Proteomic analysis of a plastid gene encoding RPS4 mutant in Chinese cabbage (Brassica campestris L. ssp. pekinensis).

Plastids are important plant cell organelles containing a genome and bacterial-type 70S ribosomes-primarily composed of plastid ribosomal proteins and ribosomal RNAs. In this study, a chlorophyll-deficient mutant (cdm) obtained from double-haploid Chinese cabbage 'FT' was identified as a plastome mutant with an A-to-C base substitution in the plastid gene encoding the ribosomal protein RPS4. To further elucidate the function and regulatory mechanisms of RPS4, a comparative proteomic analysis was conducted between cdm and its wild-type 'FT' plants by isobaric tags and a relative and absolute quantitation (iTRAQ)-based strategy. A total of 6,245 proteins were identified, 540 of which were differentially abundant proteins (DAPs) in the leaves of cdm as compared to those of 'FT'-including 233 upregulated and 307 downregulated proteins. Upregulated DAPs were mainly involved in translation, organonitrogen compound biosynthetic process, ribosomes, and spliceosomes. Meanwhile, downregulated DAPs were mainly involved in photosynthesis, photosynthetic reaction centres, photosynthetic light harvesting, carbon fixation, and chlorophyll binding. These results indicated an important role of RPS4 in the regulation of growth and development of Chinese cabbage, possibly by regulating plastid translation activity by affecting the expression of specific photosynthesis- and cold stress-related proteins. Moreover, a multiple reaction monitoring (MRM) test and quantitative real-time polymerase chain reaction analysis confirmed our iTRAQ results. Quantitative proteomic analysis allowed us to confirm diverse changes in the metabolic pathways between cdm and 'FT' plants. This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in Chinese cabbage.

Physiological, Agronomical, and Proteomic Studies Reveal Crucial Players in Rice Nitrogen Use Efficiency under Low Nitrogen Supply.

Excessive use of nitrogenous fertilizers to enhance rice productivity has become a significant source of nitrogen (N) pollution and reduced sustainable agriculture. However, little information about the physiology of different growth stages, agronomic traits, and associated genetic bases of N use efficiency (NUE) are available at low-N supply. Two rice (Oryza sativa L.) cultivars were grown with optimum N (120 kg ha-1) and low N (60 kg ha-1) supply. Six growth stages were analyzed to measure the growth and physiological traits, as well as the differential proteomic profiles, of the rice cultivars. Cultivar Panvel outclassed Nagina 22 at low-N supply and exhibited improved growth and physiology at most of the growth stages and agronomic efficiency due to higher N uptake and utilization at low-N supply. On average, photosynthetic rate, chlorophyll content, plant biomass, leaf N content, and grain yield were decreased in cultivar Nagina 22 than Panvel was 8%, 11%, 21%, 19%, and 22%, respectively, under low-N supply. Furthermore, proteome analyses revealed that many proteins were upregulated and downregulated at the different growth stages under low-N supply. These proteins are associated with N and carbon metabolism and other physiological processes. This supports the genotypic differences in photosynthesis, N assimilation, energy stabilization, and rice-protein yield. Our study suggests that enhancing NUE at low-N supply demands distinct modifications in N metabolism and physiological assimilation. The NUE may be regulated by key identified differentially expressed proteins. These proteins might be the targets for improving crop NUE at low-N supply.

Proteomics and Proteogenomics Analysis of Sweetpotato ( Ipomoea batatas) Leaf and Root.

Two complementary protein extraction methodologies coupled with an automated proteomic platform were employed to analyze tissue-specific proteomes and characterize biological and metabolic processes in sweetpotato. A total of 74 255 peptides corresponding to 4321 nonredundant proteins were successfully identified. Data were compared to predicted protein accessions for Ipomoea species and mapped on the sweetpotato transcriptome and haplotype-resolved genome. The two methodologies exhibited differences in the number and class of the unique proteins extracted. Overall, 39 916 peptides mapped to 3143 unique proteins in leaves, and 34 339 peptides mapped to 2928 unique proteins in roots. Primary metabolism and protein translation processes were enriched in leaves, whereas genetic pathways associated with protein folding, transport, sorting, as well as pathways in the primary carbohydrate metabolism were enriched in storage roots. A proteogenomics analysis successfully mapped 90.4% of the total uniquely identified peptides against the sweetpotato transcriptome and genome, predicted 741 new protein-coding genes, and specified 2056 loci where gene annotations can be further improved. The proteogenomics results provide evidence for the translation of new open reading frames (ORFs), alternative ORFs, exon extensions, and intronic ORF sequences. Data are available via ProteomeXchange with identifier PXD012999.

Histological, Physiological, and Comparative Proteomic Analyses Provide Insights into Leaf Rolling in Brassica napus.

Moderate leaf rolling is important in ideotype breeding, as it improves photosynthetic efficiency and therefore increases crop yields. To understand the regulatory network of leaf rolling in Brassica napus, a down-curved leaf mutant ( Bndcl1) has been investigated. Physiological analyses indicated that the chlorophyll contents and antioxidant enzyme activities were remarkably increased and the photosynthetic performance was significantly improved in Bndcl1. Consistent with these findings, 943 differentially accumulated proteins (DAPs) were identified in the Bndcl1 mutant and its wild-type plants using iTRAQ-based comparative proteomic analyses. Enrichment analysis of proteins with higher abundance in Bndcl1 revealed that the functional category "photosynthesis" was significantly overrepresented. Moreover, proteins associated with oxidative stress response and photosystem II repairing were also up-accumulated in Bndcl1, which might help the mutant to sustain the photosynthetic efficiency under unfavorable conditions. Histological observation showed that the mutant displayed defects in adaxial-abaxial patterning. Important DAPs associated with leaf polarity establishment were detected in Bndcl1, including ribosomal proteins, proteins involved in post-transcriptional gene silencing, and proteins related to brassinosteroid. Together, our findings may help clarify the mechanisms underlying leaf rolling and its physiological effects on plants and may facilitate ideotype breeding in Brassica napus.

Proteomic profiling of mature leaves from oil palm (Elaeis guineensis Jacq.).

Oil palm is one of the most productive oil bearing crops grown in Southeast Asia. Due to the dwindling availability of agricultural land and increasing demand for high yielding oil palm seedlings, clonal propagation is vital to the oil palm industry. Most commonly, leaf explants are used for in vitro micropropagation of oil palm and to optimize this process it is important to unravel the physiological and molecular mechanisms underlying somatic embryo production from leaves. In this study, a proteomic approach was used to determine protein abundance of mature oil palm leaves. To do this, leaf proteins were extracted using TCA/acetone precipitation protocol and separated by 2DE. A total of 191 protein spots were observed on the 2D gels and 67 of the most abundant protein spots that were consistently observed were selected for further analysis with 35 successfully identified using MALDI TOF/TOF MS. The majority of proteins were classified as being involved in photosynthesis, metabolism, cellular biogenesis, stress response, and transport. This study provides the first proteomic assessment of oil palm leaves in this important oil crop and demonstrates the successful identification of selected proteins spots using the Malaysian Palm Oil Board (MPOB) Elaeis guineensis EST and NCBI-protein databases. The MS data have been deposited in the ProteomeXchange Consortium database with the data set identifier PXD001307.

Leaf proteome rebalancing in Nicotiana benthamiana for upstream enrichment of a transiently expressed recombinant protein.

A key factor influencing the yield of biopharmaceuticals in plants is the ratio of recombinant to host proteins in crude extracts. Postextraction procedures have been devised to enrich recombinant proteins before purification. Here, we assessed the potential of methyl jasmonate (MeJA) as a generic trigger of recombinant protein enrichment in Nicotiana benthamiana leaves before harvesting. Previous studies have reported a significant rebalancing of the leaf proteome via the jasmonate signalling pathway, associated with ribulose 1,5-bisphosphate carboxylase oxygenase (RuBisCO) depletion and the up-regulation of stress-related proteins. As expected, leaf proteome alterations were observed 7 days post-MeJA treatment, associated with lowered RuBisCO pools and the induction of stress-inducible proteins such as protease inhibitors, thionins and chitinases. Leaf infiltration with the Agrobacterium tumefaciens bacterial vector 24 h post-MeJA treatment induced a strong accumulation of pathogenesis-related proteins after 6 days, along with a near-complete reversal of MeJA-mediated stress protein up-regulation. RuBisCO pools were partly restored upon infiltration, but most of the depletion effect observed in noninfiltrated plants was maintained over six more days, to give crude protein samples with 50% less RuBisCO than untreated tissue. These changes were associated with net levels reaching 425 µg/g leaf tissue for the blood-typing monoclonal antibody C5-1 expressed in MeJA-treated leaves, compared to less than 200 µg/g in untreated leaves. Our data confirm overall the ability of MeJA to trigger RuBisCO depletion and recombinant protein enrichment in N. benthamiana leaves, estimated here for C5-1 at more than 2-fold relative to host proteins.

Improved tolerance to drought stress after anthesis due to priming before anthesis in wheat (Triticum aestivum L.) var. Vinjett.

Drought stress occurring during the reproductive growth stage leads to considerable reductions in crop production and has become an important limiting factor for food security globally. In order to explore the possible role of drought priming (pre-exposure of the plants to mild drought stress) on the alleviation of a severe drought stress event later in development, wheat plants were subjected to single or double mild drought episodes (soil relative water content around 35-40%) before anthesis and/or to a severe drought stress event (soil relative water content around 20-25%) 15 d after anthesis. Here, single or double drought priming before anthesis resulted in higher grain yield than in non-primed plants under drought stress during grain filling. The photosynthesis rate and ascorbate peroxidase activity were higher while malondialdehyde content was lower in primed plants than in the non-primed plants under drought stress during grain filling. Proteins in flag leaves differently expressed by the priming and drought stress were mainly related to photosynthesis, stress defence, metabolism, molecular chaperone, and cell structure. Furthermore, the protein abundance of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) small subunit, Rubisco activase and ascorbate peroxidase were upregulated in primed plants compared with non-primed plants under drought stress during grain filling. In conclusion, the altered protein expression and upregulated activities of photosynthesis and ascorbate peroxidase in primed plants may indicate their potential roles in alleviating a later-occurring drought stress episode, thereby contributing to higher wheat grain yield under drought stress during grain filling.

Identification of allergen encoding sequences in a novel food ingredient from Moringa oleifera leaves.

Alternative sources of edible proteins are required to feed the world's growing population, such as Moringa oleifera leaves, a protein source with a balanced amino acid composition. Since Moringa leaf proteins is a novel food in the EU and UK, an assessment of their potential allergenicity of is required. Proteins from Moringa leaf powder were characterised using traditional proteomic approaches. The proteins identified were evaluated for their allergenic potential using in-silico tools. The main proteins identified belonged to photosynthetic and metabolic pathways. In-silico analysis of the leaf proteome identified moritides as potential allergens by homology with a latex allergen implicated in fruit-latex syndrome. This analysis also identified a nsLTP, a major panallergen in food. The presence of these putative allergens was confirmed by de-novo sequencing. Our study allowed identification of putative allergens, Morintides and nsLTP. Further in-vitro and in-vivo investigations are required to confirm their allergenic potential.

Proteomic analysis response of rice (Oryza sativa) leaves to ultraviolet-B radiation stress.

Rice (Oryza sativa) is a human staple food and serves as a model organism for genetic and molecular studies. Few studies have been conducted to determine the effects of ultraviolet-B (UV-B) stress on rice. UV-B stress triggers morphological and physiological changes in plants. However, the underlying mechanisms governing these integrated responses are unknown. In this study, we conducted a proteomic response of rice leaves to UV-B stress using two-dimensional gel electrophoresis and identified the selected proteins by mass spectrometry analysis. Four levels of daily biologically effective UV-B radiation intensities were imposed to determine changes in protein accumulation in response to UV-B stress: 0 (control), 5, 10, and 15 kJ m-2 d-1in two cultivars, i.e., IR6 and REX. To mimic the natural environment, we conducted this experiment in Sunlit Soil-Plant-Atmosphere-Research (SPAR) chambers. Among the identified proteins, 11% of differentially expressed proteins were found in both cultivars. In the Rex cultivar, only 45% of proteins are differentially expressed, while only 27.5% were expressed in IR6. The results indicate that REX is more affected by UV-B stress than IR6 cultivars. The identified protein TSJT1 (spot 16) in both cultivars plays a crucial role in plant growth and development during stress treatment. Additionally, we found that UV-B stress altered many antioxidant enzymes associated with redox homeostasis and cell defense response. Another enzyme, the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), has been identified as spot 15, which plays an essential role in glycolysis and cellular energy production. Another vital protein identified is glycosyl hydrolase (GH) as spot 9, which catalyzes the hydrolysis of glycosidic bonds in cell wall polymers and significantly affects cell wall architecture. Some identified proteins are related to photosynthesis, protein biosynthesis, signal transduction, and stress response. The findings of our study provide new insights into understanding how rice plants are tailored to UV-B stress via modulating the expression of UV-B responsive proteins, which will help develop superior rice breeds in the future to combat UV-B stress. Data are available via ProteomeXchange with identifier PXD032163.

Proteomic analysis of drought stress response mechanism in soybean (Glycine max L.) leaves.

Knowledge of the physiological and molecular mechanisms of drought responses is fundamental for developing genetically drought tolerant and high yielding crops. To understand molecular mechanism of drought tolerance of soybean (Glycine max L.), we compared leaf proteome patterns of in two genotypes GN-3074 (drought tolerant) and GN-2032 (drought-sensitive) under drought stress during vegetative stage. Proteins were extracted from leaves of well-watered and drought-treated plants by using the trichloroacetic acid (TCA)-acetone precipitation method and analyzed by two-dimensional polyacrylamide gel electrophoresis. Out 488 reproducibly detected and analyzed on two-dimensional electrophoresis gels, 26 proteins showed significant changes in at least one genotype. The identification of 20 differentially expressed proteins using mass spectrometry revealed a coordinated expression of proteins involved in cellular metabolisms including photosynthesis, oxidative stress defense, respiration, metabolism process, signal transduction, phosphorus transduction, and methyl transduction which enable plant to cope with drought conditions. The most identified proteins involved in photosynthesis and oxidative stress defense system. The up-regulation of several photosynthetic proteins and also high abundance of oxidative stress defense proteins in GN-3074 genotypes as compare to GN-2032 genotypes might reflect the fact that drought tolerance of GN-3074 is due to effective photosynthetic machinery and more defense against oxidative stress. Our results suggest that soybean plant might response to drought stress by applying efficiently stay-green mechanism through coordinated gene expression during vegetative stage.

Differential proteome analysis of the leaves of lead hyperaccumulator, Rhoeo discolor (L. Her.) Hance.

The present study investigated Rhoeo discolor (L. Her.) Hance for its ability to accumulate Pb, which is of relevance to phytoremediation applications. Based on this analysis, plants were found to accumulate greater than 10 mg/g (0.1%) of dry weight Pb in the shoots, which classifies the plant a Pb hyperaccumulator. Further, changes in the leaf proteome profiles in response to Pb stress were investigated. Wild-type plants were subjected to a high concentration of Pb(NO3 )2 , and the levels of Pb that accumulated in different plant tissues were determined using atomic absorption spectrophotometry. Using 2D-difference gel electrophoresis, 181 protein spots were detected to be differentially abundant in response to Pb stress and selected spots exhibiting the strongest differential abundance suggested an impairment of the photosynthetic apparatus of the plant under Pb stress. Subsequently, a more extensive, proteome wide analysis utilizing label-free quantitation further identified a predominant decrease in protein levels and a significant effect on the nuclear proteome, as well as photosynthesis, carbon fixation and metabolism, providing insight into the Pb tolerance of this system in a potential phytoremediation context.

Photoprotective Acclimation of the Arabidopsis thaliana Leaf Proteome to Fluctuating Light.

Plants are subjected to strong fluctuations in light intensity in their natural growth environment, caused both by unpredictable changes due to weather conditions and movement of clouds and upper canopy leaves and predictable changes during day-night cycle. The mechanisms of long-term acclimation to fluctuating light (FL) are still not well understood. Here, we used quantitative mass spectrometry to investigate long-term acclimation of low light-grown Arabidopsis thaliana to a FL condition that induces mild photooxidative stress. On the third day of exposure to FL, young and mature leaves were harvested in the morning and at the end of day for proteome analysis using a stable isotope labeling approach. We identified 2,313 proteins, out of which 559 proteins exhibited significant changes in abundance in at least one of the four experimental groups (morning-young, morning-mature, end-of-day-young, end-of-day-mature). A core set of 49 proteins showed significant responses to FL in three or four experimental groups, which included enhanced accumulation of proteins involved in photoprotection, cyclic electron flow around photosystem I, photorespiration, and glycolysis, while specific glutathione transferases and proteins involved in translation and chlorophyll biosynthesis were reduced in abundance. In addition, we observed pathway- and protein-specific changes predominantly at the end of day, whereas few changes were observed exclusively in the morning. Comparison of the proteome data with the matching transcript data revealed gene- and protein-specific responses, with several chloroplast-localized proteins decreasing in abundance despite increased gene expression under FL. Together, our data shows moderate but widespread alterations of protein abundance during acclimation to FL and suggests an important role of post-transcriptional regulation of protein abundance.

Proteomics towards the understanding of elicitor induced resistance of grapevine against downy mildew.

Elicitors are known to trigger plant defenses in response to biotic stress, but do not systematically lead to effective resistance to pathogens. The reasons explaining such differences remain misunderstood. Therefore, elicitation and induced resistance (IR) were investigated through the comparison of two modified ß-1,3 glucans applied on grapevine (Vitis vinifera) leaves before and after inoculation with Plasmopara viticola, the causal agent of downy mildew. The sulfated (PS3) and the shortened (H13) forms of laminarin are both known to elicit defense responses whereas only PS3 induces resistance against downy mildew. The analysis of the 2-DE gel electrophoresis revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Our results point out that the PS3-induced resistance is associated with the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could be considered as useful markers of induced resistance. SIGNIFICANCE: One strategy to reduce the application of fungicides is the use of elicitors which induce plant defense responses. Nonetheless, the elicitors do not systematically lead to resistance against pathogens. The lack of correlation between plant defense activation and induced resistance (IR) requires the investigation of what makes the specificity of elicitor-IR. In this study, the two ß-glucans elicitors, sulfated (PS3) and short (H13) laminarins, were used in the grapevine/Plasmopara viticola interaction since only the first one leads to resistance against downy mildew. To disclose IR specificity, proteomic approach has been employed to compare the two treatments before and after P. viticola inoculation. The analysis of the 2-DE revealed that PS3 and H13 induced distinct proteomic profiles after treatment and pathogen inoculation. Significant increase of the number of proteins regulated by PS3, relative to both H13 and time-points, is correlated with the resistance process establishment. Our results point that the PS3-induced resistance requires the activation of the primary metabolism especially on amino acids and carbohydrates pathways. In addition, few proteins, such as the 12-oxophytodienoate reductase (OPR-like) related to the OPDA pathway, and an Arsenite-resistance protein (Serrate-like protein) could constitute useful markers of PS3 induced resistance.

Corrigendum: Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants.

[This corrects the article on p. 903 in vol. 6, PMID: 26579153.].

Physiological and proteome studies of responses to heat stress during grain filling in contrasting wheat cultivars.

Experiments to explore physiological and biochemical differences of the effects of heat stress in ten wheat (Triticum aestivum L.) cultivars have been performed. Based on the response of photosynthesis rates, cell membrane lipid peroxide concentrations and grain yield to heat, six cultivars were clustered as heat-tolerant (cv. '579', cv. '810', cv. '1110', cv. Terice, cv. Taifun and cv. Vinjett) and four as heat-sensitive (cv. '490', cv. '633', cv. '1039' and cv. '1159'). Higher rates of photosynthetic carbon- and light-use were accompanied by lower damage to cell membranes in leaves of tolerant compared to sensitive cultivars under heat stress. The tolerant cv. '810' and the sensitive cv. '1039' were selected for further proteome analysis of leaves. Proteins related to photosynthesis, glycolysis, stress defence, heat shock and ATP production were differently expressed in leaves of the tolerant and sensitive cultivar under heat stress in relation to the corresponding control. The abundance of proteins related to signal transduction, heat shock, photosynthesis, and antioxidants increased, while the abundance of proteins related to nitrogen metabolism decreased in the tolerant cv. '810' under heat stress as compared to the control. Collectively, the results indicate that primarily changes in both the amount and activities of enzymes involved in photosynthesis and antioxidant activities in leaves contributed to higher heat tolerance in the cv. '810' compared to the heat sensitive cv. '1039'.

iTRAQ-based quantitative proteomics analysis of Brassica napus leaves reveals pathways associated with chlorophyll deficiency.

Photosynthesis, the primary source of plant biomass, is important for plant growth and crop yield. Chlorophyll is highly abundant in plant leaves and plays essential roles in photosynthesis. We recently isolated a chlorophyll-deficient mutant (cde1) from ethyl methanesulfonate (EMS) mutagenized Brassica napus. Herein, quantitative proteomics analysis using the iTRAQ approach was conducted to investigate cde1-induced changes in the proteome. We identified 5069 proteins from B. napus leaves, of which 443 showed differential accumulations between the cde1 mutant and its corresponding wild-type. The differentially accumulated proteins were found to be involved in photosynthesis, porphyrin and chlorophyll metabolism, biosynthesis of secondary metabolites, carbon fixation, spliceosome, mRNA surveillance and RNA degradation. Our results suggest that decreased abundance of chlorophyll biosynthetic enzymes and photosynthetic proteins, impaired carbon fixation efficiency and disturbed redox homeostasis might account for the reduced chlorophyll contents, impaired photosynthetic capacity and increased lipid peroxidation in this mutant. Epigenetics was implicated in the regulation of gene expression in cde1, as proteins involved in DNA/RNA/histone methylation and methylation-dependent chromatin silencing were up-accumulated in the mutant. Biological significance Photosynthesis produces more than 90% of plant biomass and is an important factor influencing potential crop yield. The pigment chlorophyll plays essential roles in light harvesting and energy transfer during photosynthesis. Mutants deficient in chlorophyll synthesis have been used extensively to investigate the chlorophyll metabolism, development and photosynthesis. However, limited information is available with regard to the changes of protein profiles upon chlorophyll deficiency. Here, a combined physiological, histological, proteomics and molecular analysis revealed several important pathways associated with chlorophyll deficiency. This work provides new insights into the regulation of chlorophyll biosynthesis and photosynthesis in higher plants and these findings may be applied to genetic engineering for high photosynthetic efficiency in crops.

Effect of temperature on the pathogenesis, accumulation of viral and satellite RNAs and on plant proteome in peanut stunt virus and satellite RNA-infected plants.

Temperature is an important environmental factor influencing plant development in natural and diseased conditions. The growth rate of plants grown at C27°C is more rapid than for plants grown at 21°C. Thus, temperature affects the rate of pathogenesis progression in individual plants. We have analyzed the effect of temperature conditions (either 21°C or 27°C during the day) on the accumulation rate of the virus and satellite RNA (satRNA) in Nicotiana benthamiana plants infected by peanut stunt virus (PSV) with and without its satRNA, at four time points. In addition, we extracted proteins from PSV and PSV plus satRNA-infected plants harvested at 21 dpi, when disease symptoms began to appear on plants grown at 21°C and were well developed on those grown at 27°C, to assess the proteome profile in infected plants compared to mock-inoculated plants grown at these two temperatures, using 2D-gel electrophoresis and mass spectrometry approaches. The accumulation rate of the viral RNAs and satRNA was more rapid at 27°C at the beginning of the infection and then rapidly decreased in PSV-infected plants. At 21 dpi, PSV and satRNA accumulation was higher at 21°C and had a tendency to increase further. In all studied plants grown at 27°C, we observed a significant drop in the identified proteins participating in photosynthesis and carbohydrate metabolism at the proteome level, in comparison to plants maintained at 21°C. On the other hand, the proteins involved in protein metabolic processes were all more abundant in plants grown at 27°C. This was especially evident when PSV-infected plants were analyzed, where increase in abundance of proteins involved in protein synthesis, degradation, and folding was revealed. In mock-inoculated and PSV-infected plants we found an increase in abundance of the majority of stress-related differently-regulated proteins and those associated with protein metabolism. In contrast, in PSV plus satRNA-infected plants the shift in the temperature barely increased the level of stress-related proteins.

Method optimization for proteomic analysis of soybean leaf: Improvements in identification of new and low-abundance proteins.

The most critical step in any proteomic study is protein extraction and sample preparation. Better solubilization increases the separation and resolution of gels, allowing identification of a higher number of proteins and more accurate quantitation of differences in gene expression. Despite the existence of published results for the optimization of proteomic analyses of soybean seeds, no comparable data are available for proteomic studies of soybean leaf tissue. In this work we have tested the effects of modification of a TCA-acetone method on the resolution of 2-DE gels of leaves and roots of soybean. Better focusing was obtained when both mercaptoethanol and dithiothreitol were used in the extraction buffer simultaneously. Increasing the number of washes of TCA precipitated protein with acetone, using a final wash with 80% ethanol and using sonication to ressuspend the pellet increased the number of detected proteins as well the resolution of the 2-DE gels. Using this approach we have constructed a soybean protein map. The major group of identified proteins corresponded to genes of unknown function. The second and third most abundant groups of proteins were composed of photosynthesis and metabolism related genes. The resulting protocol improved protein solubility and gel resolution allowing the identification of 122 soybean leaf proteins, 72 of which were not detected in other published soybean leaf 2-DE gel datasets, including a transcription factor and several signaling proteins.