The Individual Research Projects (IRP)

Each Research Work Package represents one major topic of the project. The young researchers will work interdisciplinary in one or several RWPs in this project as defined by the Individual Research Package (IRP).

Research Project IRP1 - PhD position ESR1

Host: Enrico Schleiff, Goethe University Frankfurt am Main (GUF)

Protein homeostasis during pollen development at higher temperatures

The regulation of protein and membrane homeostasis is interlinked to environmental conditions. For example, abundance and efficiency of transport complexes in the different endomembranes is dependent on environmental conditions and heat inducible HSPs are involved in recycling of preproteins under stress. In parallel, most transport systems have a multi-component architecture, and often more than one gene is found to encode for the individual components. The project aims to decipher the components involved in adaptation of protein distribution and homeostasis at high temperatures in pollen. This will include the analysis of the intracellular protein distribution system in pollen in general. Thereby, the objectives are:

1) Identification of translocon components involved in transport and translocation processes in pollen,
2) analyzing their response to environmental changes, and
3) description of their functional interplay with the regulatory network in pollen thermotolerance.

This work will include molecular, biochemical and cell biological methods.

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Research Project IRP2 - PhD position ESR2

Host: Ivo Rieu, University of Nijmegen (RUN)

The role of epigenetic modification in acclimation to heat stress in tomato

Induced tolerance to heat stress (HS) has been shown to be heritable within and over generations, suggesting the involvement of epigenetic control. Understanding epigenetic processes in pollen thermotolerant plants requires the description of the changes of stress-induced gene expression associated with alterations in DNA methylation and histone modifications; the acquirement of these changes during first stress experience; and the relation of the changes to later stress incidents. Thus, the project will focus on the question which epigenetic modifications lead to the acquisition of HS tolerance. The objectives are:

1) to determine the relation between HS regime and heritability of the induced tolerance,
2) to identify mitotically and meiotically stable HS-induced DNA methylation patterns, and
3) to correlate methylation patterns to the observed changes in gene expression and heat tolerance.

In this project high-throughput genomic techniques and other molecular biology tools will be applied.

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Research Project IRP3 - PhD position ESR3

Host: Celestina Mariani, University of Nijmegen (RUN)

Hsf-Hsp interaction and function in tomato anthers and pollen

The interaction between the regulatory intracellular networks is a major element of stress responses leading to pollen thermotolerance (PTT), but the function of components involved is not well characterised. The project will focus on generation of GM plants with pollen-specific expression or knock-down of particular genes involved in PTT. The objectives are:

1) to prove the activity of suitable promoters in planta, with respect to cell/tissue specificity and HSR,
2) to analyze the influence of altered Hsf and Hsp expression on PTT specifically, and
3) to investigate the influence on plant growth, development and stress tolerance in general.

Analysis of phenotypical and physiological parameters requires the application of genetic, molecular biological and physiological approaches.

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Research Project IRP4 - PhD position ESR4

Host: Nurit Firon, The Agricultural Research Organisation of Israel - The Volcani Centre (ARO)

The role of hormones and pollen-specific heat stress response genes in pollen thermotolerance

HSFs, chaperones and genes involved in hormone signaling and carbohydrate metabolism are involved in developing pollen HSR. Several of the identified pollen-expressed HSR genes are homologues of Arabidopsis genes that are expressed exclusively or mainly in pollen. These include a specific member of the Hsp90 chaperone family, genes that are involved in Ca2+-dependent signaling and vesicle trafficking, suggesting that maturing microspores may recruit available factors, including signaling network components used for maintaining pollen-specific functions. The project will target the activity of Hsfs during thermotolerance acquisition and their crosstalk with other, hormone based signaling pathways. The objective is to characterize the involvement of

1) Pollen-specific genes recruited for coping with heat stress.
2) Crosstalk between the HSF regulatory cascade and hormone (ethylene) signaling.

The project involves methods of molecular biology, plant physiology and microscopy.

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Research Project IRP5 - PhD position ESR5

Host: Rina Iannacone, Metapontum Agrobios S.R.L. (MAB)

Production and analysis of mutants in HSF and HSP genes

The over-expression of HSP genes in transgenic organisms was demonstrated to improve the tolerance to several abiotic stresses, but the effect of heat stress on pollen vitality produced by transgenic plants over-expressing HSP genes was poorly investigated so far. Thus, the project will aim at identification and analysis with respect to pollen thermotolerance of HSF and HSP mutants in tomato. The objectives are:

1) to verify that the genetic modifications will give beneficial effects on pollen under elevated heat stress and
2) to identify new allelic variants of HSFs or HSPs by the TILLING approach and to explore the influence of these mutants on PTT.

The project involves plant genetics, plant molecular biology and plant physiology, including high-throughput screening approaches.

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Research Project IRP6 - PhD position ESR6

Host: Arnaud Bovy, Wageningen Unversity (WUR)

Metabolomics of thermotolerant tomato lines

Environmental stress leads to an altered or even disrupted metabolism in plants. Reprogramming of gene expression during heat acclimation leads to production of proteins and metabolites required to achieve a new state of homeostasis; and changes of three types of metabolites can be envisaged: i) signal transduction molecules, ii) compounds involved in acclimation; iii) products of disrupted pathways. Remarkably little is known of the metabolic changes associated with thermotolerance in crops or in developing anthers and pollen. A selection of tomato cultivars with contrasting pollen thermotolerance and specific mutants will be analysed for metabolic changes associated with heat acclimation to obtain insight in metabolic processes in developing tomato anthers and pollen. The objectives are

1) to elucidate the metabolic and genetic basis underlying pollen thermotolerance,
2) generation of Near Isogenic lines (NILs) by crossing with wild relatives of tomato and analyzing metabolomic profiles and pollen thermotolerance in populations derived from this.

The project involves plant genetics, molecular biology, plant physiology, and advanced metabolomic profiling approaches.

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Research Project IRP7 - PhD position ESR7

Host: Wolfram Weckwerth, University Vienna (UNV)

Proteomics of pollen thermotolerance - high throughput screening for protein markers and protein modifications

The identification of proteins as BIOMARKER is a major goal of molecular methods assisting plant breeding. The requirements for this strategy are a high sample throughput for the initial discovery and a subsequent high throughput analysis of the BIOMARKER in hundreds or thousands of plant lines. The PhD student will investigate proteomic differences in pollen of distinct tomato lines and establish methods for this analysis. A focus will be placed on the analysis of protein modifications. The objectives are:

1) to optimize and apply this platform to anthers and pollen tissues to establish a proteome map of developing pollen.
2) genome-scale quantification of proteins in response to temperature treatment, including establishing of thorough pre-fractionation techniques (SDS-PAGE, FPLC, HPLC or IF),
3) establishment of rapid Mass Western for specific target proteins applicable for high-throughput analysis of many plant lines,
4) integration of proteomics metabolomics and transcriptomics data (GXP,WUR,UNF) and
5) analysis of histone modifications and protein phosphorylation using proteomics profiling. The project involves plant molecular biology, biochemistry, and advanced proteomics approaches.

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Research Project IRP8 - PhD position ESR8

Host: Maria Luisa Chiusano, University of Studies of Naples Frederico II (UNF)

Development of a gene network for modelling of pollen thermotolerance

All high-throughput approaches require the management and organization of the data produced and the integration of the manifold existing resources for the identication of variations with respect to environment or genetics. The project will include the development of an integrated database with metabolomic, transcriptomic and proteomic data associated to information related to the many gene and gene families involved. The objectives are

1) development of an efficient data management system,
2) integration of proteomics, metabolomics, and transcriptomics data derived in the different projects,
3) modelling of the regulatory network implied in the HSR, and
4) comparative analysis among heat sensitive and tolerant genotypes to provide support for the functional analysis of HSF and HSP gene families, and the comparative analysis of data from cultivars and mutants with differential heat tolerance.

The project involves data management, data mining and bioinformatics.

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Research Project IRP9 - Postdoc position ER1

Host: Klaus-Dieter Scharf, Goethe University Frankfurt am Main (GUF)

Regulation and interactions of pollen-specific Hsf and chaperone networks

The control of the cellular heat stress response is linked to the activity of Hsfs and chaperones acting as regulators of Hsfs, and interactions within and between the two families play a key role for adaptation of heat stress gene expression. Differences in transcript and protein levels of Hsfs and Hsps in developing pollen of heat-sensitive and heat-tolerant tomato cultivars have been reported. The project will focus on the functional analyses of the pollen-specific Hsf and Hsp networks. The objectives are:

1) to describe the regulatory cascades involved in the developmental control of Hsf and Hsp expression in pollen,
2) to verify the specific function of individual factors and the role of their interplay in pollen heat stress response, and
3) to identify factor-specific differences mirroring the temperature limits in pollen thermotolerance.

The project involves biochemical, cell biological and physiological approaches. Thereby, it is expected that the postdoc will bring expertise into the project, but he will also find optimal conditions to be trained in the complementary techniques.

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Research Project IRP10 - Postdoc position ER2

Host: Peter Winter, GenXPro GmbH (GXP)

Transcriptomes and epigenomes of germplasm and breeding lines contrasting in their response to heat stress

Plant responses to environmental stress involve concerted re-regulation of transcription of thousands of genes. Reports on the contribution of small RNAs and Natural Antisense Transcripts (NATs) to stress tolerance are only emerging and the epigenetic control of transcription and its regulation has been described for only a few loci in model plants. Consequently, the systems biology of stress tolerance requiring such knowledge is still in its infancy. The project includes the high throughput analysis of the transcriptome and epigenetic modifications in different cell types based on advanced deep-sequencing and transcriptome profiling techniques. The objectives are

1) dissecting the genetic factors underlying pollen thermotolerance (PTT) into individual components and
2) identifying cytosin methylation/demethylation patterns that may predispose to PPT.

The project requires experiences in appropriate molecular and bioinformatic techniques.

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