N-vironment - The role of the N-end rule in plant response to the environment

Manipulation of plants to provide stability of yield under unpredictable growth conditions will be essential to respond to the effect of climate change in increasing the uncertainty of the agricultural environment. Selective and conditional removal of regulator proteins by proteolysis is emerging as a major regulatory principle in plants. The aim of the N-vironment project is to provide a complete mechanistic understanding of the role of the N-end rule pathway of targeted proteolysis in controlling plant responses to the environment. To achieve this goal consortium partners have been selected that represent internationally leading European teams with experience in the pathways multiple facets, including the biochemical basis of the pathway, hypoxia and plant development, transcriptional regulation of stress and metabolic signalling. The project will bring together six research groups with complementary expertise in fundamental molecular plant science, biochemistry and chemistry, in four institutions. The research programme of the N-vironment consortium will be achieved through six inter-related Work Packages carried out by the four consortium partners (three funded by ERA-CAPS, one associated laboratory). The objectives of these work packages are related to three fundamental questions that arose after integration of the recent discoveries by consortium members of the role of the N-end rule pathway of targeted proteolysis as a major regulator of plant development and response to the environment. Question 1: What are the protein substrates and enzymatic components of the N-end rule pathway? Question 2: How is the Nend rule pathway integrated into cellular signaling pathways? Question 3: What is the extent of the role of the N-end rule pathway in plant response to the biotic and abiotic environment? The proposed research has highly innovative measurable outcomes that address this newly discovered area of plant biology, and will uncover: New mechanisms regulating protein stability, new mechanisms of environmental stress sensing, new functions for proteins in stress sensing, the importance of the N-end rule in a key EU crop, tomato. This fast-developing area of plant molecular science is led by N-vironment members (including discovery of substrates, methods of entry into the N-end rule pathway, biochemical components of the pathway), and together with the availability of a large number of unique resources within the consortium, makes the N-vironment proposal highly novel and timely. The synergistic value of this collaborative programme will be the development and exploitation of a highly novel area of plant biology of key importance to agriculture, in which Europe has the capacity to take a world lead through ERA-CAPS funding.

The project >The role of the N-end rule in controlling plant response to the environment (N- vironment)< was initiated as an ERA-CAPS EU project. Groups from the UK, Germany and Italy collaborated with the applicant to analyze the role of the N-end rule pathway of protein turnover in plant responses to the environment. We could show that the N-end rule degradation pathway is not only instrumental in measuring oxygen concentration in plant tissues, but also for sensing nitric oxide (NO). As a result, environmental parameters are linked to transcriptional control, in order to mediate survival of plants under the stressful conditions of flooding (oxygen shortage) and other stresses (that lead to low intracellular nitric oxide concentrations). The applicants group could contribute to elucidation of the role of nitric oxide in transcriptional control. Furthermore, mutants in the N-end rule pathway were characterized and made available to other groups for their experiments. Arabidopsis lines with reporter constructs for monitoring N-end rule protein turnover in plants were also made available. A major effort went into investigation of the enzymology of the N-end rule pathway. This pathway consists of a set of enzymes that recognize amino-terminal amino acids of proteins. Depending on the nature of the first amino acid, the pathway initiates proteolytic destruction of the protein. Recognition of proteins with amino-terminal Leucin is not yet understood molecularly. Mutants in turnover of a reporter protein with amino-terminal Leucin were analyzed. We could identify one factor involved in the degradation, but it turned out that the novel pathway encompasses additional proteins that remain to be characterized in a follow- up project. In parallel to the characterization of mutants, we elucidated alternative methods for studying protein interactions in the N-end rule pathway. In addition, proteins were investigated that contain domains with known ability to bind to amino-termini of proteins. Arabidopsis encodes several candidate proteins, which were investigated for a role in the N-end rule pathway of protein degradation.