Before their death, plant tissues undergo the essential process of senescence. Senescence is characterized by a coordinated recovery of nutrients and their retranslocation to surviving structures, such as seeds of annual plants. In monocarpic crops (e.g., maize, wheat, and barley), timing and efficiency of senescence can impact yield and grain quality. However, our understanding of senescence regulation and nutrient remobilization is limited, and protein- and metabolite-level analyses of the process are scarce, particularly in crops. To improve understanding of physiology in barley ( Hordeum vulgare L.) leaf senescence, a systems-level comparison of near-isogenic germplasm, late-senescing/low-grain protein content variety 'Karl' and an early-senescing/high-grain protein content line ('10_11'), was performed. Protein levels in flag leaves (topmost leaves) of 'Karl' and '10_11' were compared at 14 and 21 days past anthesis (dpa) using both two-dimensional fluorescence difference gel electrophoresis (2-D DIGE) and shotgun proteomic approaches. Conspicuously, proteins with roles in plant pathogen defense were present at higher levels in '10_11' as compared to 'Karl'. These included membrane receptors, glucanases, pathogenesis-related and disease resistance proteins. Proteins involved in protein degradation and organic acid/amino acid metabolism were upregulated in line '10_11' as compared to 'Karl', expectedly in early-senescing leaves involved in nitrogen remobilization. Metabolite levels were compared in the same plant material as protein levels except that analyses were also performed at anthesis (0 dpa), using mass spectrometry-based non-targeted metabolic profiling techniques. Metabolites with higher abundance in early-senescing line '10_11' included gibberellin catabolites, Yang cycle intermediates and intermediates of jasmonic acid biosynthesis. These differences were mostly observed at 0 dpa, indicating an early shift in phytohormone metabolism that may be important for senescence regulation and plant disease resistance between 'Karl' and '10_11' during the senescence phase, as jasmonic acid and ethylene have roles in plant pathogen defense. Overall, proteomic and metabolomic analyses performed here shed new light on the regulation of the senescence process, on the importance of plant defense against pathogens during senescence, and possibly on crosstalk between senescence regulation and pathogen defense. Proteins and metabolites identified in this study may become targets for ongoing efforts at improving crop yield, quality and environmental stress resistance.
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