Mitochondrial regulation through thiol-switching in plants

Abstract

The seed represents a key stage in the lifecycle of spermatophytes (seed plants). Dry seeds can overcome unfavourable conditions, such as drought, heat or cold, by maintaining their metabolism in a quiescent state. Yet, the reactivation of the metabolism is an important prerequisite for germination to provide energy and organic compounds, and is initiated by imbibition with water. How exactly the restart of metabolism occurs and how it is regulated to drive germination while operating in a resource efficient manner is insufficiently understood. Since de novo gene expression is energy demanding and starts comparably late in germination, posttranslational protein modifications have been implicated as a key regulatory mechanism in quiescence release. <br /> In this work the reestablishment and the regulation of the (respiratory) energy metabolism was investigated. A methodology was developed to monitor rearrangements of energy and redox metabolism in seeds live and with subcellular specificity and very early at imbibition. That was possible by optimising and employing different genetically encoded fluorescent probes, including a probe for monitoring hydrogen peroxide (roGFP2-Orp1), targeted to the mitochondrial matrix and the cytosol in seeds of Arabidopsis thaliana. Rapid rebooting of ATP homeostasis and thiol redox systems within the first hour of imbibition was observed and independently confirmed by established analytical methods. To generate a framework of redox regulation of mitochondrial proteins during this restart, a model system of isolated, functional mitochondria was established. Monitoring of the glutathione redox potential dynamics by roGFP2-Grx1 in the matrix showed that the addition of specific substrates led to a sharp activation of the matrix thiol redox systems, similar to what had been observed in imbibed seeds. Cysteine residues of proteins underwent specific reduction mediated by the endogenous thiol redox machinery of the mitochondrial matrix, while the thermodynamic, kinetic and structural constraints that provide specificity to thiol redox switching in vivo were largely maintained. In situ targets of the mitochondrial redox machinery were identified and the degree of redox-switching was quantified by iodoacetyl tandem mass tag (iodoTMT)-based thiol redox proteomics. Proteins involved in mitochondrial energy metabolism, such as of different respiratory complexes and the tricarboxylic acid cycle, and of the amino acid metabolism, were identified as targets of redox switching. To dissect target specificity of the individual redox machineries and their potential impact on the<br /> seed physiology during germination, different mutants of the individual mitochondrial redox systems were investigated. Mutants impaired in either the glutathione-/ or thioredoxin-linked redox machineries of the mitochondrial matrix showed lowered germination vigour, especially in aged seeds. Their seeds showed increased respiration rates at imbibition, while the reestablishment of their adenine nucleotide energy charge was not affected, but intermediates of the tricarboxylic acid cycle were altered. Only a small subset of proteins was found to be affected in their thiol redox status in the different mitochondrial redox mutants, as investigated in the isolated mitochondria model by redox proteomics. <br /> Taken together, the observations of this work suggest that redox and energy physiology of seeds are reactivated immediately at imbibition. A global reductive shift of specific protein cysteine residues includes enzymes of the respiratory metabolism, which redox-switching may trigger (de-)activation, allowing fine-tuning of activities at imbibition to optimise germination efficiency.