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Decker, Daniel
Publications (10 of 11) Show all publications
Decker, D. & Kleczkowski, L. A. (2019). UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions. Frontiers in Plant Science, 9, Article ID 1822.
Open this publication in new window or tab >>UDP-Sugar Producing Pyrophosphorylases: Distinct and Essential Enzymes With Overlapping Substrate Specificities, Providing de novo Precursors for Glycosylation Reactions
2019 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 9, article id 1822Article, review/survey (Refereed) Published
Abstract [en]

Nucleotide sugars are the key precursors for all glycosylation reactions and are required both for oligo- and polysaccharides synthesis and protein and lipid glycosylation. Among all nucleotide sugars, UDP-sugars are the most important precursors for biomass production in nature (e.g., synthesis of cellulose, hemicellulose, and pectins for cell wall production). Several recent studies have already suggested a potential role for UDP-Glc in plant growth and development, and UDP-Glc has also been suggested as a signaling molecule, in addition to its precursor function. In this review, we will cover primary mechanisms of formation of UDP-sugars, by focusing on UDP-sugar metabolizing pyrophosphorylases. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase), and UDP-N-acetyl glucosamine pyrophosphorylase (UAGPase), which can be distinguished both by their amino acid sequences and by differences in substrate specificity. Substrate specificities of these enzymes are discussed, along with structure-function relationships, based on their crystal structures and homology modeling. Earlier studies with transgenic plants have revealed that each of the pyrophosphorylases is essential for plant survival, and their loss or a decrease in activity results in reproductive impairment. This constitutes a problem when studying exact in vivo roles of the enzymes using classical reverse genetics approaches. Thus, strategies involving the use of specific inhibitors (reverse chemical genetics) are also discussed. Further characterization of the properties/roles of pyrophosphorylases should address fundamental questions dealing with mechanisms and control of carbohydrate synthesis and may allow to identify targets for manipulation of biomass production in plants.

Place, publisher, year, edition, pages
Frontiers Media S.A., 2019
Keywords
carbohydrate biosynthesis, chemical genetics, nucleotide sugar synthesis, enzyme substrate specificity, UDP-glucose pyrophosphorylase, UDP-N-acetylglucosamine pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-155633 (URN)10.3389/fpls.2018.01822 (DOI)000454879300001 ()
Funder
Swedish Research CouncilThe Kempe FoundationsLars Hierta Memorial Foundation
Available from: 2019-01-25 Created: 2019-01-25 Last updated: 2019-01-25Bibliographically approved
Decker, D., Öberg, C. & Kleczkowski, L. A. (2018). The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases. Plant Signalling & Behavior, 13(8), Article ID e1507406.
Open this publication in new window or tab >>The structure-activity relationship of the salicylimide derived inhibitors of UDP-sugar producing pyrophosphorylases
2018 (English)In: Plant Signalling & Behavior, ISSN 1559-2316, E-ISSN 1559-2324, Vol. 13, no 8, article id e1507406Article in journal (Refereed) Published
Abstract [en]

UDP-sugars are key precursors for biomass production in nature (synthesis of cellulose, hemicellulose, etc.). They are produced de novo by distinct UDP-sugar producing pyrophosphorylases. Studies on the roles of these enzymes using genetic knockouts were hampered by sterility of the mutants and by functional-complementation from related enzyme(s), hindering clear interpretation of the results. In an attempt to override these difficulties, we turned to the reverse chemical genetics approaches to identify compounds which interfere with the activity of those enzymes in vivo. Hit expansion on one of such compounds, a salicylimide derivative, allowed us to identify several inhibitors with a range of activities. The present study provides a structure-activity relationship for these compounds.

Place, publisher, year, edition, pages
Taylor & Francis, 2018
Keywords
Enzyme inhibition, reverse chemical genetics, UDP-N-acetyl glucosamine pyrophosphorylase, UDP- ucose pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Biochemistry and Molecular Biology Botany
Identifiers
urn:nbn:se:umu:diva-152288 (URN)10.1080/15592324.2018.1507406 (DOI)000444498100025 ()30125142 (PubMedID)
Available from: 2018-10-01 Created: 2018-10-01 Last updated: 2018-10-01Bibliographically approved
Decker, D., Öberg, C. & Kleczkowski, L. A. (2017). Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies. The Plant Journal, 90(6), 1093-1107
Open this publication in new window or tab >>Identification and characterization of inhibitors of UDP-glucose and UDP-sugar pyrophosphorylases for in vivo studies
2017 (English)In: The Plant Journal, ISSN 0960-7412, E-ISSN 1365-313X, Vol. 90, no 6, p. 1093-1107Article in journal (Other academic) Published
Abstract [en]

UDP-sugars serve as ultimate precursors in hundreds of glycosylation reactions (e.g. for protein and lipid glycosylation, synthesis of sucrose, cell wall polysaccharides, etc.), underlying an important role of UDP-sugar-producing enzymes in cellular metabolism. However, genetic studies on mechanisms of UDP-sugar formation were frequently hampered by reproductive impairment of the resulting mutants, making it difficult to assess an in vivo role of a given enzyme. Here, a chemical library containing 17 500 compounds was separately screened against purified UDP-glucose pyrophosphorylase (UGPase) and UDP-sugar pyrophosphorylase (USPase), both enzymes representing the primary mechanisms of UDP-sugar formation. Several compounds have been identified which, at 50 μm, exerted at least 50% inhibition of the pyrophosphorylase activity. In all cases, both UGPase and USPase activities were inhibited, probably reflecting common structural features of active sites of these enzymes. One of these compounds (cmp #6), a salicylamide derivative, was found as effective inhibitor of Arabidopsis pollen germination and Arabidopsis cell culture growth. Hit optimization on cmp #6 yielded two analogs (cmp #6D and cmp #6D2), which acted as uncompetitive inhibitors against both UGPase and USPase, and were strong inhibitors in the pollen test, with apparent inhibition constants of less than 1 μm. Their effects on pollen germination were relieved by addition of UDP-glucose and UDP-galactose, suggesting that the inhibitors targeted UDP-sugar formation. The results suggest that cmp #6 and its analogs may represent useful tools to study in vivo roles of the pyrophosphorylases, helping to overcome the limitations of genetic approaches.

Place, publisher, year, edition, pages
John Wiley & Sons, 2017
Keywords
chemical library screening, inhibitors, UDP-sugar synthesis, pyrophosphorylases, pollen germination, Arabidopsis cell culture, enzyme kinetics, Arabidopsis thaliana, reverse chemical genetics
National Category
Biochemistry and Molecular Biology Pharmaceutical Sciences Plant Biotechnology
Identifiers
urn:nbn:se:umu:diva-134159 (URN)10.1111/tpj.13531 (DOI)000403881500006 ()28273406 (PubMedID)
Note

Originally included in thesis in manuscript form.

Available from: 2017-04-27 Created: 2017-04-27 Last updated: 2018-06-25Bibliographically approved
Decker, D. & Kleczkowski, L. (2017). Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases. Frontiers in Plant Science, 8, Article ID 1610.
Open this publication in new window or tab >>Substrate Specificity and Inhibitor Sensitivity of Plant UDP-Sugar Producing Pyrophosphorylases
2017 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 8, article id 1610Article in journal (Refereed) Published
Abstract [en]

UDP-sugars are essential precursors for glycosylation reactions producing cell wall polysaccharides, sucrose, glycoproteins, glycolipids, etc. Primary mechanisms of UDP sugar formation involve the action of at least three distinct pyrophosphorylases using UTP and sugar-1-P as substrates. Here, substrate specificities of barley and Arabidopsis (two isozymes) UDP-glucose pyrophosphorylases (UGPase), Arabidopsis UDP-sugar pyrophosphorylase (USPase) and Arabidopsis UDP-N-acetyl glucosamine pyrophosphorylase2 (UAGPase2) were investigated using a range of sugar-1-phosphates and nucleoside-triphosphates as substrates. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Fru-1-P and Fru-2-P (Km values over 10 mM). Contrary to an earlier report, their activity with Gal-1-P was extremely low. USPase reacted with a range of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P (K-m of 1.3 mM), beta-L-Ara-1-P and alpha-D-Fuc-1-P (K-m of 3.4 mM), but not beta-L-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P (K-m of 1 mM) and, to some extent, D-Glc-1-P (Km of 3.2 mM). Generally, different conformations/substituents at C2, C4, and C5 of the pyranose ring of a sugar were crucial determinants of substrate specificity of a given pyrophosphorylase. Homology models of UDP-sugar binding to UGPase, USPase and UAGPase2 revealed more common amino acids for UDP binding than for sugar binding, reflecting differences in substrate specificity of these proteins. UAGPase2 was inhibited by a salicylate derivative that was earlier shown to affect UGPase and USPase activities, consistent with a common structural architecture of the three pyrophosphorylases. The results are discussed with respect to the role of the pyrophosphorylases in sugar activation for glycosylated end-products.

Place, publisher, year, edition, pages
FRONTIERS MEDIA SA, 2017
Keywords
enzyme structure-function analyses, enzyme substrate specificity, nucleotide sugar synthesis, UDP- uctose, UDP-fucose, UDP-N-acetyl glucosamine pyrophosphorylase, UDP-sugar pyrophosphorylase
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-140459 (URN)10.3389/fpls.2017.01610 (DOI)000411190900001 ()
Available from: 2017-10-26 Created: 2017-10-26 Last updated: 2018-06-09Bibliographically approved
Decker, D. (2017). UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions. (Doctoral dissertation). Umeå: Umeå Universitet
Open this publication in new window or tab >>UDP-sugar metabolizing pyrophosphorylases in plants: formation of precursors for essential glycosylation-reactions
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

UDP-sugar metabolizing pyrophosphorylases provide the primary mechanism for de novo synthesis of UDP-sugars, which can then be used for myriads of glycosyltranferase reactions, producing cell wall carbohydrates, sucrose, glycoproteins and glycolipids, as well as many other glycosylated compounds. The pyrophosphorylases can be divided into three families: UDP-Glc pyrophosphorylase (UGPase), UDP-sugar pyrophosphorylase (USPase) and UDP-N-acety lglucosamine pyrophosphorylase (UAGPase), which can be discriminated both by differences in accepted substrate range and amino acid sequences.

This thesis focuses both on experimental examination (and re-examination) of some enzymatic/ biochemical properties of selected members of the UGPases and USPases and UAGPase families and on the design and implementation of a strategy to study in vivo roles of these pyrophosphorylases using specific inhibitors. In the first part, substrate specificities of members of the Arabidopsis UGPase, USPase and UAGPase families were comprehensively surveyed and kinetically analyzed, with barley UGPase also further studied with regard to itspH dependency, regulation by oligomerization, etc. Whereas all the enzymes preferentially used UTP as nucleotide donor, they differed in their specificity for sugar-1-P. UGPases had high activity with D-Glc-1-P, but could also react with Frc-1-P, whereas USPase reacted with arange of sugar-1-phosphates, including D-Glc-1-P, D-Gal-1-P, D-GalA-1-P, β-L-Ara-1-P and α-D-Fuc-1-P. In contrast, UAGPase2 reacted only with D-GlcNAc-1-P, D-GalNAc-1-P and, to some extent, with D-Glc-1-P. A structure activity relationship was established to connect enzyme activity, the examined sugar-1-phosphates and the three pyrophosphorylases. The UGPase/USPase/UAGPase active sites were subsequently compared in an attempt to identify amino acids which may contribute to the experimentally determined differences in substrate specificities.

The second part of the thesis deals with identification and characterization of inhibitors of the pyrophosphorylases and with studies on in vivo effects of those inhibitors in Arabidopsis-based systems. A novel luminescence-based high-throughput assay system was designed, which allowed for quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The assay was then used to screen a chemical library (which contained 17,500 potential inhibitors) to identify several compounds affecting UGPase and USPase. Hit-optimization on one of the compounds revealed even stronger inhibitors of UGPase and USPase which also strongly inhibited Arabidopsis pollen germination, by disturbing UDP-sugar metabolism. The inhibitors may represent useful tools to study in vivo roles of the pyrophosphorylases, as a complement to previous genetics-based studies.

The thesis also includes two review papers on mechanisms of synthesis of NDP-sugars. The first review covered the characterization of USPase from both prokaryotic and eukaryotic organisms, whereas the second review was a comprehensive survey of NDP-sugar producing enzymes (not only UDP-sugar producing and not only pyrophosphorylases). All these enzymes were discussed with respect to their substrate specificities and structural features (if known) and their proposed in vivo functions.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet, 2017. p. 51
Keywords
Chemical library screening, Cell wall synthesis, Glycosylation, Nucleotide sugars, Oligomerization, Protein structure, Reverse chemical genetics, Sugar activation, UDP-sugar synthesis
National Category
Plant Biotechnology Microbiology Pharmaceutical Chemistry
Research subject
biochemical pharmacology; Biochemistry; biology; Molecular Biology
Identifiers
urn:nbn:se:umu:diva-134087 (URN)978-91-7601-713-5 (ISBN)
Public defence
2017-05-22, KB.E3.01, Lilla Hörsalen, KBC-huset, Umeå Universitet, Umeå, 10:15 (English)
Opponent
Supervisors
Available from: 2017-04-28 Created: 2017-04-26 Last updated: 2018-06-09Bibliographically approved
Kleczkowski, L. A. & Decker, D. (2015). Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants. Journal of Applied Glycoscience, 62(2), 25-36
Open this publication in new window or tab >>Sugar activation for production of nucleotide sugars as aubstrates for glycosyltransferases in plants
2015 (English)In: Journal of Applied Glycoscience, ISSN 1344-7882, Vol. 62, no 2, p. 25-36Article, review/survey (Refereed) Published
Abstract [en]

In order to serve as a glycosyl donor, a sugar or a sugar derivative (e.g. GlcA) needs to be “activated” to a highly energetic state of a nucleotide-sugar. This activation requires the involvement of specific enzymes which produce NDP-sugars (or, in one case, NMP-sugar), using NTP or NDP as substrate. The present review provides concise survey of distinct plant nucleotide-sugar pyrophosphorylases (all using NTP as one of the substrates and differing in sugar specificity) as well as nucleotide-sugar phosphorylases and sucrose synthase (all using NDP as one of substrates). The pyrophosphorylases discussed include UGPase, USPase, UAGPase, AGPase, GMPase (VTC1), and FKGP, whereas phosphorylases include ADP-Glc phosphorylase and GDP-Gal phosphorylase (VTC2/VTC5). We also discuss the activation mechanism of 3-deoxy-D-manno-octulosonic acid (Kdo) by CKS, leading to the formation of a unique NMP-linked sugar (CMP-Kdo).

Place, publisher, year, edition, pages
Tokyo: The Japanese Society of Applied Glycoscience, 2015
Keywords
Cell wall formation, glycosyltransferases, nucleotide sugars, sugar activation
National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:umu:diva-118718 (URN)10.5458/jag.jag.JAG-2015_003 (DOI)
Funder
Swedish Research Council
Available from: 2016-03-30 Created: 2016-03-30 Last updated: 2018-06-07Bibliographically approved
Decker, D., Lindberg, S., Eriksson, J. & Kleczkowski, L. A. (2014). A luminescence-based assay of UDP-sugar producing pyrophosphorylases. Analytical Methods, 6(1), 57-61
Open this publication in new window or tab >>A luminescence-based assay of UDP-sugar producing pyrophosphorylases
2014 (English)In: Analytical Methods, ISSN 1759-9660, E-ISSN 1759-9679, Vol. 6, no 1, p. 57-61Article in journal (Refereed) Published
Abstract [en]

A coupled luminescence assay was applied to monitor pyrophosphate (PPi) production by either purified barley UDP-glucose pyrophosphorylase (UGPase) or purified Leishmania UDP-sugar pyrophosphorylase (USPase). In the assay, the PPi produced by the pyrophosphorylases was converted to ATP by ATP-sulfurylase, and the ATP produced was linked to luminescent light formation through the action of firefly luciferase. The assay allowed for a quantitative measurement of UGPase and USPase activities, down to a pmol per min level. The activities were linear with time and proportional to the amount of the enzyme added, and were neither affected by Pi nor by DTT. For UGPase, K-m values with UTP and Glc-1-P were 0.14 and 0.26 mM, respectively, whereas for USPase the respective K-m values with UTP, Glc-1-P and Gal-1-P were 0.4, 2.9 and 3.9 mM. Possible applications of the luminescence-based assay for not only UDP-sugar producing pyrophosphorylases, but also other types of pyrophosphorylases are discussed.

National Category
Chemical Sciences Botany
Identifiers
urn:nbn:se:umu:diva-85622 (URN)10.1039/C3AY41811a (DOI)000329071500004 ()
Funder
Swedish Research Council
Available from: 2014-02-10 Created: 2014-02-07 Last updated: 2018-06-08Bibliographically approved
Wang, J., Kucukoglu, M., Zhang, L., Chen, P., Decker, D., Nilsson, O., . . . Zheng, B. (2013). The Arabidopsis LRR-RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF-PXY/TDR-WOX4 signaling pathway. BMC Plant Biology, 13, 94
Open this publication in new window or tab >>The Arabidopsis LRR-RLK, PXC1, is a regulator of secondary wall formation correlated with the TDIF-PXY/TDR-WOX4 signaling pathway
Show others...
2013 (English)In: BMC Plant Biology, ISSN 1471-2229, E-ISSN 1471-2229, Vol. 13, p. 94-Article in journal (Refereed) Published
Abstract [en]

Background: Although a number of leucine-rich repeat receptor-like kinase-encoding genes (LRR-RLKs) have been identified in plants, a functional role has been determined for only a few. Recent studies have demonstrated that an LRR-RLK, PXY/TDR, is important for the process of secondary vascular development. Other studies have indicated that PXY/TDR is unlikely to be the sole LRR-RLK involved in this complex process.

Results: In this study, in silico analyses led to the identification of three Arabidopsis LRR-RLK genes (PXY-correlated; PXC1, 2, 3) with transcript accumulation profiles that correlated strongly with several key regulators of vascular development, including PXY/TDR, HB-8, REV, and CLE41. Expression profiling using qPCR and promoter: reporter lines indicated that all three PXC genes are associated with the vasculature. One in particular, PXC1 (At2g36570), had a strong correlation with PXY/TDR. Shifting pxc1 mutants from long-days to short-days showed that loss of the gene led to a dramatic reduction in secondary wall formation in xylem fibers. Transcript analysis of mutants for a variety of secondary cell wall-associated genes, including PXY/TDR indicated that the pathways mediated by PXC1 connect with those mediated by the TDIF-PXY/TDR-WOX4 system.

Conclusions: The data indicate that the LRR-RLK, PXC1 is involved in secondary cell wall formation in xylem fibers. Whereas further study is needed to identify the ligands and mode of action of the PXC1 protein, it is clear from this work that similarly to the shoot apical meristem (SAM), secondary vascular development requires contributions from a number of LRR-RLKs.

Keywords
LRR-RLK, Arabidopsis, Secondary Wall Formation, TDIF-PXY/TDR-WOX4 Signaling
National Category
Botany
Identifiers
urn:nbn:se:umu:diva-79255 (URN)10.1186/1471-2229-13-94 (DOI)000321846300001 ()
Available from: 2013-09-05 Created: 2013-08-13 Last updated: 2018-06-08Bibliographically approved
Decker, D., Meng, M., Gornicka, A., Hofer, A., Wilczynska, M. & Kleczkowski, L. A. (2012). Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase. Phytochemistry, 79, 39-45
Open this publication in new window or tab >>Substrate kinetics and substrate effects on the quaternary structure of barley UDP-glucose pyrophosphorylase
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2012 (English)In: Phytochemistry, ISSN 0031-9422, E-ISSN 1873-3700, Vol. 79, p. 39-45Article in journal (Refereed) Published
Abstract [en]

UDP-Glc pyrophosphorylase (UGPase) is an essential enzyme responsible for production of UDP-Glc, which is used in hundreds of glycosylation reactions involving addition of Glc to a variety of compounds. In this study, barley UGPase was characterized with respect to effects of its substrates on activity and quaternary structure of the protein. Its K(m) values with Glc-1-P and UTP were 0.33 and 0.25 mM, respectively. Besides using Glc-1-P as a substrate, the enzyme had also considerable activity with Gal-1-P; however, the K(m) for Gal-1-P was very high (>10 mM), rendering this reaction unlikely under physiological conditions. UGPase had a relatively broad pH optimum of 6.5-8.5, regardless of the direction of reaction. The enzyme equilibrium constant was 0.4, suggesting slight preference for the Glc-1-P synthesis direction of the reaction. The quaternary structure of the enzyme, studied by Gas-phase Electrophoretic Mobility Macromolecule Analysis (GEMMA), was affected by addition of either single or both substrates in either direction of the reaction, resulting in a shift from UGPase dimers toward monomers, the active form of the enzyme. The substrate-induced changes in quaternary structure of the enzyme may have a regulatory role to assure maximal activity. Kinetics and factors affecting the oligomerization status of UGPase are discussed.

Keywords
Cell wall synthesis, Oligomerization, Protein structure, Sucrose metabolism, Sucrose synthase, Sugar activation, UDP-sugar synthesis
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:umu:diva-58610 (URN)10.1016/j.phytochem.2012.04.002 (DOI)000307031800003 ()22552276 (PubMedID)
Available from: 2012-09-04 Created: 2012-09-04 Last updated: 2018-06-08Bibliographically approved
Kleczkowski, L. A., Decker, D. & Wilczynska, M. (2011). UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation. Plant Physiology, 156(1), 3-10
Open this publication in new window or tab >>UDP-sugar pyrophosphorylase: a new old mechanism for sugar activation
2011 (English)In: Plant Physiology, ISSN 0032-0889, E-ISSN 1532-2548, Vol. 156, no 1, p. 3-10Article in journal (Refereed) Published
Abstract [en]

Recent developments in studies on properties and functions of UDP-sugar pyrophosphorylase (USPase) in metabolism are presented. The protein was characterized from plants and protozoans (Leishmania, Trypanosoma), but apparently it is also present in bacteria. In plants, USPase deficiency leads to male-sterility. USPase produces a variety of UDP-sugars and their analogs required for cell wall biosynthesis as well as for protein and lipid glycosylation, among other functions. Substrate specificity of USPases from different sources is reviewed, and their function/ structure properties are discussed, based on recent crystallization of the protein, with emphasis on common structural blueprint with some other pyrophosphorylases. Some strategies for future research on USPase are discussed.

National Category
Biochemistry and Molecular Biology Botany
Identifiers
urn:nbn:se:umu:diva-41618 (URN)10.1104/pp.111.174706 (DOI)000290207100001 ()21444645 (PubMedID)
Available from: 2011-03-30 Created: 2011-03-30 Last updated: 2018-06-08Bibliographically approved
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