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ESR6 (INRA)

Project title: Nitrogen remobilization and autophagy in senescing leaves and in response to nitrogen availability

Objectives: The objective of ESR6 is to determine whether autophagy is a key mechanism in the control of nitrogen remobilization and chloroplast degradation in barley, and to study whether the senescence barley mutants identified by CAU are impaired in nitrogen remobilization and autophagy. As for ESR1, the final goal is to select barley lines for breeding.

Description of work: Previous work at INRA had identified in Arabidopsis molecular, enzymatic and metabolic markers of N remobilization and autophagy. ESR6 will be in charge of monitoring the timetable of senescence, autophagy and N remobilisation events during leaf ageing of barley grown upon plentiful nitrate supply or under nitrate limiting conditions using the same kind of markers as used in Arabidopsis. ESR6 will design probes for quantitative real-time RT-PCR. ESR6 will be in charge of producing in vitro GFP/RFP::ATG8 fusions for barley transformation. In collaboration with ESR9 (UL), ESR6 will monitor autophagosome, stromule and vesicle production in WT, and in senescence HvWhirly and HvSV40 mutants in response to nitrogen, ageing and darkness. ESR6 will produce HvATG5 and HvATG2 RNAi constructions. Transgenic barley lines will be prepared at UA and used to estimate the role of autophagy in chloroplast degradation and nitrogen remobilisation in barley. ESR6 will characterize the phenotype of autophagy-defective plants upon high and low nitrate supplies. In collaboration with ESR9, ESR6 will examine the protease, protease inhibitor and chloroplast proteins present in the leaves of autophagy mutants barley grown at high and low nitrate supplies. ESR6 will monitor nitrogen remobilisation to the seeds in the various barley genotypes (HvWhirly, HvSV40, HvATG) using 15N tracing and will analyse seed contents.

Deliverables: Molecular markers for autophagy; transgenic barley plants with HvATG RNAi constructs and UBQ::GFP/RFP::HvATG8 constructs