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Sugar-modulated gene expression and cell division in cell culture and seedlings of A. thaliana
Umeå University, Faculty of Science and Technology, Umeå Plant Science Centre (UPSC). Umeå University, Faculty of Science and Technology, Department of Plant Physiology. (Leszek A. Kleczkowski)
2014 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Throughout their life cycle, plants adjust growth in response to their developmental and environmental situation within the limits of their energetic capacities. This capacity is defined by the local sugar availability, which is constantly modulated through synthesis, transport and consumption of sugar. The monitoring of sugar presence is carried out by a complex signalling network in which simple sugars (e.g. glucose, fructose and sucrose) act as metabolic signals for the modulation of physiological processes. However, often it remains unclear whether the regulation is induced by the simple sugars themselves or by their derivatives generated during sugar metabolism. This thesis focuses on the dissection of distinct sugar signals, their generation, perception and impact on the modulation of gene expression and cell division both in cell culture and young seedlings.

Based on a stem-cell-like A. thaliana cell culture, which could be sustained in a hormone-free media, a new biological system, supplied with Xyl as the only carbon source was developed. The performance of a variety of sugar and sugar analogue treatments in this novel system allowed for the identification of sugar-responsive candidate genes, which were specifically regulated by glucose, fructose and sucrose. For several genes (e.g. bZIP63, AT5g22920, TPS9, MGD2 and BT2), this regulation required both sugar transport into the cytosol and metabolisation for the generation of the signal. Furthermore, gene expression analyses in young A. thaliana seedlings indicated the requirement for the catalytic activity of hexokinase 1 in the regulation of bZIP63, Atg22920 and BT2 under conditions of a perturbed carbohydrate balance. These findings have been combined in a proposed model for the transcriptional regulation of bZIP63, AT5g22920, TPS9, MGD2 and BT2, which further proposes a function of those genes in the regulation of cell division.

The optimisation of a protocol for long-term real-time live-cell imaging provided a valuable tool to show that, similar to gene expression, the progression of cell division depended on a sugar-type-specific regulation at the single-cell level; this regulation was most likely caused by prolongation of the interphase. Together with the observation of cell death and growth arrest of the primary root in intact seedlings in response to the glucose analogue 2dog, this led to the conclusion that sugar signals themselves were sufficient to induce cell division. However, the continuation of cell cycle progression and consequently organ growth over long-time required the availability of the energy contained in the sugar.

Place, publisher, year, edition, pages
Umeå: Umeå University , 2014. , 59 p.
Keyword [en]
Arabidopsis thaliana, carbohydrates, cell culture, cell division, gene expression, homeostasis, live cell imaging, sugar-signals, sugar-analogues
National Category
Biochemistry and Molecular Biology Botany Cell Biology
Research subject
Physiological Botany
Identifiers
URN: urn:nbn:se:umu:diva-96580ISBN: 978-91-7601-174-4 (print)OAI: oai:DiVA.org:umu-96580DiVA: diva2:765472
Public defence
2014-12-17, KB3A9; KBC huset, Umeå universitet, Umeå, 10:00 (English)
Opponent
Supervisors
Available from: 2014-11-26 Created: 2014-11-24 Last updated: 2015-04-29Bibliographically approved
List of papers
1. Functional Dissection of Sugar Signals Affecting Gene Expression in Arabidopsis thaliana
Open this publication in new window or tab >>Functional Dissection of Sugar Signals Affecting Gene Expression in Arabidopsis thaliana
2014 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 9, no 6, e100312- p.Article in journal (Refereed) Published
Abstract [en]

Background: Sugars modulate expression of hundreds of genes in plants. Previous studies on sugar signaling, using intact plants or plant tissues, were hampered by tissue heterogeneity, uneven sugar transport and/or inter-conversions of the applied sugars. This, in turn, could obscure the identity of a specific sugar that acts as a signal affecting expression of given gene in a given tissue or cell-type. Methodology/Principal Findings: To bypass those biases, we have developed a novel biological system, based on stem-cell-like Arabidopsis suspension culture. The cells were grown in a hormone-free medium and were sustained on xylose as the only carbon source. Using functional genomics we have identified 290 sugar responsive genes, responding rapidly (within 1 h) and specifically to low concentration (1 mM) of glucose, fructose and/or sucrose. For selected genes, the true nature of the signaling sugar molecules and sites of sugar perception were further clarified using non-metabolizable sugar analogues. Using both transgenic and wild-type A. thaliana seedlings, it was shown that the expression of selected sugar-responsive genes was not restricted to a specific tissue or cell type and responded to photoperiod-related changes in sugar availability. This suggested that sugar-responsiveness of genes identified in the cell culture system was not biased toward heterotrophic background and resembled that in whole plants. Conclusions: Altogether, our research strategy, using a combination of cell culture and whole plants, has provided an unequivocal evidence for the identity of sugar-responsive genes and the identity of the sugar signaling molecules, independently from their inter-conversions or use for energy metabolism.

National Category
Genetics
Identifiers
urn:nbn:se:umu:diva-91274 (URN)10.1371/journal.pone.0100312 (DOI)000338276300062 ()
Available from: 2014-07-28 Created: 2014-07-28 Last updated: 2017-12-05Bibliographically approved
2. Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis
Open this publication in new window or tab >>Hexokinase 1 is required for glucose-induced repression of bZIP63, At5g22920, and BT2 in Arabidopsis
2015 (English)In: Frontiers in Plant Science, ISSN 1664-462X, E-ISSN 1664-462X, Vol. 6, 525Article in journal (Refereed) Published
Abstract [en]

Simple sugars, like glucose (Glc) and sucrose (Suc), act as signals to modulate the expression of hundreds of genes in plants. Frequently, however, it remains unclear whether this regulation is induced by the sugars themselves or by their derivatives generated in the course of carbohydrate (CH) metabolism. In the present study, we tested the relevance of different CH metabolism and allocation pathways affecting expression patterns of five selected sugar-responsive genes (bZIP63, At5g22920, BT2, MGD2, and TPS9) in Arabidopsis thaliana. In general, the expression followed diurnal changes in the overall sugar availability. However, under steady growth conditions, this response was hardly impaired in the mutants for CH metabolizing/transporting proteins (adg1, sex1, sus1-4, sus5/6, and tpt2), including also hexokinase1 (HXK1) loss- and gain-of-function plants—gin2.1 and oe3.2, respectively. In addition, transgenic plants carrying pbZIP63::GUS showed no changes in reporter-gene-expression when grown on sugar under steady-state conditions. In contrast, short-term treatments of agar-grown seedlings with 1% Glc or Suc induced pbZIP63::GUS repression, which became even more apparent in seedlings grown in liquid media. Subsequent analyses of liquid-grown gin2.1 and oe3.2 seedlings revealed that Glc -dependent regulation of the five selected genes was not affected in gin2.1, whereas it was enhanced in oe3.2 plants for bZIP63, At5g22920, and BT2. The sugar treatments had no effect on ATP/ADP ratio, suggesting that changes in gene expression were not linked to cellular energy status. Overall, the data suggest that HXK1 does not act as Glc sensor controlling bZIP63, At5g22920, and BT2 expression, but it is nevertheless required for the production of a downstream metabolic signal regulating their expression.

Keyword
glucose sensing, hexokinase, BT2 expression, bZIP63 expression, At5g22920 expression, diurnal regulation of expression, sugar regulation of gene expression
National Category
Biochemistry and Molecular Biology Botany
Research subject
Physiological Botany
Identifiers
urn:nbn:se:umu:diva-96578 (URN)10.3389/fpls.2015.00525 (DOI)000358589400001 ()26236323 (PubMedID)
Note

Originally published in thesis in manuscript form with the title: The metabolic activity of HEXOKINASE 1 is required for glucose-induced repression of bZIP63, At5g22920 and BT2 in Arabidopsis

Available from: 2014-11-24 Created: 2014-11-24 Last updated: 2017-12-05Bibliographically approved
3. Monitoring the role of distinct sugars on cell division in Arabidopsis plant cells and seedlings
Open this publication in new window or tab >>Monitoring the role of distinct sugars on cell division in Arabidopsis plant cells and seedlings
(English)Manuscript (preprint) (Other academic)
Abstract [en]

Within the last decades, research on sugar-dependent plant growth provided evidence for a directregulation of cell division by sugars. Recently we showed, in A. thaliana cell suspension cultures, thatdistinct sugars differentially regulate a rapid transcriptional response of genes, some of which functionduring the cell cycle progression. In order to assess the relationship between sugar species and celldivision, we developed a new methodology for long-term real-time live-cell imaging on dividing A.thaliana cell suspension cultures. This technique, using cells grown in hormone-free media, allowed toestimate the cell cycle synchronicity and efficiency of an entire cell population and to monitor thedynamics and geometry of cell division in single cells in response to a given sugars. A marker cell lineconstitutively expressing TUA::GFP, a protein that labels microtubule-based structures hallmarkingprogression of the mitotic division, was used to measure the sugar-dependent efficiency andsynchronicity of the cell cycle progression. Altogether, we were able to confirm the distinct relationshipsof specific sugar molecules on the cell cycle progression at a single cell level. Cell division efficiencyand synchronicity were altered when grown on the different sugars sucrose, glucose and xylose.Interestingly, the progression of the mitotic division appeared unaffected by the sugar species supplied,indicating that length of the interphase is likely to control the cell division rate. In contrast, treatment ofA. thaliana cell cultures and seedlings with the Glc-analogue 2-deoxy-glucose (2dog) led to growtharrest and to cell death during long exposure. The growth resulting from 2dog removal in cell culturesand seedlings showed the unique feature of plants to induce new active zones of cell division.

Keyword
Cell suspension culture, cell division, long-term real-time live cell imaging, sugar regulation of gene expression and cellular processes
National Category
Cell Biology Botany Biochemistry and Molecular Biology
Research subject
Physiological Botany; cellforskning
Identifiers
urn:nbn:se:umu:diva-96579 (URN)
Available from: 2014-11-24 Created: 2014-11-24 Last updated: 2014-11-25Bibliographically approved

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