associated events during tomato ripening, providing new insights into the molecular biology of ethylene-mediated ripening regulatory networks.

A secreted effector protein (SNE1) from Phytophthora infestans is a broadly acting suppressor of programmed cell death.

Evasion or active suppression of host defenses are critical strategies employed by biotrophic phytopathogens and hemibiotrophs whose infection mechanism includes sequential biotrophic and necrotrophic stages. Although defense suppression by secreted effector proteins has been well studied in bacteria, equivalent systems in fungi and oomycetes are poorly understood. We report the characterization of SNE1 (suppressor of necrosis 1), a gene encoding a secreted protein from the hemibiotrophic oomycete Phytophthora infestans that is specifically expressed at the transcriptional level during biotrophic growth within the host plant tomato (Solanum lycopersicum). Using transient expression assays, we show that SNE1 suppresses the action of secreted cell death-inducing effectors from Phytophthora that are expressed during the necrotrophic growth phase, as well as programmed cell death mediated by a range of Avr-R protein interactions. We also report that SNE1 contains predicted NLS motifs and translocates to the plant nucleus in transient expression studies. A conceptual model is presented in which the sequential coordinated secretion of antagonistic effectors by P. infestans first suppresses, but then induces, host cell death, thereby providing a highly regulated means to control the transition from biotrophy to necrotrophy.

Plant glycosyl hydrolases and biofuels: a natural marriage.

Much of what is currently known about the structure, properties and biochemical activities of glycosyl hydrolases (GHs) has resulted from detailed studies of microbial enzymes. Conversely, such information is sparse in the plant GH literature, where the focus has traditionally been on studying expression and biological function. However, the current resurgence of interest in lignocellulosic biofuels is catalyzing new interest in this field, and recent reports suggest that some plant GH families have more in common with their microbial counterparts than was previously suspected. The repertoires of plant GHs, with their associated catalytic activities and polysaccharide binding affinities, may have valuable applications in modifying plant cell wall architecture and in the development and characterization of new bioenergy feedstocks.

Structure of the Glucanase Inhibitor Protein (GIP) Family from Phytophthora Species Suggests Coevolution with Plant Endo-β-1,3-Glucanases

During invasion of their plant hosts, species of the oomycete genus Phytophthora secrete glucanase inhibitor proteins (GIPs) into the plant apoplast, which bind and inhibit the activity of plant extracellular endo-beta-1,3-glucanases (EGases). GIPs show structural homology to the chymotrypsin class of serine proteases (SP) but lack proteolytic activity due to the absence of an intact catalytic triad and, thus, belong to a broader class of proteins called serine protease homologs (SPH). To study the evolutionary relationship between GIPs and functional SP, database searches were used to identify 48 GIP homologs in the P. sojae, P. ramorum, and P. infestans genomes, composing GIPs, SPH, and potentially functional SP. Analyses of P. infestans-inoculated tomato leaves showed that P. infestans GIPs and tomato EGases are present in the apoplast and form stable complexes in planta. Studies of the temporal expression of a four-membered GIP family from P. infestans (PiGIP1 to PiGIP4) further revealed that the genes show distinctly different patterns during an infection timecourse. Codon evolution analyses of GIP homologs identified several positively selected peptide sites and structural modeling revealed them to be in close proximity to rapidly evolving EGase residues, suggesting that the interaction between GIPs and EGases has the hallmarks of a coevolving molecular arms race.

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.