| 1. |
- Arias, Carolina, et al.
(författare)
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Nuclear proteome analysis of Chlamydomonas with response to CO2 limitation
- 2020
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Ingår i: Algal Research. - : Elsevier. - 2211-9264. ; 46
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Tidskriftsartikel (refereegranskat)abstract
- Chlamydomonas reinhardtii is a unicellular green alga that can survive at a wide range of inorganic carbon (Ci) concentrations by regulating the activity of a CO2-concentrating mechanism (CCM) as well as other cellular functions. Under CO2 limited conditions, C. reinhardtii cells display a wide range of adaptive responses including changes in photosynthetic electron transport, mitochondria localization in the cells, the structure of the pyrenoid starch sheath, and primary metabolism. In addition to these functional and structural changes, gene and protein expression are also affected. Several physiological aspects of the CO2 response mechanism have been studied in detail. However, the regulatory components (transcription factors and transcriptional regulators) involved in this process are not fully characterized. Here we report a comprehensive analysis of the C. reinhardtii nuclear proteome using liquid chromatography electrospray ionization spectrometry (LC-ESI-MS). The study aims to identify the proteins that govern adaptation to varying CO2 concentrations in Chlamydomonas. The nuclear proteome of C. reinhardtii cells grown in the air at high (5%) and low (0.04%) CO2 concentrations were analyzed. Using this approach, we identified 1378 proteins in total, including 90 putative transcription factors and 27 transcriptional regulators. Characterization of these new regulatory components could shed light on the molecular mechanisms underlying acclimation to CO2 stress.
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| 2. |
- Bollhöner, Benjamin, et al.
(författare)
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The function of two type II metacaspases in woody tissues of Populus trees
- 2018
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Ingår i: New Phytologist. - : Wiley. - 0028-646X .- 1469-8137. ; 217:4, s. 1551-1565
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Tidskriftsartikel (refereegranskat)abstract
- Metacaspases (MCs) are cysteine proteases that are implicated in programmed cell death of plants. AtMC9 (Arabidopsis thaliana Metacaspase9) is a member of the Arabidopsis MC family that controls the rapid autolysis of the xylem vessel elements, but its downstream targets in xylem remain uncharacterized. PttMC13 and PttMC14 were identified as AtMC9 homologs in hybrid aspen (Populustremulaxtremuloides). A proteomic analysis was conducted in xylem tissues of transgenic hybrid aspen trees which carried either an overexpression or an RNA interference construct for PttMC13 and PttMC14. The proteomic analysis revealed modulation of levels of both previously known targets of metacaspases, such as Tudor staphylococcal nuclease, heat shock proteins and 14-3-3 proteins, as well as novel proteins, such as homologs of the PUTATIVE ASPARTIC PROTEASE3 (PASPA3) and the cysteine protease RD21 by PttMC13 and PttMC14. We identified here the pathways and processes that are modulated by PttMC13 and PttMC14 in xylem tissues. In particular, the results indicate involvement of PttMC13 and/or PttMC14 in downstream proteolytic processes and cell death of xylem elements. This work provides a valuable reference dataset on xylem-specific metacaspase functions for future functional and biochemical analyses.
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| 3. |
- Businge, Edward, et al.
(författare)
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The effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce somatic embryos
- 2013
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Ingår i: Physiologia Plantarum. - : Wiley. - 0031-9317 .- 1399-3054. ; 149, s. 273-285
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Tidskriftsartikel (refereegranskat)abstract
- Somatic embryogenesis (SE) represents a useful experimental system for studying the regulatory mechanisms of embryo development. In this study, the effect of carbohydrates and osmoticum on storage reserve accumulation and germination of Norway spruce [Picea abies (L.) Karst] somatic embryos were investigated. Using time lapse photography, we monitored development from proliferation of proembryogenic masses (PEMs) to maturation of somatic embryos in two P. abies cell lines cultured on two maturation treatments. A combination of sugar assays, metabolic and proteomic analyses were used to quantify storage reserves in the mature somatic embryos. The maturation treatment containing a nonpermeating osmoticum, polyethylene glycol (PEG, 7.5%) and maltose (3%) as the carbohydrate gave significantly high maturation and low germination frequencies of somatic embryos compared to the treatment with only 3% sucrose. Somatic embryos treated with 3% sucrose contained high levels of sucrose, raffinose and late embryogenesis abundant (LEA) proteins. These compounds are known to be involved in the acquisition of desiccation tolerance during seed development and maturation. In addition the sucrose treatment significantly increased the content of starch in the somatic embryos while the maltose and PEG treatment resulted in somatic embryos with a high content of storage proteins. The high levels of sucrose, raffinose and LEA proteins in the embryos treated with 3% sucrose suggest that sucrose may improve the germination of somatic embryos by promoting the acquisition of desiccation tolerance.
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| 4. |
- Bygdell, Joakim
(författare)
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Plant biology through quantitative proteomics
- 2013
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- Over the last decade the field of mass spectrometry based proteomics has advanced from qualitative, analyses leading to publications revolving around lists of identified proteins and peptides, to addressing more biologically relevant issues requiring measurement of the abundance of identified proteins and hence quantitive mass spectrometry. The work described in this thesis addresses problems with quantitive proteomics in plant sciences, particularly complications caused by the complexity of plant proteomes (generated by genomic duplications), which makes mass spectrometry-based based proteomic analyses more difficult than in mammalian species. In order to understand complex biological processes it is vital to analyse the participating molecules with as little bias as possible. Strategies for minimizing and maximizing the acquired information in proteomic investigations of plants are presented in the appended papers and discussed in the thesis.
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| 5. |
- Bygdell, Joakim, et al.
(författare)
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Protein expression in tension wood formation monitored at high tissue resolution in Populus
- 2017
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Ingår i: Journal of Experimental Botany. - : Oxford University Press. - 0022-0957 .- 1460-2431. ; 68:13, s. 3405-3417
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Tidskriftsartikel (refereegranskat)abstract
- Tension wood (TW) is a specialized tissue with contractile properties that is formed by the vascular cambium in response to gravitational stimuli. We quantitatively analysed the proteomes of Populus tremula cambium and its xylem cell derivatives in stems forming normal wood (NW) and TW to reveal the mechanisms underlying TW formation. Phloem-, cambium-, and wood-forming tissues were sampled by tangential cryosectioning and pooled into nine independent samples. The proteomes of TW and NW samples were similar in the phloem and cambium samples, but diverged early during xylogenesis, demonstrating that reprogramming is an integral part of TW formation. For example, 14-3-3, reactive oxygen species, ribosomal and ATPase complex proteins were found to be up-regulated at early stages of xylem differentiation during TW formation. At later stages of xylem differentiation, proteins involved in the biosynthesis of cellulose and enzymes involved in the biosynthesis of rhamnogalacturonan-I, rhamnogalacturonan-II, arabinogalactan-II and fasciclin-like arabinogalactan proteins were up-regulated in TW. Surprisingly, two isoforms of exostosin family proteins with putative xylan xylosyl transferase function and several lignin biosynthesis proteins were also up-regulated, even though xylan and lignin are known to be less abundant in TW than in NW. These data provided new insight into the processes behind TW formation.
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| 6. |
- Gandla, Madhavi Latha, et al.
(författare)
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Overexpression of vesicle-associated membrane protein PttVAP27-17 as a tool to improve biomass production and the overall saccharification yields in Populus trees
- 2021
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Ingår i: Biotechnology for Biofuels. - : Springer Science and Business Media LLC. - 1754-6834. ; 14:1
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Tidskriftsartikel (refereegranskat)abstract
- Background Bioconversion of wood into bioproducts and biofuels is hindered by the recalcitrance of woody raw material to bioprocesses such as enzymatic saccharification. Targeted modification of the chemical composition of the feedstock can improve saccharification but this gain is often abrogated by concomitant reduction in tree growth. Results In this study, we report on transgenic hybrid aspen (Populus tremula x tremuloides) lines that showed potential to increase biomass production both in the greenhouse and after 5 years of growth in the field. The transgenic lines carried an overexpression construct for Populus tremula x tremuloides vesicle-associated membrane protein (VAMP)-associated protein PttVAP27-17 that was selected from a gene-mining program for novel regulators of wood formation. Analytical-scale enzymatic saccharification without any pretreatment revealed for all greenhouse-grown transgenic lines, compared to the wild type, a 20-44% increase in the glucose yield per dry weight after enzymatic saccharification, even though it was statistically significant only for one line. The glucose yield after enzymatic saccharification with a prior hydrothermal pretreatment step with sulfuric acid was not increased in the greenhouse-grown transgenic trees on a dry-weight basis, but increased by 26-50% when calculated on a whole biomass basis in comparison to the wild-type control. Tendencies to increased glucose yields by up to 24% were present on a whole tree biomass basis after acidic pretreatment and enzymatic saccharification also in the transgenic trees grown for 5 years on the field when compared to the wild-type control. Conclusions The results demonstrate the usefulness of gene-mining programs to identify novel genes with the potential to improve biofuel production in tree biotechnology programs. Furthermore, multi-omic analyses, including transcriptomic, proteomic and metabolomic analyses, performed here provide a toolbox for future studies on the function of VAP27 proteins in plants.
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| 7. |
- Miguel, Sissi, et al.
(författare)
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A GDSL lipase-like from Ipomoea batatas catalyzes efficient production of 3,5-diCQA when expressed in Pichia pastoris
- 2020
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Ingår i: Communications Biology. - : Springer Nature. - 2399-3642. ; 3:1
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Tidskriftsartikel (refereegranskat)abstract
- The synthesis of 3,5-dicaffeoylquinic acid (3,5-DiCQA) has attracted the interest of many researchers for more than 30 years. Recently, enzymes belonging to the BAHD acyltransferase family were shown to mediate its synthesis, albeit with notably low efficiency. In this study, a new enzyme belonging to the GDSL lipase-like family was identified and proven to be able to transform chlorogenic acid (5-O-caffeoylquinic acid, 5-CQA, CGA) in 3,5-DiCQA with a conversion rate of more than 60%. The enzyme has been produced in different expression systems but has only been shown to be active when transiently synthesized in Nicotiana benthamiana or stably expressed in Pichia pastoris. The synthesis of the molecule could be performed in vitro but also by a bioconversion approach beginning from pure 5-CQA or from green coffee bean extract, thereby paving the road for producing it on an industrial scale. Miguel et al. identify a new enzyme belonging to the GDSL lipase-like family that is involved in the final stage of transformation of 5-CQA into 3,5-diCQA. This enzyme is able to realize an efficient transformation by over 60%, making the transformation process a valuable technological tool that can be easily transferred on an industrial scale.
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| 8. |
- Nilsson, Robert, et al.
(författare)
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Proteomics of Plasma Membranes from Poplar Trees Reveals Tissue Distribution of Transporters, Receptors, and Proteins in Cell Wall Formation
- 2010
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Ingår i: Molecular & Cellular Proteomics. - 1535-9484 .- 1535-9476. ; 9:2, s. 368-387
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Tidskriftsartikel (refereegranskat)abstract
- By exploiting the abundant tissues available from Populus trees, 3-4 m high, we have been able to isolate plasma membranes of high purity from leaves, xylem, and cambium/phloem at a time (4 weeks after bud break) when photosynthesis in the leaves and wood formation in the xylem should have reached a steady state. More than 40% of the 956 proteins identified were found in the plasma membranes of all three tissues and may be classified as "housekeeping" proteins, a typical example being P-type H+-ATPases. Among the 213 proteins predicted to be integral membrane proteins, transporters constitute the largest class (41%) followed by receptors (14%) and proteins involved in cell wall and carbohydrate metabolism (8%) and membrane trafficking (8%). ATP-binding cassette transporters (all members of subfamilies B, C, and G) and receptor-like kinases (four subfamilies) were two of the largest protein families found, and the members of these two families showed pronounced tissue distribution. Leaf plasma membranes were characterized by a very high proportion of transporters, constituting almost half of the integral proteins. Proteins involved in cell wall synthesis (such as cellulose and sucrose synthases) and membrane trafficking were most abundant in xylem plasma membranes in agreement with the role of the xylem in wood formation. Twenty-five integral proteins and 83 soluble proteins were exclusively found in xylem plasma membranes, which identifies new candidates associated with cell wall synthesis and wood formation. Among the proteins uniquely found in xylem plasma membranes were most of the enzymes involved in lignin biosynthesis, which suggests that they may exist as a complex linked to the plasma membrane. Molecular & Cellular Proteomics 9:368-387, 2010.
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| 9. |
- Obudulu, Ogonna, et al.
(författare)
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A multi-omics approach reveals function of Secretory Carrier-Associated Membrane Proteins in wood formation of Populus trees
- 2018
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Ingår i: BMC Genomics. - : Springer Publishing Company. - 1471-2164. ; 19
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Tidskriftsartikel (refereegranskat)abstract
- Background: Secretory Carrier-Associated Membrane Proteins (SCAMPs) are highly conserved 32–38 kDa proteins that are involved in membrane trafficking. A systems approach was taken to elucidate function of SCAMPs in wood formation of Populus trees. Phenotypic and multi-omics analyses were performed in woody tissues of transgenic Populus trees carrying an RNAi construct for Populus tremula x tremuloides SCAMP3 (PttSCAMP3;Potri.019G104000).Results: The woody tissues of the transgenic trees displayed increased amounts of both polysaccharides and lignin oligomers, indicating increased deposition of both the carbohydrate and lignin components of the secondary cell walls. This coincided with a tendency towards increased wood density as well as significantly increased thickness of the suberized cork in the transgenic lines. Multivariate OnPLS (orthogonal projections to latent structures) modeling of five different omics datasets (the transcriptome, proteome, GC-MS metabolome, LC-MS metabolome and pyrolysis-GC/MS metabolome) collected from the secondary xylem tissues of the stem revealed systemic variation in the different variables in the transgenic lines, including changes that correlated with the changes in the secondary cell wall composition. The OnPLS model also identified a rather large number of proteins that were more abundant in the transgenic lines than in the wild type. Several of these were related to secretion and/or endocytosis as well as both primary and secondary cell wall biosynthesis.Conclusions: Populus SCAMP proteins were shown to influence accumulation of secondary cell wall components, including polysaccharides and phenolic compounds, in the woody tissues of Populus tree stems. Our multi-omics analyses combined with the OnPLS modelling suggest that this function is mediated by changes in membrane trafficking to fine-tune the abundance of cell wall precursors and/or proteins involved in cell wall biosynthesis and transport. The data provides a multi-level source of information for future studies on the function of the SCAMP proteins in plant stem tissues.
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| 10. |
- Obudulu, Ogonna, et al.
(författare)
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Quantitative proteomics reveals protein profiles underlying major transitions in aspen wood development
- 2016
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 17
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Tidskriftsartikel (refereegranskat)abstract
- Background: Wood development is of outstanding interest both to basic research and industry due to the associated cellulose and lignin biomass production. Efforts to elucidate wood formation (which is essential for numerous aspects of both pure and applied plant science) have been made using transcriptomic analyses and/or low-resolution sampling. However, transcriptomic data do not correlate perfectly with levels of expressed proteins due to effects of post-translational modifications and variations in turnover rates. In addition, high-resolution analysis is needed to characterize key transitions. In order to identify protein profiles across the developmental region of wood formation, an in-depth and tissue specific sampling was performed. Results: We examined protein profiles, using an ultra-performance liquid chromatography/quadrupole time of flight mass spectrometry system, in high-resolution tangential sections spanning all wood development zones in Populus tremula from undifferentiated cambium to mature phloem and xylem, including cell expansion and cell death zones. In total, we analyzed 482 sections, 20-160 mu m thick, from four 47-year-old trees growing wild in Sweden. We obtained high quality expression profiles for 3,082 proteins exhibiting consistency across the replicates, considering that the trees were growing in an uncontrolled environment. A combination of Principal Component Analysis (PCA), Orthogonal Projections to Latent Structures (OPLS) modeling and an enhanced stepwise linear modeling approach identified several major transitions in global protein expression profiles, pinpointing (for example) locations of the cambial division leading to phloem and xylem cells, and secondary cell wall formation zones. We also identified key proteins and associated pathways underlying these developmental landmarks. For example, many of the lignocellulosic related proteins were upregulated in the expansion to the early developmental xylem zone, and for laccases with a rapid decrease in early xylem zones. We observed upregulation of two forms of xylem cysteine protease (Potri.002G005700.1 and Potri.005G256000.2; Pt-XCP2.1) in early xylem and their downregulation in late maturing xylem. Our data also show that Pt-KOR1.3 (Potri.003G151700.2) exhibits an expression pattern that supports the hypothesis put forward in previous studies that this is a key xyloglucanase involved in cellulose biosynthesis in primary cell walls and reduction of cellulose crystallinity in secondary walls. Conclusion: Our novel multivariate approach highlights important processes and provides confirmatory insights into the molecular foundations of wood development.
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| 11. |
- Piltti, Juha, 1982-, et al.
(författare)
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Effects of long-term hypoxia in human chondrosarcoma cells
- 2018
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Ingår i: Journal of Cellular Biochemistry. - : John Wiley & Sons. - 0730-2312 .- 1097-4644. ; 119:2, s. 2320-2332
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Tidskriftsartikel (refereegranskat)abstract
- The cell-based therapies could be potential methods to treat damaged cartilage tissues. Instead of native hyaline cartilage, the current therapies generate mainly weaker fibrocartilage-type of repair tissue. A correct microenvironment influences the cellular phenotype, and together with external factors it can be used, e.g., to aid the differentiation of mesenchymal stem cells to defined types of differentiated adult cells. In this study, we investigated the effect of long-term exposure to 5% low oxygen atmosphere on human chondrosarcoma HCS-2/8 cells. This atmosphere is close to normal oxygen tension of cartilage tissue. The proteome was analyzed with label-free mass spectrometric method and further bioinformatic analysis. The qRT-PCR method was used to gene expression analysis, and ELISA and dimethylmethylene blue assays for type II collagen and sulfated glycosaminoglycan measurements. The hypoxic atmosphere did not influence cell proliferation, but enhanced slightly ACAN and COL2A1 gene expression. Proteomic screening revealed a number of hypoxia-induced protein level responses. Increased ones included NDUFA4L2, P4HA1, NDRG1, MIF, LDHA, PYGL, while TXNRD1, BAG2, TXN2, AQSTM1, TNFRSF1B and EPHX1 decreased during the long-term hypoxia. Also a number of proteins previously not related to hypoxia changed during the treatment. Of those S100P, RPSS26, NDUFB11, CDV3 and TUBB8 had elevated levels, while ALCAM, HLA-B, EIF1, and ACOT9 were lower in the hypoxia samples. In conclusion, low oxygen condition causes changes in the cellular amounts of several proteins.
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| 12. |
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| 13. |
- Piltti, Juha, 1982-, et al.
(författare)
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Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and change the S100 protein profile in human chondrosarcoma cells
- 2017
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Ingår i: Scientific Reports. - : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Articular chondrocytes are slowly dividing cells that tend to lose their cell type-specific phenotype and ability to produce structurally and functionally correct cartilage tissue when cultured. Thus, culture conditions, which enhance the maintenance of chondrocyte phenotype would be very useful for cartilage research. Here we show that Rho-kinase inhibition by Y-27632 under hypoxic conditions efficiently maintains and even enhances chondrocyte-specific extracellular matrix production by chondrocytic cells. The effects of long-term Y-27632 exposure to human chondrosarcoma 2/8 cell phenotype maintenance and extracellular matrix production were studied at normoxia and at a 5% low oxygen atmosphere. Y-27632 treatment at normoxia induced ACAN and COL2A1 gene up-regulation and a minor increase of sulfated glycosaminoglycans (sGAGs), while type II collagen expression was not significantly up-regulated. A further increase in expression of ACAN and COL2A1 was achieved with Y-27632 treatment and hypoxia. The production of sGAGs increased by 65.8%, and ELISA analysis revealed a 6-fold up-regulation of type II collagen. Y-27632 also induced the up-regulation of S100-A1 and S100-B proteins and modified the expression of several other S100 protein family members, such as S100-A4, S100-A6, S100-A13 and S100-A16. The up-regulation of S100-A1 and S100-B proteins is suggested to enhance the chondrocytic phenotype of these cells.
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| 14. |
- Piltti, Juha, et al.
(författare)
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Rho-kinase inhibitor Y-27632 and hypoxia synergistically enhance chondrocytic phenotype and modify S100 protein profiles in human chondrosarcoma cells
- 2017
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Ingår i: Scientific Reports. - London : Nature Publishing Group. - 2045-2322. ; 7
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Tidskriftsartikel (refereegranskat)abstract
- Articular chondrocytes are slowly dividing cells that tend to lose their cell type-specific phenotype and ability to produce structurally and functionally correct cartilage tissue when cultured. Thus, culture conditions, which enhance the maintenance of chondrocyte phenotype would be very useful for cartilage research. Here we show that Rho-kinase inhibition by Y-27632 under hypoxic conditions efficiently maintains and even enhances chondrocyte-specific extracellular matrix production by chondrocytic cells. The effects of long-term Y-27632 exposure to human chondrosarcoma 2/8 cell phenotype maintenance and extracellular matrix production were studied at normoxia and at a 5% low oxygen atmosphere. Y-27632 treatment at normoxia induced ACAN and COL2A1 gene up-regulation and a minor increase of sulfated glycosaminoglycans (sGAGs), while type II collagen expression was not significantly up-regulated. A further increase in expression of ACAN and COL2A1 was achieved with Y-27632 treatment and hypoxia. The production of sGAGs increased by 65.8%, and ELISA analysis revealed a 6-fold up-regulation of type II collagen. Y-27632 also induced the up-regulation of S100-A1 and S100-B proteins and modified the expression of several other S100 protein family members, such as S100-A4, S100-A6, S100-A13 and S100-A16. The up-regulation of S100-A1 and S100-B proteins is suggested to enhance the chondrocytic phenotype of these cells.
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| 15. |
- Shaikhali, Jehad, et al.
(författare)
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Biochemical and redox characterization of the mediator complex and its associated transcription factor GeBPL, a GLABROUS1 enhancer binding protein
- 2015
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 468:3, s. 385-400
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Tidskriftsartikel (refereegranskat)abstract
- The eukaryotic mediator integrates regulatory signals from promoter-bound transcription factors (TFs) and transmits them to RNA polymerase II (Pol II) machinery. Although redox signalling is important in adjusting plant metabolism and development, nothing is known about a possible redox regulation of mediator. In the present study, using pull-down and yeast two-hybrid assays, we demonstrate the association of mediator (MED) subunits MED10a, MED28 and MED32 with the GLABROUS1 (GL1) enhancer-binding protein-like (GeBPL), a plant-specific TF that binds a promoter containing cryptochrome 1 response element 2 (CryR2) element. All the corresponding recombinant proteins form various types of covalent oligomers linked by intermolecular disulfide bonds that are reduced in vitro by the thioredoxin (TRX) and/or glutathione/glutaredoxin (GRX) systems. The presence of recombinant MED10a, MED28 and MED32 subunits or changes of its redox state affect the DNA-binding capacity of GeBPL suggesting that redox-driven conformational changes might modulate its activity. Overall, these results advance our understanding of how redox signalling affects transcription and identify mediator as a novel actor in redox signalling pathways, relaying or integrating redox changes in combinationwith specific TFs as GeBPL.
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| 16. |
- Srivastava, Vaibhav, et al.
(författare)
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OnPLS integration of transcriptomic, proteomic and metabolomic data shows multi-level oxidative stress responses in the cambium of transgenic hipI- superoxide dismutase Populus plants
- 2013
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Ingår i: BMC Genomics. - : BioMed Central. - 1471-2164. ; 14
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Tidskriftsartikel (refereegranskat)abstract
- BACKGROUND: Reactive oxygen species (ROS) are involved in the regulation of diverse physiological processes in plants, including various biotic and abiotic stress responses. Thus, oxidative stress tolerance mechanisms in plants are complex, and diverse responses at multiple levels need to be characterized in order to understand them. Here we present system responses to oxidative stress in Populus by integrating data from analyses of the cambial region of wild-type controls and plants expressing high-isoelectric-point superoxide dismutase (hipI-SOD) transcripts in antisense orientation showing a higher production of superoxide. The cambium, a thin cell layer, generates cells that differentiate to form either phloem or xylem and is hypothesized to be a major reason for phenotypic perturbations in the transgenic plants. Data from multiple platforms including transcriptomics (microarray analysis), proteomics (UPLC/QTOF-MS), and metabolomics (GC-TOF/MS, UPLC/MS, and UHPLC-LTQ/MS) were integrated using the most recent development of orthogonal projections to latent structures called OnPLS. OnPLS is a symmetrical multi-block method that does not depend on the order of analysis when more than two blocks are analysed. Significantly affected genes, proteins and metabolites were then visualized in painted pathway diagrams.RESULTS: The main categories that appear to be significantly influenced in the transgenic plants were pathways related to redox regulation, carbon metabolism and protein degradation, e.g. the glycolysis and pentose phosphate pathways (PPP). The results provide system-level information on ROS metabolism and responses to oxidative stress, and indicate that some initial responses to oxidative stress may share common pathways.CONCLUSION: The proposed data evaluation strategy shows an efficient way of compiling complex, multi-platform datasets to obtain significant biological information.
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| 17. |
- Vaisanen, E., et al.
(författare)
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Hunting monolignol transporters: membrane proteomics and biochemical transport assays with membrane vesicles of Norway spruce
- 2020
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Ingår i: Journal of Experimental Botany. - : Oxford University Press (OUP). - 0022-0957 .- 1460-2431. ; 71:20, s. 6379-6395
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Tidskriftsartikel (refereegranskat)abstract
- Both the mechanisms of monolignol transport and the transported form of monolignols in developing xylem of trees are unknown. We tested the hypothesis of an active, plasma membrane-localized transport of monolignol monomers, dimers, and/or glucosidic forms with membrane vesicles prepared from developing xylem and lignin-forming tissuecultured cells of Norway spruce (Picea abies L. Karst.), as well as from control materials, comprising non-lignifying Norway spruce phloem and tobacco (Nicotiana tabacum L.) BY-2 cells. Xylem and BY-2 vesicles transported both coniferin and p-coumaryl alcohol glucoside, but inhibitor assays suggested that this transport was through the tonoplast. Membrane vesicles prepared from lignin-forming spruce cells showed coniferin transport, but the K-m value for coniferin was much higher than those of xylem and BY-2 cells. Liquid chromatography-mass spectrometry analysis of membrane proteins isolated from spruce developing xylem, phloem, and lignin-forming cultured cells revealed multiple transporters. These were compared with a transporter gene set obtained by a correlation analysis with a selected set of spruce monolignol biosynthesis genes. Biochemical membrane vesicle assays showed no support for ABC-transporter-mediated monolignol transport but point to a role for secondary active transporters (such as MFS or MATE transporters). In contrast, proteomic and co-expression analyses suggested a role for ABC transporters and MFS transporters.
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| 18. |
- Xu, Hao, et al.
(författare)
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Yeast Elongator protein Elp1p does not undergo proteolytic processing in exponentially growing cells
- 2015
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Ingår i: MicrobiologyOpen. - : Wiley. - 2045-8827. ; 4:6, s. 867-878
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Tidskriftsartikel (refereegranskat)abstract
- In eukaryotic organisms, Elongator is a six-subunit protein complex required for the formation of 5-carbamoylmethyl (ncm5) and 5-methylcarboxymethyl (mcm5) side chains on uridines present at the wobble position (U34) of tRNA. The open reading frame encoding the largest Elongator subunit Elp1p has two in-frame 5′ AUG methionine codons separated by 48 nucleotides. Here, we show that the second AUG acts as the start codon of translation. Furthermore, Elp1p was previously shown to exist in two major forms of which one was generated by proteolysis of full-length Elp1p and this proteolytic cleavage was suggested to regulate Elongator complex activity. In this study, we found that the vacuolar protease Prb1p was responsible for the cleavage of Elp1p. The cleavage occurs between residues 203 (Lys) and 204 (Ala) as shown by amine reactive Tandem Mass Tag followed by LC-MS/MS (liquid chromatography mass spectrometry) analysis. However, using a modified protein extraction procedure, including trichloroacetic acid, only full-length Elp1p was observed, showing that truncation of Elp1p is an artifact occurring during protein extraction. Consequently, our results indicate that N-terminal truncation of Elp1p is not likely to regulate Elongator complex activity.
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| 19. |
- Zhang, Xueyang, et al.
(författare)
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Cellulose Synthase Stoichiometry in Aspen Differs from Arabidopsis and Norway Spruce
- 2018
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Ingår i: Plant Physiology. - : Oxford University Press (OUP). - 0032-0889 .- 1532-2548. ; 177, s. 1096-1107
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Tidskriftsartikel (refereegranskat)abstract
- Cellulose is synthesized at the plasma membrane by cellulose synthase complexes (CSCs) containing cellulose synthases (CESAs). Genetic analysis and CESA isoform quantification indicate that cellulose in the secondary cell walls of Arabidopsis (Arabidopsis thaliana) is synthesized by isoforms CESA4, CESA7, and CESA8 in equimolar amounts. Here, we used quantitative proteomics to investigate whether the CSC model based on Arabidopsis secondary cell wall CESA stoichiometry can be applied to the angiosperm tree aspen (Populus tremula) and the gymnosperm tree Norway spruce (Picea abies). In the developing xylem of aspen, the secondary cell wall CESA stoichiometry was 3:2:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b, while in Norway spruce, the stoichiometry was 1:1:1, as observed previously in Arabidopsis. Furthermore, in aspen tension wood, the secondary cell wall CESA stoichiometry changed to 8:3:1 for PtCESA8a/b:PtCESA4:PtCESA7a/b. PtCESA8b represented 73% of the total secondary cell wall CESA pool, and quantitative polymerase chain reaction analysis of CESA transcripts in cryosectioned tension wood revealed increased PtCESA8b expression during the formation of the cellulose-enriched gelatinous layer, while the transcripts of PtCESA4, PtCESA7a/b, and PtCESA8a decreased. A wide-angle x-ray scattering analysis showed that the shift in CESA stoichiometry in tension wood coincided with an increase in crystalline cellulose microfibril diameter, suggesting that the CSC CESA composition influences microfibril properties. The aspen CESA stoichiometry results raise the possibility of alternative CSC models and suggest that homomeric PtCESA8b complexes are responsible for cellulose biosynthesis in the gelatinous layer in tension wood.
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