Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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A Plausible Microtubule-Based Mechanism for Cell Division Orientation in Plant Embryogenesis
Chakrabortty, Bandan ; Willemsen, Viola ; Zeeuw, Thijs de; Liao, Che Yang ; Weijers, Dolf ; Mulder, Bela ; Scheres, Ben - \ 2018
Current Biology 28 (2018)19. - ISSN 0960-9822 - p. 3031 - 3043.e2.
arabidopsis - auxin - cell shape - computational modeling - cytokinesis - division orientation - embryogenesis - microtubules - systems biology

Oriented cell divisions are significant in plant morphogenesis because plant cells are embedded in cell walls and cannot relocate. Cell divisions follow various regular orientations, but the underlying mechanisms have not been clarified. We propose that cell-shape-dependent self-organization of cortical microtubule arrays is able to provide a mechanism for determining planes of early tissue-generating divisions and may form the basis for robust control of cell division orientation in the embryo. To show this, we simulate microtubules on actual cell surface shapes, from which we derive a minimal set of three rules for proper array orientation. The first rule captures the effects of cell shape alone on microtubule organization, the second rule describes the regulation of microtubule stability at cell edges, and the third rule includes the differential effect of auxin on local microtubule stability. These rules generate early embryonic division plane orientations and potentially offer a framework for understanding patterned cell divisions in plant morphogenesis. Chakrabortty et al. show that a computational model for dynamic self-organization of cortical microtubules on experimentally extracted cell shapes provides a plausible molecular mechanism for division plane orientation in the first four divisions of early stage A. thaliana embryos, in WT as well as two developmental mutants bodenlos and clasp.

How virtual shade sheds light on plant plasticity
Bongers, Franca J. - \ 2017
Wageningen University. Promotor(en): Niels Anten, co-promotor(en): R. Pierik; Jochem Evers. - Wageningen : Wageningen University - ISBN 9789463432047 - 140
planten - fenotypen - fenotypische variatie - modellen - arabidopsis - natuurlijke selectie - schaduw - reacties - concurrentie tussen planten - licht - plants - phenotypes - phenotypic variation - models - natural selection - shade - responses - plant competition - light

Phenotypic plasticity is the ability of a genotype to express multiple phenotypes in accordance with different environments. Although variation in plasticity has been observed, there is limited knowledge on how this variation results from natural selection. This thesis analyses how variation in the level of plasticity influences light competition between plants and how this variation could result from selection, driven by light competition, in various environments. As an exemplary case of phenotypic plasticity, this thesis focusses on phenotypic responses of the annual rosette plant Arabidopsis thaliana (Brassicaceae) in response to the proximity of neighbour plants, as signalled through the red : far—red (R:FR) ratio, which are responses associated with the shade avoidance syndrome (SAS).

Plant experiments were conducted to measure variation in these plastic responses and a functional-structural plant (FSP) model was created that simulates plant structures in 3D and includes these organ-level plastic responses while simulating explicitly a heterogeneous light environment. Simulating individual plants that explicitly compete for light, while their phenotype changes through plasticity, gave insights in the role of the level of phenotypic plasticity and site of signal perception on plant competitiveness. In addition, an analysis on how natural selection in different environments acts on the level of plasticity was performed by combining FSP simulations and evolutionary game theoretical (EGT) principles.

Enrichment proteomics challenges and perspectives : analysis of the N-glycoproteome and plasma membrane proteome in glycosylation mutants and plant-pathogen interactions
Song, Wei - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol; Twan America. - Wageningen : Wageningen University - ISBN 9789462578722 - 172
proteomics - glycoproteins - arabidopsis - plant-animal interactions - plant pathogens - plasma membranes - eiwitexpressieanalyse - glycoproteïnen - plant-dier interacties - plantenziekteverwekkers - plasmamembranen

This thesis is based on two technology projects from the Centre for BioSystems Genomics (CBSG), entitled ‘Comparative proteomics on Plant Pathogen interactions through enrichment of the N-glycoproteome and tagged-glycoproteome’ (TD7) and ‘Plasma Membrane proteomics for Plant Pathogen interactions’ (TD5). In the former project we developed the protocol for isolation, identification and quantification of N-linked glycoproteins from plants and used it to obtain a comprehensive inventory of glycan-occupancy of Arabidopsis glycoproteins. In the second project, a protocol for the enrichment of plasma membrane (PM) fraction from plant material was developed and applied to study the role of the PM proteome in the interaction of plants with the plant pathogen Phytophthora infestans. Combined these activities have resulted in a thesis devoted to technical developments in label-free comparative enrichment proteomics, with validation in a number of different biological systems.

Mechanistic dissection of plant embryo initiation
Radoeva, T.M. - \ 2016
Wageningen University. Promotor(en): Dolf Weijers, co-promotor(en): Sacco de Vries. - Wageningen : Wageningen University - ISBN 9789462578135 - 183 p.
embryogenesis - embryos - plants - auxins - genes - genomics - arabidopsis - cell suspensions - in vivo experimentation - zygotes - monozygotic twins - embryogenese - embryo's - planten - auxinen - genen - genomica - celsuspensies - in vivo experimenten - zygoten - monozygote tweelingen

Land plants can reproduce sexually by developing an embryo from a fertilized egg cell, the zygote. After fertilization, the zygote undergoes several rounds of controlled cell divisions to generate a mature embryo. However, embryo formation can also be induced in a variety of other cell types in many plant species. These non-zygotic embryos go through analogous developmental phases and are morphologically similar to the zygotic embryo. Despite its fundamental importance and enormous application potential, the mechanisms that alter cell fate from non-embryonic to embryonic are elusive. In the past decades, a variety of different model systems have been used to identify regulators of embryo induction, but it is unclear if these act in a common network. We recently found that inhibition of auxin response in the extra-embryonic suspensor cells cell-autonomously and predictably triggers a switch towards embryo identity. In my thesis I have used the suspensor-derived embryogenesis as a uniform model system to study the crucial first reprogramming step of embryo initiation process.

Through genome-wide transcriptional profiling upon local (suspensor-specific) auxin response inhibition (Chapter 2) and through testing the ability of fifteen known embryogenesis inducers to promote embryo formation in suspensor cells (Chapter 3), we suggest that suspensor to embryo transformation requires a defined set of genetic regulators. The results obtained in my thesis provide essential tools and basis for further research and are a step forward to understanding the first step of embryo initiation process and to unravel the mystery of totipotency in plants.

Stem cell organization in Arabidopsis : from embryos to roots
Wendrich, J.R. - \ 2016
Wageningen University. Promotor(en): Dolf Weijers, co-promotor(en): Bert de Rybel. - Wageningen : Wageningen University - ISBN 9789462577350 - 192 p.
arabidopsis - stem cells - roots - plant embryos - morphogenesis - biological development - cellular biology - plant cell biology - stamcellen - wortels - plantenembryo's - morfogenese - biologische ontwikkeling - celbiologie - plantencelbiologie

Growth of plant tissues and organs depends on continuous production of new cells, by niches of stem cells. Stem cells typically divide to give rise to one differentiating daughter and one non-differentiating daughter. This constant process of self-renewal ensures that the niches of stem cells or meristems stay active throughout plant-life. Specification of stem cells occurs very early during development of the emrbyo and they are maintained during later stages. The Arabidopsis embryo is a highly predictable and relatively simple model to study several developmental processes. Chapter 1 discusses the Arabidopsis embryo as a model for development and morphogenesis and describes the currently known factors involved in these processes.

Molecular cloning is a vital technique of today’s plant biological research. The ability to quickly produce reliable constructs for follow-up analyses can greatly accelerate biological research. In Chapter 2, we describe the optimization of a highly efficient Ligation Independent Cloning method. This method makes use of sticky overhangs that enable in vivo ligation of cloning products. We present a step-by-step protocol that enables generating plant transformation-ready constructs in a semi-high-throughput manner, within two to three days. This method can for example facilitate follow-up analysis of genome-wide approaches.

Proteins regularly function as part of larger protein-complexes and their interaction partners can often be indicative of functionality. Unbiased, in vivo analysis of protein complexes can therefore be very informative for the functional characterization of a protein of interest. In Chapter 3, we describe an optimized method for immunoprecipitation followed by tandem mass-spectrometry. By performing mass-spectrometry measurements on at least three biological replicates, relative abundance of proteins in GFP-tagged sample compared to background controls can be statistically evaluated to identify high-confidence interactors. In this step-by-step protocol we detail the entire procedure from plant material to data analysis and visualization.

The establishment of distinct cellular identities is of critical importance for multicellular organisms. The first step that leads to cell identity is the activation of a unique set of transcripts and this often exploited in order to infer cell identity. In Chapter 4, we have generated 12 gene expression marker lines and describe their expression domain in the Arabidopsis embryo. We divided them into four different categories based on their expression domain: (I) ground tissue; (II) root stem cell; (III) shoot apical meristem; and (IV) post-embryonic. In addition, we used two stem cell markers to show their use as marker lines in genetic studies.

A central player in development of the Arabidopsis root meristem is the AUXIN RESPONSE FACTOR5/MONOPTEROS (MP). Several downstream targets of this transcription factor have been characterized, but the main focus has been on targets that were themselves transcription factors. An open question remains, therefore, how MP can orchestrate cellular responses during development. Chapter 5 describes the in-depth functional and biochemical characterization of a group of IQ-domain proteins. We show that their expression is regulated by the hormone auxin and that they bind microtubules and Calmodulins, in vivo. In addition, we show that the subcellular localization of IQD18 is cell cycle dependent. Loss- and gain-of-function analysis resulted in differential auxin- and calcium-signaling output, suggesting these proteins may form a bridge between these two major signaling pathways. Furthermore, this indicates a mode for how MP may be affecting cellular responses, during root development.

In Chapter 6, we take a step back and re-evaluate the currently prevailing model for stem cell organization in the Arabidopsis (embryonic) root. Using different gene expression markers, we were able to generate non-cell type specific and cell type specific transcriptomic datasets from systematically obtained ontogenetic cell populations in the root meristem. Follow-up analyses give support for an extended model for stem cell organization in the root.

Finally, in Chapter 7, we discuss the novel findings of this thesis and suggestions are made for future research directions.

Metabolic engineering of biosynthesis and sequestration of artemisinin
Wang, B. - \ 2016
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Sander van der Krol. - Wageningen : Wageningen University - ISBN 9789462576728 - 210 p.
artemisinin - nicotiana benthamiana - arabidopsis - biosynthesis - malaria - drugs - genetic engineering - metabolism - artemisinine - biosynthese - geneesmiddelen - genetische modificatie - metabolisme

The sesquiterpenoid artemisinin (AN) is the most important medicine for the treatment of malaria in humans. The industrial production of AN still mainly depends on extraction from the plant Artemisia annua. However, the concentration of AN in A. annua is low. Although different engineering strategies have been used in both A. annua and heterologous plant and yeast production platforms, the worldwide capacity and production costs for AN are not in balance with its demand (Chapter 1). Although the genes encoding for the entire AN biosynthesis pathway (AN-PW) of the AN precursor dihydroartemisinic acid (DHAA) have been identified, the application of these genes in pathway engineering seem to be limited by lack of control over product transport and sequestration. At the onset of this thesis project there was no information on transport in the AN-PW. However, it was known that DHAA is converted into AN outside the glandular trichome cells of A. annua. Therefore, in this thesis I tried to gain more knowledge on transport within the AN-PW and the use of different metabolic engineering strategies to improve the production of AN.

At the onset of my PhD project, the AN-PW genes from two different A. annua chemotypes were compared to understand the basis of different relative activities in the two branches of the AN-PW (Chapter 2). For these assays we used transient expression in N. benthamiana. In the AN-PW, artemisinic aldehyde (AAA) is at a branch point as it can be converted to artemisinic acid (AA) by amorphadiene oxidase (AMO), or to dehydroartemisinic aldehyde (DHAAA) by artemisinic aldehyde Δ11 (13) reductase (DBR2). AA is the precursor for arteannuin B (AB) while DHAAA may be converted by a CYP71AV1 or an ALDH1 to dehydroartemisinic acid (DHAA), the precursor for AN. In this chapter we demonstrate that the CYP71AV1 from a high AN production (HAP) chemotype has reduced activity in the AB branch of the pathway compared to the CYP71AV1 from a low AN production (LAP) chemotype. In addition, we show that the relative expression levels of DBR2 and ALDH1 also affect the AN/AB chemotype. The low catalytic efficiency of AMO from the HAP chemotype may be caused by a deletion of seven amino acids at the N-terminus of the protein compared to CYP71AV1 from LAP. Ectopic expression of the AN-PW genes in N. benthamiana showed that the bulk of the PW products are modified by glycosylation and glutathione conjugations. These side reactions therefore compete with the biosynthesis flux towards the AN precursor DHAA. At this point in my thesis the ectopic expression of AN-PW genes in N. benthamiana had not yielded any AN. At a later stage it became clear that this was due to harvest of leaves at 5-7 days post agro-infiltration (dpi), while AN in N. benthamiana leaves expressing AN-PW genes only becomes detectable after 7 dpi.

Glycosylation of the bulk of the AN-PW products in N. benthamiana stresses the need for an efficient transport of (DH)AA to the outside of cells in order to escape from the glycosylation reactions. In Chapter 3, transport and sequestration of AN precursors was investigated by studying the effect of membrane transporters (PDRs) and lipid transfer proteins (LTPs). Hereto, two membrane transporters with activity towards AN-PW products were made available by the group of Prof. Marc Boutry and we isolated three LTP genes from Artemisia annua which showed expression in the glandular trichomes. In this chapter we show that AaLTP3 displays specific activity, together with AaPDR2 towards transport of (DH)AA to the apoplast in N. benthamiana. Moreover, infiltration experiments with (DH)AA in N. benthamiana leaves revealed that these compounds are rapidly taken up by the cells and that inside the cells there is a strong reverse flux in the AN-PW by conversion of (DH)AA towards (DH)AAA and (DH)AAOH. Subsequently we demonstrated that AaLTP3 has a stronger activity in keeping products in the apoplast than the AaPDR2 membrane transporter. Therefore, I suggest that by removal of (DH)AA from the cytosol through transport over the plasma membrane by AaPDR2 and subsequent sequestration in the apoplast by AaLTP3, AaLTP3 creates sink activity which prevents reflux of (DH)AA from the apoplast back into the cells. AaLTP3 therefore contributes to a directional flux through the AN-PW towards the end product (DH)AA. Finally, in this work we could also for the first time detect AN and AB in N. benthamiana leaves by extraction of necrotic leaves at 13 dpi.

Because in A. annua glandular trichome cells both the AN sesquiterpene biosynthesis pathway and the flavonoid biosynthesis pathway are active, we explored whether there is a functional interaction between these two major secondary metabolite biosynthesis pathways. In Chapter 4 we describe how we manipulate the flavonoid biosynthesis pathway in N. benthamiana leaves using the Antirhinum majus transcription factor Rosea1 (ROS) and test coexpression of ROS with AN-PW genes. The co-expression of ROS stimulates AN-PW product accumulation. Subsequent analysis indicates that this is most likely from transcriptional activation of the enzyme Mevalonate Kinase (MVK) in the mevalonate pathway, which provides precursors for the sesquiterpene biosynthesis pathway. In addition, we demonstrate that production of flavonoids competes with AN-PW product accumulation, as co-expression of AN-PW genes with ROS, but simultaneous inhibition of chalcone synthase (CHS) by a CHSRNAi construct, results in higher AN-PW product levels. However, accumulation of the end products AN and AB was not affected significantly. Finally, the combined expression of AN-PW+ROS+AaPDR2+AaLTP3+ CHSRNAi results in highest sequestration of (DH)AA in the apoplast and highest accumulation of the end products AN and AB in N. benthamiana.

During my thesis work, in a related project it was found that expression of another sesquiterpene biosynthesis gene (caryophyllene synthase; CST) in transgenic Arabidopsis resulted in higher caryophyllene emission for a transformant expressing a genomic DNA of CST, compared with a similar transformant expressing a CST cDNA described in literature. This suggested that ectopic expression of intron containing genes is more efficient than ectopic expression of cDNAs. To test whether in the context of metabolic engineering the use of genomic (intron-containing) genes is more efficient than the use of the corresponding cDNA we generated a set of stable transformed Arabidopsis lines with either genomic CST (gCST), cDNA CST (cCST), genomic amorphadiene synthesis (gADS) and cDNA ADS (cADS). In chapter 5 we show that indeed the lines with overexpression of the genomic clones yield higher levels of the anticipated products (caryophyllene or amorphadiene) than the lines with overexpression of the corresponding cDNAs. Transcript analysis showed that for gCST the increase in caryophyllene production was higher than can be explained solely by the increase in CST transcription. In the context of transient expression in N. benthamiana leaves the intron-mediated-enhancement effect was less pronounced.

In the final discussion chapter 6 I review limitations and potential solutions to metabolic engineering of the AN-PW in plants, and I discuss the impact of our findings on AN production capacity using transient expression versus natural production in A. annua. Moreover, I discuss how the finding of this thesis go beyond just insights into the AN-PW as especially the identification of the role of LTPs in sesquestration of (sesqui)terpenes into the apoplast may have an impact on the metabolic engineering efforts of many other (sesqui)terpene pathways. Because some plant hormones are also terpenoid products the newly identified role of LTPs may also have impact on a deeper understanding of hormone signalling in plants. I have already started exploring this path by generating a set of Arabidopsis plants with overexpression of different Arabidopsis LTP genes to test whether any hormone related traits are altered (Chapter 6). Preliminary results do indeed confirm a role of LTPs in endogenous plant hormone balance, something worthwhile to be further explored in future research.

Moleculaire inzichten vergroten sturingsmogelijkheden wortelgroei : Auxine speelt een sleutelrol
Scheres, B.J.G. ; Heuvelink, E. - \ 2015
Onder Glas 12 (2015)12. - p. 16 - 17.
botany - agricultural research - arabidopsis - auxins - proteins - roots - shoots - growth regulators - growth promoters - plantkunde - landbouwkundig onderzoek - auxinen - eiwitten - wortels - scheuten - groeiregulatoren - groeibevorderaars
Het wortelstelsel van gewassen is vaak ‘de verborgen helft’ genoemd. Lange tijd bestond er een enorm gebrek aan kennis over wortelgroei. Dat maakt het moeilijk er op te veredelen of om er in de teelt anders mee om te gaan. De afgelopen jaren is het inzicht belangrijk gegroeid, met name op genetisch en moleculair niveau.
Adaptation and acclimation of seed performance
Souza Vidigal, D. De - \ 2015
Wageningen University. Promotor(en): Harro Bouwmeester, co-promotor(en): Leonie Bentsink; Henk Hilhorst. - Wageningen University - ISBN 9789462575943 - 156
zaden - zaadkwaliteit - zaadkieming - klimaatverandering - adaptatie - arabidopsis - kiemrust - klimaatadaptatie - seeds - seed quality - seed germination - climatic change - adaptation - seed dormancy - climate adaptation
Functional analyses of AtCHR12 and AtCHR23 : plant growth responses upon over-expression of chromatin remodeling ATPase genes
Folta, A. - \ 2015
Wageningen University. Promotor(en): Ton Bisseling, co-promotor(en): Ludmila Mlynarova. - Wageningen : Wageningen University - ISBN 9789462575561 - 144
planten - groei - genexpressie - chromatine - atp - arabidopsis - solanum lycopersicum - zaadkieming - groeiregulatoren - plants - growth - gene expression - chromatin - seed germination - growth regulators

Living organisms have to deal with changing environmental conditions during their whole life cycle. In contrast to animals, plants are sessile organisms. Therefore they have evolved multiple regulatory mechanisms that help them to cope with changing conditions. One of the first responses to stress conditions is reduction or arrest of growth. Therefore regulation of growth and development is essential to successfully complete their life cycle. To correctly time their development, plants need to integrate various environmental signals with intrinsic developmental programs. In this integration, regulation of gene expression plays a major role.

The genetic information of an organism is stored in DNA sequence. DNA forms a complex with histones and other proteins, which is called chromatin. Chromatin is a highly dynamic complex and modification of the chromatin structure makes DNA more or less accessible to the transcriptional machinery and other regulatory proteins. The modification of chromatin organization is called chromatin remodeling and it involves both covalent modifications of DNA and histone tails, and non-covalent modification of chromatin structure by ATP-dependent chromatin remodeling complexes. ATP-dependent chromatin remodeling complexes comprise multiple protein subunits with SNF2 ATPase as a catalytic subunit. Depending on the subunit composition, the complexes can perform different tasks at various places of chromatin, and can be active at different developmental stages (Chapter 1).

The SNF2 ATPases are conserved from yeast to plants. In Arabidopsis, 41 SNF2 ATPases have been identified. The focus of this thesis is on two of those ATPases – AtCHR12 and AtCHR23. It has been shown previously that AtCHR12 is involved in growth responses to environmental cues. We have extended these studies to its paralog AtCHR23 (Chapter 2). In contrast to over-expression of AtCHR12, which affects growth only during reproductive stage of development, over-expression of AtCHR23 leads to smaller seedlings and reduced vegetative growth. Upon application of mild abiotic stress, the growth reduction is stronger than in wild-type plants. Moreover, the transgenic plants manifest increased variability of growth. The increased growth variability correlates with increased expression variability of genes associated with stress. The results indicate that accurate and controlled expression of AtCHR23 is required for stability and robustness of growth, as well as gene expression.

Regulation of growth is important not only during vegetative or reproductive stage of development, but also during embryo development. The growth of the embryo is interrupted during the embryo maturation phase and it was suggested that AtCHR12 may be involved in this temporary growth arrest. Here we have shown that both AtCHR12 and AtCHR23 are expressed during embryo development, and that over-expression of AtCHR12 or AtCHR23 affects the embryo maturation phase with consequences on two important developmental transitions in plant life – germination and transition to flowering.

Over-expression of AtCHR12 or AtCHR23 leads to reduced seed germination, which is more pronounced under stress conditions (Chapter 3). The reduced germination of over-expressing lines is associated with increased transcript levels of seed maturation genes and reduced degradation of their mRNAs in germinating seeds. The results indicate that repression of AtCHR12 and AtCHR23 in germinating seeds is required for full germination.

The connection between embryo development and flowering time control was observed in transgenic lines over-expressing AtCHR12 (Chapter 4). Over-expression of AtCHR12 results in early flowering under both long- and short-day conditions. The early flowering phenotype correlates with reduced expression of the main flowering repressor, FLC. The reduced FLC expression correlates with increased levels of repressive histone mark H3K27me3 on the FLC locus. Additionally, FLC expression was affected already during FLC reprogramming, which takes place during embryo development. This leads to reduced FLC expression in mature embryos. The results show that AtCHR12 over-expression affects flowering time by different mechanisms than other Snf2-subfamily ATPases. In contrast to AtCHR12, BRAHMA was shown to regulate flowering time via the photoperiod pathway, while SPLAYED affects flowering time by repressing FT expression.

We have observed that over-expression of AtCHR12 or AtCHR23 affects plant growth in response to stress, and play a role in germination and transition to flowering. These traits are also important for agriculture, and such genes are potentially interesting targets for breeding programs. To test, if such genes have a similar role in crops, we have studied the effect of the tomato ortholog of AtCHR12 and AtCHR23 on tomato growth.

Tomato (Solanum lycopersicum), as well as other crops, have only one ortholog of AtCHR12 and AtCHR23, which was suggested to possess a role of both ATPases. We have successfully cloned the tomato ortholog and over-expressed it in tomato plants (Chapter 5). The transgenic tomato plants have reduced vegetative growth and compacted reproductive structures, resembling the phenotype of AtCHR23 and AtCHR12 over-expression, respectively. However, in contrast to Arabidopsis, the tomato plants responded to abiotic stress similarly as wild-type, and they flowered later than wild-type plants. The results indicate that modification of expression of AtCHR12 and AtCHR23 orthologs could be used to develop novel methods to control plant growth.

Taken together, the research described in this thesis identifies AtCHR12 and AtCHR23 as regulators of plant growth, especially in response to environment, as well as of the seed maturation program with clear effects on seed germination and flowering time, and we show that such genes can be potentially interesting for agriculture and horticulture practice.

Effect of temperature on biomass allocation in seedlings of two contrasting genotypes of the oilseed crop Ricinus communis
Ribeiro de Jesus, P.R. ; Zanotti, R.F. ; Deflers, C. ; Fernandez, L.G. ; Castro, R.D. De; Ligterink, W. ; Hilhorst, H.W.M. - \ 2015
Journal of Plant Physiology 185 (2015). - ISSN 0176-1617 - p. 31 - 39.
abiotic stress tolerance - plant-responses - heat-stress - castor-oil - acid gaba - growth - metabolomics - arabidopsis - pathways - moisture
Ricinus communis is becoming an important crop for oil production, and studying the physiological and biochemical aspects of seedling development may aid in the improvement of crop quality and yield. The objective of this study was to assess the effect of temperature on biomass allocation in two R. communis genotypes. Biomass allocation was assessed by measuring dry weight of roots, stems, and cotyledons of seedlings grown at three different temperatures. Root length of each seedling was measured. Biomass allocation was strongly affected by temperature. Seedlings grown at 25 ¿C and 35 ¿C showed greater biomass than seedlings grown at 20 ¿C. Cotyledon and stem dry weight increased for both genotypes with increasing temperature, whereas root biomass allocation showed a genotype-dependent behavior. Genotype MPA11 showed a continuous increase in root dry weight with increasing temperature, while genotype IAC80 was not able to sustain further root growth at higher temperatures. Based on metabolite and gene expression profiles, genotype MPA11 increases its level of osmoprotectant molecules and transcripts of genes encoding for antioxidant enzymes and heat shock proteins to a higher extent than genotype IAC80. This might be causal for the ability to maintain homeostasis and support root growth at elevated temperatures in genotype MPA11.
On the Origin of SERKs: Bioinformatics Analysis of the Somatic Embryogenesis Receptor Kinases
Toorn, M. aan den; Albrecht, C. ; Vries, S.C. de - \ 2015
Molecular Plant 8 (2015)5. - ISSN 1674-2052 - p. 762 - 782.
epidermal-growth-factor - leucine-rich repeat - molecular characterization - evolutionary conservation - expression analysis - gene family - phosphorylation sites - duplicated genes - protein-kinases - arabidopsis
Somatic embryogenesis receptor-like kinases (SERKs) are leucine-rich repeat receptor-like kinases involved in several, seemingly unrelated, plant-signaling pathways. In Arabidopsis thaliana, functional and genetic analysis of four SERK proteins has indicated that they are only partly redundant; their functions overlap but each performs a specific subset of signaling roles. The molecular basis for the functional specificity within this highly homologous protein family is currently not known. Sequence analysis of SERK proteins from different plant species indicates that the SERKs are a highly conserved protein family present in monocots, dicots, and non-vascular plants. Residues in the extracellular domain that are important for interaction with other receptor kinases are highly conserved, even among SERK members without a function in the corresponding pathways. SERK2, for instance, does not function in the brassinosteroid pathway, does not interact with BRI1, but is conserved in its BRI1-interacting domain. Further sequence analysis indicates that SERK3/BAK1 and SERK4/BKK1 have diverged from the original SERK protein in both their extracellular and cytoplasmic domains. Functional analysis of chimeric SERK proteins shows that different domains provide the SERK proteins with different functional specificity. For instance, the SERK1 or SERK2 extracellular domains are essential for SERK function in male sporogenesis, while the SERK3 extracellular and cytoplasmic domains are essential for SERK3 activity in brassinosteroid and flagellin signaling. The emerging picture is that SERKs are ancient genes, whose products have been recruited as co-receptors in the newly evolved signaling pathways. The SERK ligand-binding and protein–protein interaction domains are highly conserved, allowing all SERKs to form complexes, albeit with different affinity. However, specific functional residues must have been altered, in both the extracellular and intracellular domains, to allow for the observed differences in functionality.
Reporters for sensitive and quantitative measurement of auxin response
Liao, C.Y. ; Smet, W.M.S. ; Brunoud, G. ; Yoshida, S. ; Vernoux, T. ; Weijers, D. - \ 2015
Nature Methods : techniques for life scientists and chemists 12 (2015). - ISSN 1548-7091 - p. 207 - 210.
apical-basal axis - box protein tir1 - aux/iaa proteins - arabidopsis - transcription - expression - transport - specificity - sufficient - perception
The visualization of hormonal signaling input and output is key to understanding how multicellular development is regulated. The plant signaling molecule auxin triggers many growth and developmental responses, but current tools lack the sensitivity or precision to visualize these. We developed a set of fluorescent reporters that allow sensitive and semiquantitative readout of auxin responses at cellular resolution in Arabidopsis thaliana. These generic tools are suitable for any transformable plant species.
The butterfly plant arms-race escalated by gene and genome duplications
Edger, P.P. ; Heidel-Fischer, H.M. ; Bekaert, K.M. ; Rota, J. ; Glockner, G. ; Platts, A.E. ; Heckel, D.G. ; Der, J.P. ; Wafula, E.K. ; Tang, M. ; Hofberger, J.A. ; Smithson, A. ; Hall, J.C. ; Blanchette, M. ; Bureau, T.E. ; Wright, S.I. ; dePamphilis, C.W. ; Schranz, M.E. ; Conant, G.C. ; Barker, M.S. ; Wahlberg, N. ; Vogel, H. ; Pires, J.C. ; Wheat, C.W. - \ 2015
Proceedings of the National Academy of Sciences of the United States of America 112 (2015)27. - ISSN 0027-8424 - p. 8362 - 8366.
evolutionaire genetica - co-evolutie - diversificatie - brassica - pieridae - papilionidae - glucosinolaten - fylogenie - evolutionary genetics - coevolution - diversification - glucosinolates - phylogeny - diversity - defense - cytochrome-p450 - polymorphism - arabidopsis - metabolism - expression - speciation
Coevolutionary interactions are thought to have spurred the evolution of key innovations and driven the diversification of much of life on Earth. However, the genetic and evolutionary basis of the innovations that facilitate such interactions remains poorly understood. We examined the coevolutionary interactions between plants (Brassicales) and butterflies (Pieridae), and uncovered evidence for an escalating evolutionary arms-race. Although gradual changes in trait complexity appear to have been facilitated by allelic turnover, key innovations are associated with gene and genome duplications. Furthermore, we show that the origins of both chemical defenses and of molecular counter adaptations were associated with shifts in diversification rates during the arms-race. These findings provide an important connection between the origins of biodiversity, coevolution, and the role of gene and genome duplications as a substrate for novel traits.
Karyotype evolution in apomictic Boechera and the origin of the aberrant chromosomes
Mandáková, T. ; Schranz, M.E. ; Sharbel, T.F. ; Jong, J.H.S.G.M. de; Lysak, M. - \ 2015
The Plant Journal 82 (2015)5. - ISSN 0960-7412 - p. 785 - 793.
holboellii complex - genus boechera - genome sequence - centric fission - brassicaceae - arabidopsis - centromere - phylogeny - arabis - reproduction
Chromosome rearrangements may result in both decrease and increase of chromosome numbers. Here we have used comparative chromosome painting (CCP) to reconstruct the pathways of descending and ascending dysploidy in the genus Boechera (tribe Boechereae, Brassicaceae). We describe the origin and structure of three Boechera genomes and establish the origin of the previously described aberrant Het and Del chromosomes found in Boechera apomicts with euploid (2n = 14) and aneuploid (2n = 15) chromosome number. CCP analysis allowed us to reconstruct the origin of seven chromosomes in sexual B. stricta and apomictic B. divaricarpa from the ancestral karyotype (n = 8) of Brassicaceae lineage I. Whereas three chromosomes (BS4, BS6, and BS7) retained their ancestral structure, five chromosomes were reshuffled by reciprocal translocations to form chromosomes BS1-BS3 and BS5. The reduction of the chromosome number (from x = 8 to x = 7) was accomplished through the inactivation of a paleocentromere on chromosome BS5. In apomictic 2n = 14 plants, CCP identifies the largely heterochromatic chromosome (Het) being one of the BS1 homologues with the expansion of pericentromeric heterochromatin. In apomictic B. polyantha (2n = 15), the Het has undergone a centric fission resulting in two smaller chromosomes – the submetacentric Het' and telocentric Del. Here we show that new chromosomes can be formed by a centric fission and can be fixed in populations due to the apomictic mode of reproduction.
Detection of induced mutations in CaFAD2 genes by next generation sequencing leading to the production of improved oil composition in Crambe abyssinica
Cheng, J. ; Salentijn, E.M.J. ; Huang Bangquan, ; Denneboom, C. ; Dechesne, A.C. ; Krens, F.A. ; Visser, R.G.F. ; Loo, E.N. van - \ 2015
Plant Biotechnology Journal 13 (2015)4. - ISSN 1467-7644 - p. 471 - 481.
induced point mutations - crop improvement - reverse genetics - oleic-acid - functional genomics - fad2 gene - discovery - arabidopsis - populations - cloning
Crambe abyssinica is a hexaploid oil crop for industrial applications. An increase of erucic acid (C22:1) and reduction of polyunsaturated fatty acid (PUFA) contents in crambe oil is a valuable improvement. An increase in oleic acid (C18:1), a reduction in PUFA and possibly an increase in C22:1 can be obtained by down-regulating the expression of fatty acid desaturase2 genes (CaFAD2), which code for the enzyme that converts C18:1 into C18:2. We conducted EMS-mutagenesis in crambe, followed by Illumina sequencing, to screen mutations in three expressed CaFAD2 genes. Two novel analysis strategies were used to detect mutation sites. In the first strategy, mutation detection targeted specific sequence motifs. In the second strategy, every nucleotide position in a CaFAD2 fragment was tested for the presence of mutations. Seventeen novel mutations were detected in 1100 one-dimensional pools (11 000 individuals) in three expressed CaFAD2 genes, including non-sense mutations and mis-sense mutations in CaFAD2-C1, -C2 and -C3. The homozygous non-sense mutants for CaFAD2-C3 resulted in a 25% higher content of C18:1 and 25% lower content of PUFA compared to the wild type. The mis-sense mutations only led to small changes in oil composition. Concluding, targeted mutation detection using NGS in a polyploid was successfully applied and it was found that a non-sense mutation in even a single CaFAD2 gene can lead to changes in crambe oil composition. Stacking the mutations in different CaFAD2 may gain additional changes in C18:1 and PUFA contents.
BABY BOOM-induced somatic embryogenesis in Arabidopsis
Horstman, A. - \ 2015
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kim Boutilier. - Wageningen : Wageningen University - ISBN 9789462572317 - 233
arabidopsis - somatische embryogenese - in vitro kweek - cellen - weefselkweek - plantengroeiregulatoren - somatische embryo's - transcriptie - somatic embryogenesis - in vitro culture - cells - tissue culture - plant growth regulators - somatic embryos - transcription

Under appropriate tissue culture conditions, somatic plant cells can be induced to form embryos in a process called somatic embryogenesis (SE). SE provides a way to clonally propagate desirable plants and is therefore an important plant breeding tool. SE has also fascinated scientists for decades as an expression of plant ‘totipotency’, the ability to regenerate a whole new individual through embryogenesis. This thesis aims to obtain a deeper understanding of somatic embryo induction in Arabidopsis by the transcription factor BABY BOOM (BBM), through identification and functional analysis of BBM-binding proteins and BBM target genes.

Chapter 1 introduces the concept of somatic embryogenesis, describes the different SE systems in Arabidopsis, and discusses the role of the plant hormone auxin and chromatin modifying proteins in this process. An overview is presented on the current knowledge on SE-induction through ectopic overexpression of certain transcription factor genes. These include BBM, as well as other genes that are studied in this thesis in relation to BBM.

BBM is part of the eight member AIL subfamily of AP2/ERF domain transcription factors. Chapter 2 reviews the role of AIL proteins during embryogenesis, stem cell niche specification, meristem maintenance and organ positioning and growth. We summarize the gene regulatory networks in which AILs function and describe how these transcription factors integrate multiple hormonal inputs, with special emphasis on the interactions between AILs and auxin. Finally, we conclude that although the functions of AILs in plant development are well described, knowledge on the molecular mode of action of AIL proteins and the identity of AIL target genes is still limited.

Transcription factors function in protein complexes and in Chapter 3 we show that members of the HOMEODOMAIN GLABROUS (HDG) transcription factor family physically interact with BBM and other AILs. HDG genes are expressed in the epidermis, the outer cell layer of the plant, where they promote differentiation of cells into specialized epidermal cell types, such as trichomes or stomata. We show that ectopic overexpression of HDG1 leads to loss of root and shoot meristems, phenotypes that had previously been reported for loss-of-function ail mutants. Conversely, down-regulation of HDG genes led to reduced cell differentiation, enhanced cell proliferation and SE phenotypes, phenotypes that resemble those found in AIL overexpression lines. Moreover, we found that co-overexpression of BBM and HDG1 reduces the overexpression phenotypes of both proteins. These results suggest opposite functions of AIL and HDG transcription factors, with AILs stimulating cell proliferation and HDGs stimulating cell differentiation, with the ratio between the two proteins determining the developmental outcome. Finally, we show that HDGs and AILs regulate each other on a transcriptional level and that they share common target genes.

A variety of AIL overexpression phenotypes has been described in the literature, with BBM and PLT5/AIL5 being the only known AILs that induce SE upon overexpression. We show in Chapter 4 that all AIL proteins except AIL1 and ANT are able to induce SE, but that this phenotype relies on a high AIL protein dosage. Using BBM and PLT2 as AIL representatives, we show that an intermediate AIL concentration induces organogenesis (ectopic root and shoot formation) and that a low concentration inhibits cellular differentiation. In addition, we show that BBM and PLT2 induce direct SE when activated at seed germination, while post-germination activation leads to indirect SE from callus. The LEAFY COTYLEDON (LEC)/LAFL genes, which also encode SE-inducing transcription factors, are direct targets of BBM/PLT2 during direct SE, showing that these two SE pathways are linked. Using LAFL gene mutants, we show that the LAFL pathway is an important downstream component of BBM-mediated SE.

Chapter 5 presents the in vivo, genome-wide analysis of BBM DNA binding sites in somatic embryos using chromatin immunoprecipitation followed by sequencing (ChIP-seq). Our ChIP-seq and gene expression analysis reveal that BBM binds and positively regulates auxin biosynthesis genes and the recently discovered positive regulators of SE, the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED (AHL) genes. Knock-out of either pathway reduced BBM-mediated SE, showing that auxin biosynthesis and the AHL genes are important components of the BBM pathway. We also show that BBM binds to a consensus DNA motif that resembles the reported ANT binding motif.

Chapter 6 reviews methods for identifying the direct target genes of a plant transcription factor using microarrays, as was done for HDG1 (Chapter 4). We describe which different systems can be used to control transcription factor activity, and how these can be combined with microarray analysis to identify target genes. In addition, we provide guidelines for the statistical analysis of microarray data and for the confirmation of candidate target genes.

In plant biology, protein-protein interactions are often studied using bimolecular fluorescence complementation (BiFC) or split-YFP. In my BBM-HDG interaction studies I encountered problems using this method, which lead to the cautionary note on the use of BiFC presented in Chapter 7. BiFC is based on the restoration of fluorescence after the two non-fluorescent halves of a fluorescent protein are brought together by a protein-protein interaction event. However, because the fluorescent protein halves are prone to self-assembly, it is crucial to use proper controls and a quantitative read-out of fluorescence to avoid false positive interactions. We present a guideline for the setup of a BiFC experiment, discussing each step in the protocol.

Chapter 8 discusses how the results presented in this thesis contribute to our knowledge on AIL transcription factors and somatic embryo induction, as well as the questions that still remain. An extended model of dose-dependent AIL function is proposed, as well as mechanisms by which the AIL-HDG interaction could function at the molecular level. Finally, an overview is provided of the molecular-genetic intersection between the different transcription factor-induced SE pathways.

MADS dynamics : gene regulation in flower development by changes in chromatin structure and MADS-domain protein binding
Pajoro, A. - \ 2015
Wageningen University. Promotor(en): Gerco Angenent, co-promotor(en): Kerstin Kaufmann. - Wageningen : Wageningen University - ISBN 9789462572669 - 219
bloemen - plantenontwikkeling - genregulatie - chromatine - apicale meristemen - arabidopsis - moleculaire biologie - flowers - plant development - gene regulation - chromatin - apical meristems - molecular biology


During the life cycle, a plant undergoes a series of developmental phase changes. The first phase change is the transition from the initial juvenile vegetative stage into the adult vegetative phase. During the juvenile phase plants produce leaves and axillary buds, whereas during the adult phase the initiation of reproductive structures occurs. The next developmental change is the switch from vegetative to reproductive growth, when the shoot apical meristem acquires the identity of an inflorescence meristem that will then produce floral meristems. Arabidopsis floral meristems produce four concentric whorls of floral organs: sepals, petals, stamens and carpels. Each developmental change is controlled by coordinated network of regulators, known as gene regulatory networks (GRNs), which determine the transcription of a specific set of genes. The aim of the study presented in this thesis was to understand the dynamics of GRNs during floral organ development in Arabidopsis and correlate the binding of key regulatory MADS domain transcription factors with the accessibility of the chromatin in a genome-wide context.

In chapter 1 and 2 we reviewed the current knowledge on the regulation of transcription in the model plant Arabidopsis thaliana. In chapter 1 we mainly focus on how the view of the GRN underling flower development has changed during the last decades, while in chapter 2 we more broadly revised the mechanisms that control developmental switches in plants. The recent introduction of next-generation sequencing and genome-wide approaches has changed our view on gene regulation and GRNs. We moved from linear genetic interactions towards global highly connected gene networks. The high numbers of interactions that were detected in protein-DNA binding profiles revealed a much higher network complexity than previously anticipated and demonstrated that master regulators of development not only control another layer of regulators, but also genes encoding structural proteins, enzymes and signalling proteins. Moreover, most transcription factors bind to their own locus, highlighting that auto-regulatory loops are a common mechanism of regulation.

The discovery of interactions between transcriptional master regulators with epigenetic factors provides new insights into general transcriptional regulatory mechanisms. Switches of developmental programmes and cell fates in complex organisms are controlled at the level of gene expression by the combined action of chromatin regulators and transcription factors.

Although many master regulators of meristem and organ identities have been identified, it is still not well understood how they act at the molecular level and how they can switch an entire developmental program in which thousands of genes are involved. Using flower development as a model system, in chapters 3 and 4 we investigated general concepts of transcription regulation by analysing the dynamics of protein-DNA binding, chromatin accessibility and gene expression.

Using an inducible system for synchronised flower formation, we characterised DNA-binding profiles of two MADS-domain transcription factors, APETALA1 (AP1) and SEPALLATA3 (SEP3), at three stages of flower development. Our study revealed that these MADS-domain proteins, select their binding sites, and thereby their target genes, in a partly stage-specific fashion. By combining the information from DNA-binding and gene expression data, we proposed models of stage-specific GRNs in flower development. Since developmental control of gene expression is tightly linked with dynamic changes in chromatin accessibility, we identified DNase I hypersensitive sites (DHSs, chapter 3) and we characterised nucleosome occupancy (chapter 4) at different stages of flower development. We observed dynamics in chromatin landscape manifested in increasing and decreasing DHSs as well as in changes in nucleosome occupancy and position.

Next, we addressed the question how MADS-domain protein stage-specific binding is achieved at the molecular level in a chromatin context. In the nucleus the DNA is wrapped around histone octamers to form nucleosomes, which are then packed into highly dense structures, and hence transcription factor binding sites may not be easily accessible. A result of the combined analysis of MADS-domain binding and chromatin dynamics is that MADS-domain proteins bind prevalently to nucleosome depleted regions, and that binding of AP1 and SEP3 to DNA precedes opening of the chromatin, which suggests that these MADS-domain transcription factors may act as so-called “pioneer factors”.

The isolation and analysis of developing flowers of specific stages increased the specificity of our genome-wide experiments, enabling the identification of novel actors in the GRN that regulates flower development. In this thesis we characterised the role of some novel regulators in more detail: in chapter 3 we focussed on the GROWTH REGULATING FACTOR (GRF) family genes; in chapter 5 we investigated the action of STERILE APETALA (SAP); and in chapter 6 we elucidated the regulation and the role of a member of the WUSCHEL-related homeobox (WOX) family, WOX12. GRF family genes are dynamically bound by AP1 and SEP3 at the different stages of flower development. All family members are bound by SEP3, while only a subset of the genes is bound by AP1. The defects in floral organs observed upon down-regulation of these genes highlight their role down-stream of MADS-domain transcription factors. In addition to AP1 and SEP3, SAP is also a target of other MADS-domain proteins, such as APETALA3 (AP3), PISTILLATA (PI), and AGAMOUS (AG). SAP is strongly expressed in meristems and loss of function of SAP causes strong aberrations in flowers, such as a reduction in petal and stamen numbers. We found that SAP interacts with proteins of the SCF ubiquitin ligase complex, suggesting that SAP could act in the ubiquitination pathway.

WOX12 down-regulation leads to defects in floral organ identity specification with the formation of stamenoid-petals, while ectopic expression of WOX12 leads to an opposite effect: it causes the formation of petaloid-stamens in the third whorl. WOX12 acts downstream of AP1. Ectopic expression of WOX12 leads to reduction of AG expression, suggesting a role for WOX12 in the antagonistic interplay between the homeotic genes AP1 and AG.

In chapter 7 we discuss the findings of this thesis. Taken together, the work performed in this thesis increased our knowledge on the GRN that regulates flower development and on the mode of action of MADS-domain transcription factors. We hypothesise that MADS-domain proteins may act as pioneer factors, proteins that access and remodel condensed chromatin. However, differently from other pioneer factors, MADS-domain transcription factors do not actively deplete nucleosomes, but instead they interact with chromatin remodelers to shape chromatin landscape. Given the important roles of MADS-domain proteins as master regulators of developmental switches, their pioneer behaviour represents an intriguing mode of action.

High-throughput phenotyping of plant resistance to aphids by automated video tracking
Kloth, K.J. ; Broeke, C.J.M. ten; Thoen, H.P.M. ; Hanhart-van den Brink, M. ; Wiegers, G.L. ; Krips, O.E. ; Noldus, L.P.J.J. ; Dicke, M. ; Jongsma, M.A. - \ 2015
Plant Methods 11 (2015). - ISSN 1746-4811 - 14 p.
green peach aphid - nasonovia-ribisnigri - glucosinolate accumulation - signaling pathways - defense responses - feeding-behavior - myzus-persicae - lettuce aphid - arabidopsis - herbivores
Background: Piercing-sucking insects are major vectors of plant viruses causing significant yield losses in crops.Functional genomics of plant resistance to these insects would greatly benefit from the availability of highthroughput, quantitative phenotyping methods. Results: We have developed an automated video tracking platform that quantifies aphid feeding behaviour on leaf discs to assess the level of plant resistance. Through the analysis of aphid movement, the start and duration of plant penetrations by aphids were estimated. As a case study, video tracking confirmed the near-complete resistance of lettuce cultivar ‘Corbana’ against Nasonovia ribisnigri (Mosely), biotype Nr:0, and revealed quantitative resistance in Arabidopsis accession Co-2 against Myzus persicae (Sulzer). The video tracking platform was benchmarked against Electrical Penetration Graph (EPG) recordings and aphid population development assays. The use of leaf discs instead of intact plants reduced the intensity of the resistance effect in video tracking, but sufficiently replicated experiments resulted in similar conclusions as EPG recordings and aphid population assays. One video tracking platform could screen 100 samples in parallel. Conclusions: Automated video tracking can be used to screen large plant populations for resistance to aphids and other piercing-sucking insects.
Bodembacterie helpt plant tegen rupsenvraat
Sikkema, A. ; Pangesti, N.P.D. - \ 2015
Wageningen : St. voor Duurzame Ontwikkeling
arabidopsis - bodembacteriën - rizosfeerbacteriën - pseudomonas - gewasbescherming - rupsen - plaagresistentie - biologische bestrijding - landbouwkundig onderzoek - soil bacteria - rhizosphere bacteria - plant protection - caterpillars - pest resistance - biological control - agricultural research
Bodembacteriën die in het wortelmilieu van planten leven, verminderen de vatbaarheid van planten voor rupsenvraat. Dat blijkt uit onderzoek van Wageningse entomologen. In de modelplant Arabidopsis konden ze aantonen dat rhizobacteriën de plant in verhoogde staat van paraatheid brengen.
The cell size distribution of tomato fruit can be changed by overexpression of CDKA1
Czerednik, A. ; Busscher, M. ; Angenent, G.C. ; Maagd, R.A. de - \ 2015
Plant Biotechnology Journal 13 (2015)2. - ISSN 1467-7644 - p. 259 - 268.
cyclin-dependent kinase - lycopersicon-esculentum mill - plant development - arabidopsis - endoreduplication - growth - gene - expression - division - dna
Tomato is one of the most cultivated vegetables in the world and an important ingredient of the human diet. Tomato breeders and growers face a continuous challenge of combining high quantity (production volume) with high quality (appearance, taste and perception for the consumers, processing quality for the processing industry). To improve the quality of tomato, it is important to understand the regulation of fruit development and of fruit cellular structure, which is in part determined by the sizes and numbers of cells within a tissue. The role of the cell cycle therein is poorly understood. Plant cyclin-dependent kinases (CDKs) are homologues of yeast cdc2, an important cell cycle regulator conserved throughout all eukaryotes. CDKA1 is constitutively expressed during the cell cycle and has dual functions in S- and M-phase progression. We have produced transgenic tomato plants with increased expression of CDKA1 under the control of the fruit-specific TPRP promoter, which despite a reduced number of seeds and diminished amount of jelly, developed fruits with weight and shape comparable to that of wild-type fruits. However, the phenotypic changes with regard to the pericarp thickness and placenta area were remarkable. Fruits of tomato plants with the highest expression of CDKA1 had larger septa and columella (placenta), compared with wild-type fruits. Our data demonstrate the possibility of manipulating the ratio between cell division and expansion by changing the expression of a key cell cycle regulator and probably its activity with substantial effects on structural traits of the harvested fruit.
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