Abstract
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ1-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD+ as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.
Highlights
Intracellular accumulation of high proline levels as a stress protectant has long been reported in many plant species in response to a wide array of abiotic stress conditions (Verbruggen and Hermans, 2008), ranging from drought, excess salt, and cold (Hayat et al, 2012) to the treatment with heavy metals (Sharma and Dietz, 2006)
Expression of Rice P5CDH under Hyperosmotic Stress Conditions, and in Response to the Exogenous Supply of Metabolically Related Amino Acids To investigate the regulation of OsP5CDH during stress-induced proline accumulation, rice suspension cultured cells were treated with increasing concentrations of either NaCl or polyethylene glycol (PEG) to induce salt or osmotic stress conditions, respectively
Rice pyrroline5-carboxylic acid (P5C) Dehydrogenase is Constitutively Expressed and High Intracellular Levels of Proline Increase only Slightly its mRNA Abundance, whereas its Transcript Level Seems not Subject to Environmental Control Proline oxidation to glutamate takes place in the mitochondrion by means of a short pathway in which a membrane-bound proline dehydrogenase converts proline into P5C, which in turn is further oxidized by a soluble P5C dehydrogenase (Forlani et al, 1997a)
Summary
Intracellular accumulation of high proline levels as a stress protectant has long been reported in many plant species in response to a wide array of abiotic stress conditions (Verbruggen and Hermans, 2008), ranging from drought, excess salt, and cold (Hayat et al, 2012) to the treatment with heavy metals (Sharma and Dietz, 2006). High concentrations of proline in the cell increase in turn reactive oxygen species (ROS) formation in the mitochondrion through the activity of a proline dehydrogenase [EC 1.4.3] that is believed to transfer electrons directly to the respiratory chain (Liang et al, 2013). The need to balance ROS production and scavenging to maintain optimal signaling levels for reinstating metabolic homeostasis during stress situations has been well-established (Türkan and Demiral, 2009). During the recovery from stress, this short catabolic pathway provides energy, reducing power and precursors for nucleotide synthesis to allow the resumption of cell division (Hare and Cress, 1997)
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