Proteomics profiling of fiber development and domestication in upland cotton (Gossypium hirsutum L.)

Comparative proteomic analyses were performed to detail the evolutionary consequences of strong directional selection for enhanced fiber traits in modern upland cotton (Gossypium hirsutum L.). Using two complementary proteomic approaches, 2-DE and iTRAQ LC–MS/MS, fiber proteomes were examined for four representative stages of fiber development. Approximately 1,000 protein features were characterized using each strategy, collectively resulting in the identification and functional categorization of 1,223 proteins. Unequal contributions of homoeologous proteins were detected for over a third of the fiber proteome, but overall expression was balanced with respect to the genome-of-origin in the allopolyploid G. hirsutum. About 30 % of the proteins were differentially expressed during fiber development within wild and domesticated cotton. Notably, domestication was accompanied by a doubling of protein developmental dynamics for the period between 10 and 20 days following pollination. Expression levels of 240 iTRAQ proteins and 293 2-DE spots were altered by domestication, collectively representing multiple cellular and metabolic processes, including metabolism, energy, protein synthesis and destination, defense and stress response. Analyses of homoeolog-specific expression indicate that duplicated gene products in cotton fibers can be differently regulated in response to selection. These results demonstrate the power of proteomics for the analysis of crop domestication and phenotypic evolution.     

Dissecting Pistil Responses to Incompatible and Compatible Pollen in Self-Incompatibility <Emphasis Type="Italic">Brassica oleracea</Emphasis> Using Comparative Proteomics

Angiosperms have developed self-incompatibility (SI) systems to reject self-pollen, thereby promoting outcrossing. The Brassicaceae belongs to typical sporophytic system, having a single S-locus controlled SI response, and was chosen as a model system to study SI-related intercellular signal transduction. In this regard, the downstream factor of EXO70A1 was unknown. Here, protein two-dimensional electrophoresis (2-DE) method and coupled with matrix-assisted laser desorption ionization/time of flight of flight mass spectrometry (MALDI-TOF -MS) and peptide mass fingerprinting (PMF) was used to further explore the mechanism of SI responses in Brassica oleracea L. var. capitata L. at protein level. To further confirm the time point of protein profile change, total proteins were collected from B. oleracea pistils at 0 min, 1 h, and 2 h after self-pollination. In total 902, 1088 and 1023 protein spots were separated in 0 min, 1 h and 2 h 2-DE maps, respectively. Our analyses of self-pollination profiles indicated that proteins mainly changed at 1 h post-pollination in B. oleracea. Moreover, 1077 protein spots were separated in cross-pollinated 1 h (CP) pistil 2-DE map. MALDI-TOF-MS and PMF successfully identified 34 differentially-expressed proteins (DEPs) in SP and CP 1 h 2-DE maps. Gene ontology and KEGG analysis revealed an array of proteins grouped in the following categories: stress and defense response (35%), protein metabolism (18%), carbohydrate and energy metabolism (12%), regulation of translation (9%), pollen tube development (12%), transport (9%) and cytoskeletal (6%). Sets of DEPs identified specifically in SP or only up-regulated expressed in CP pistils were chosen for funther investigating in floral organs and during the process of self- and cross-pollination. The function of these DEPs in terms of their potential involvement in SI in B. oleracea is discussed.     

Effects of human and porcine bile on the proteome of Helicobacter hepaticus

Background

Helicobacter hepaticus colonizes the intestine and liver of mice causing hepatobiliary disorders such as hepatitis and hepatocellular carcinoma, and has also been associated with inflammatory bowel disease in children. In its habitat, H. hepaticus must encounter bile which has potent antibacterial properties. To elucidate virulence and host-specific adaptation mechanisms of H. hepaticus modulated by human or porcine bile, a proteomic study of its response to the two types of bile was performed employing two-dimensional gel electrophoresis (2-DE) and mass spectrometry.

Results

The 2-DE and mass spectrometry analyses of the proteome revealed that 46 proteins of H. hepaticus were differentially expressed in human bile, 18 up-regulated and 28 down-regulated. In the case of porcine bile, 32 proteins were differentially expressed of which 19 were up-regulated, and 13 were down-regulated. Functional classifications revealed that identified proteins participated in various biological functions including stress response, energy metabolism, membrane stability, motility, virulence and colonization. Selected genes were analyzed by RT-PCR to provide internal validation for the proteomic data as well as provide insight into specific expressions of motility, colonization and virulence genes of H. hepaticus in response to human or porcine bile.

Conclusions

Overall, the data suggested that bile is an important factor that determines virulence, host adaptation, localization and colonization of specific niches within host environment.     

Proteomics of methyl jasmonate induced defense response in maize leaves against Asian corn borer

Background

Jasmonic acid (JA) and methyl jasmonate (MeJA) regulate plant development, resistance to stress, and insect attack by inducing specific gene expression. However, little is known about the mechanism of plant defense against herbivore attack at a protein level. Using a high-resolution 2-D gel, we identified 62 MeJA-responsive proteins and measured protein expression level changes.

Results

Among these 62 proteins, 43 proteins levels were increased while 11 proteins were decreased. We also found eight proteins uniquely expressed in response to MeJA treatment. Data are available via ProteomeXchange with identifier PXD001793. The proteins identified in this study have important biological functions including photosynthesis and energy related proteins (38.4%), protein folding, degradation and regulated proteins (15.0%), stress and defense regulated proteins (11.7%), and redox-responsive proteins (8.3%). The expression levels of four important genes were determined by qRT-PCR analysis. The expression levels of these proteins did not correlate well with their translation levels. To test the defense functions of the differentially expressed proteins, expression vectors of four protein coding genes were constructed to express in-fusion proteins in E. coli. The expressed proteins were used to feed Ostrinia furnacalis, the Asian corn borer (ACB). Our results demonstrated that the recombinant proteins of pathogenesis-related protein 1 (PR1) and thioredoxin M-type, chloroplastic precursor (TRXM) showed the significant inhibition on the development of larvae and pupae.

Conclusions

We found MeJA could not only induce plant defense mechanisms to insects, it also enhanced toxic protein production that potentially can be used for bio-control of ACB.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1363-1) contains supplementary material, which is available to authorized users.     

Comparative proteomic analysis reveals the roots response to low root-zone temperature in <Emphasis Type="Italic">Malus baccata</Emphasis>

Soil temperature is known to affect plant growth and productivity. In this study we found that low root-zone temperature (LRT) inhibited the growth of apple (Malus baccata Borkh.) seedlings. To elucidate the molecular mechanism of LRT response, we performed comparative proteome analysis of the apple roots under LRT for 6 days. Total proteins of roots were extracted and separated by two-dimensional gel electrophoresis (2-DE) and 29 differentially accumulated proteins were successfully identified by MALDI-TOF/TOF mass spectrometry. They were involved in protein transport/processing/degradation (21%), glycometabolism (20%), response to stress (14%), oxidoreductase activity (14%), protein binding (7%), RNA metabolism (7%), amino acid biosynthesis (3%) and others (14%). The results revealed that LRT inhibited glycometabolism and RNA metabolism. The up-regulated proteins which were associated with oxidoreductase activity, protein metabolism and defense response, might be involved in protection mechanisms against LRT stress in the apple seedlings. Subsequently, 8 proteins were selected for the mRNA quantification analysis, and we found 6 of them were consistently regulated between protein and mRNA levels. In addition, the enzyme activities in ascorbate–glutathione (AsA–GSH) cycle were determined, and APX activity was increased and GR activity was decreased under LRT, in consistent with the protein levels. This study provides new insights into the molecular mechanisms of M. baccata in responding to LRT.     

Proteomic profiling of proteins associated with the rejuvenation of Sequoia sempervirens (D. Don) Endl

Background

Hepatitis B virus (HBV) is a major cause of liver infection in human. Because of the lack of an appropriate cell culture system for supporting HBV infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. Duck heptatitis B virus (DHBV) can naturally infect primary duck hepatocytes (PDHs) that provide valuable model systems for studying hepadnavirus infection in vitro. In this report, we explored global changes in cellular protein expression in DHBV infected PDHs by two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF/TOF tandem mass spectrometry (MS/MS).

Results

The effects of hepadnavirus infection on hepatocytes were investigated in DHBV infected PDHs by the 2-DE analysis. Proteomic profile of PDHs infected with DHBV were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected PDHs, and 75 differentially expressed protein spots were revealed by 2-DE analysis. Among the selected protein spots, 51 spots were identified corresponding to 42 proteins by MS/MS analysis; most of them were matched to orthologous proteins of Gallus gallus, Anas platyrhynchos or other avian species, including alpha-enolase, lamin A, aconitase 2, cofilin-2 and annexin A2, etc. The down-regulated expression of beta-actin and annexin A2 was confirmed by Western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed.

Conclusions

Differentially expressed proteins of DHBV infected PDHs revealed by 2-DE, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis.     

Salinity induced changes in the chloroplast proteome of the aquatic pteridophyte <Emphasis Type="Italic">Azolla microphylla</Emphasis>

The growth of the nitrogen fixing aquatic pteridophyte Azolla microphylla is severely affected by salinity. Salinity exposure (0.5%) resulted in significant reduction in chlorophyll a and b content, altered chl a/b ratio and photosynthetic efficiency (Fv/Fm). Chloroplasts maintain photosynthesis but are highly sensitive to salinity stress. Chloroplast proteins extracted from A. microphylla was separated by two-dimensional electrophoresis (2DE) and approximately 200 proteins were observed on each gel. Forty two differentially expressed protein spots were detected and out of this 17 could be identified through MALDI-TOF-MS/MS analysis. Out of the 17 identified proteins, 15 were found to be down regulated and 2 proteins were up regulated. Most of the down regulated proteins were associated with Calvin cycle, ATP synthesis, oxygen evolution, photosystem I and ROS scavenging. The results show changes in proteome dynamics of the chloroplasts of A. microphylla and such changes may lead to reduction in growth and metabolism. The primary target of salinity in A. microphylla is photosynthesis and the changes in the proteome dynamics of the chloroplasts lead to reduced growth.     

Expression and characteristics of rice xylanase inhibitor OsXIP,a member of a new class of antifungal proteins

It has been hypothesized that xylanase inhibitors play important roles in plant defense against microbial pathogens. Currently, there is little information available about xylanase inhibitor OsXIP in rice and its gene expression. We cloned a xylanase inhibitor gene OsXIP from rice (Oryza sativa L. cv. Nipponbare) genomic DNA. To determine the function of OsXIP, we generated OsXIP-overexpressing transgenic rice plants. The transgenic plants had significantly higher OsXIP expression and showed enhanced defense response to Magnaporthe oryzae compared to the wild-type plants. The results also showed that the increased OsXIP expression was accompanied by the up-regulation of pathogenesisrelated genes. To clarify the OsXIP expression pattern, a ProOsXIP::GUS vector was constructed and transgenic plants were obtained. GUS staining results revealed that OsXIP showed organ-specific expressions in rice plants. OsXIP was primarily expressed in the roots and in the veins, but it was weakly expressed in the leaves. Analyses of the OsXIP expression in response to biotic and abiotic stresses indicated that it was drastically induced by biotic stresses and methyl jasmonate treatment. OsXIP, a member of a new class of antifungal proteins, may function as a barrier that prevents the cell wall degradation by xylanases excreted by fungal pathogens. The OsXIP was found to be a stressresponsive gene and it could take part in plant defense via a JA-mediated signaling pathway.     

Physiological and proteomic analysis of mycorrhizal <Emphasis Type="Italic">Pinus massoniana</Emphasis> inoculated with <Emphasis Type="Italic">Lactarius insulsus</Emphasis> under drought stress

This study aimed to investigate physiological and protein expression alterations of mycorrhizal Pinus massoniana Lamb. inoculated with Lactarius insulsus in response to drought stress. The P. massoniana seedlings were inoculated with L. insulsus (Li group) and ectomycorrhized fungal-free filtrate (control, CK group), respectively. After two and a half years, all the plants were exposed to a simulate drought condition without water for 21 days. The soil relative water content (SRWC), wilting degree (WD) and wilting rate (WR) of the plants were measured and root proteome was analyzed based on two-dimensional gel electrophoresis (2-DE), respectively at four time points as 0, 7, 14 and 21 days during the whole drought period. Finally, the electrospray ionization mass spectrometry (ESI-MS) was used to identify the differentially expressed proteins (DEPs) between Li and CK groups. The SRWC was higher, while WR and WD were lower in Li group, compared with that in CK group. Based on 2-DE and ESI-MS, 22 DEPs were identified between Li and CK groups during drought stress. Among them, four proteins had the annotated information in relevant databases, including 1,4-benzoquinone reductase, PSCHI4, ribosomal protein L16 (RPL16) and AINTEGUMENTA-like (AIL) protein. Mycorrhizal P. massoniana inoculated with L. insulsus achieved an enhanced drought resistance as compared to the non-mycorrhizal, and the altered protein expressions such as 1,4-benzoquinone reductase, PSCHI4, RPL16, and AIL might contribute to the improved resistance under drought stress.     

Comparative proteomic analysis of <Emphasis Type="Italic">Phalaenopsis</Emphasis> leaves in the vegetative and flowering phase

Phalaenopsis, an epiphytic crassulacean acid metabolism (CAM) plant, requires moderate variations of day/night temperatures for flowering. In this study, changes in chlorophyll content, chlorophyll fluorescence, sugar components, titratable acidity and soluble protein content in Phalaenopsis leaves during flowering were observed. Comparative proteomic analysis of Phalaenopsis leaves in the vegetative and flowering phase was performed for the first time using iTRAQ (isobaric tags for relative and absolute quantification). A total of 126 proteins were differentially expressed in Phalaenopsis leaves. Analysis of potential functions revealed that the major categories of predicted function of the up-regulated proteins were protein destination (27 %), photosynthesis (15.9 %), primary metabolism (14.3 %) and defense (12.7 %) in the flowering phase, while the major categories of predicted function of the down-regulated proteins were protein destination (33.3 %), primary metabolism (20.6 %), transportation (14.3 %) and signal transduction (11.1 %). Proteome profile analysis indicated that the proteome changes were consistent with changes in sugar and protein metabolites. Some novel proteins were differentially expressed, most of which were identified as signaling proteins, including 14-3-3 proteins, fibrillin, rapid alkalinization factors (RALF), the Ras-related protein RABB1c, calreticulin and calmodulin. Histone, importin alpha, multidrug resistance proteins and the ABC transporters were also differentially expressed. These results provide insights into the mechanisms that regulate flowering in complex flowering plants.     

Identification of inducible proteins in the phenanthrene degrader <Emphasis Type="Italic">Sphingobium chungbukense</Emphasis> DJ77 by 2-dimentional electrophoresis and liquid chromatography/tandem mass spectrometry

Sphingomonas chungbukensis DJ77 is a novel aromatic hydrocarbon-degrading bacterium capable of growing on phenanthrene as its sole source of carbon and energy. In this study, the protein expression profiles of S. chungbukensis DJ77 grown in the presence of phenanthrene were investigated by using two-dimensional gel electrophoresis (2-DE). Among 1000 protein spots visualized by 2-DE, the four proteins (i.e. 4-oxalocrotonate decarboxylase, 2-hydroxy-6-oxo-phenylhexa-2,4-dienoate hydrolase, glutathione S-transferase, and 2,3-dihydroxybiphenyl 1,2-dioxygenase) showing the significant upregulation by phenanthrene were identified by reversed-phase liquid chromatography–tandem mass spectrometry. Evidently, these proteins were involved in the metabolism of aromatic hydrocarbons. This can explain why S. chungbukensis DJ77 shows a significantly higher rate of phenanthrene consumption during the degradation process. The present analysis of proteomic responses and the detailed analysis results will be quite helpful to better understand the global physiology of S. chungbukensis DJ77, as proteome databases for various aromatic hydrocarbon-degrading strains have already been established.