Abstract
The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ1-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP+ were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP+ ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Å resolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.
Highlights
Among proteinogenic amino acids, proline plays an important role in protein structure, uniquely contributing to backbone folding and stability (Ge and Pan, 2009)
The kinetic mechanisms for product inhibition were elucidated, and the regulatory effects of anions and cations were differentiated. These results suggest that under physiological conditions only NADPH would act in vivo as the electron donor, and that a stress-induced increase in the cytosolic cation content and/or in the NADPH/NADP+ ratio would instantly enhance P5C reductase activity, with no need of transcriptional control
Functional Properties of Rice P5C Reductase The cDNA of the only gene coding for P5C reductase in the japonica rice genome was subcloned into the expression vector pET151 and expressed in E. coli
Summary
Proline plays an important role in protein structure, uniquely contributing to backbone folding and stability (Ge and Pan, 2009). Features of rice P5C reductase levels has been shown in response to either osmotic, oxidative, or temperature stress (Verslues and Sharma, 2010), implying a role in stress tolerance and osmoregulation (Szabados and Savouré, 2010; Hayat et al, 2012), redox balance (Liang et al, 2013), and apoptosis (Monteoliva et al, 2014) Both the level of free proline and proline metabolism in plants were hypothesized to influence the transition to flowering (Mattioli et al, 2009), as well as pollen and embryo development (Lehmann et al, 2010; Funck et al, 2012). Null mutations of P5C reductase are embryo-lethal (Funck et al, 2012) and specific inhibitors of P5C reductase exert phytotoxic effects (Forlani et al, 2007), and may represent new active principles for weed control (Forlani et al, 2008)
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