Summary Proline is a common compatible osmolyte in higher plants. Proline accumulation in response to water stress and salinity is preceded by a rapid increase of the mRNA level of Δ1‐pyrroline‐5‐carboxylate synthase (P5CS) controlling the rate‐limiting step of glutamate‐derived proline biosynthesis. P5CS is encoded by two differentially regulated genes in Arabidopsis. Gene AtP5CS1 mapped to chromosome 2‐78.5 is expressed in most plant organs, but silent in dividing cells. Gene AtP5CS2 located close to marker m457 on chromosome 3–101.3 contributes 20–40% of total P5CS mRNA in plant tissues, but is solely responsible for the synthesis of abundant P5CS mRNA in rapidly dividing cell cultures. Accumulation of AtP5CS transcripts is regulated in a tissue specific manner and inducible by drought, salinity, ABA, and to a lesser extent by auxin. Induction of AtP5CS1 mRNA accumulation in salt‐treated seedlings involves an immediate early transcriptional response regulated by ABA signalling that is not inhibited by cycloheximide, but abolished by the deficiency of ABA biosynthesis in the aba1 Arabidopsis mutant. However, inhibition of protein synthesis by cycloheximide prevents the induction of AtP5CS2 mRNA accumulation, and blocks further increase of AtP5CS1 mRNA levels during the second, slow phase of salt‐induction. Mutations abi1 and axr2, affecting ABA‐perception in Arabidopsis, reduce the accumulation of both AtP5CS mRNAs during salt‐stress, whereas ABA‐signalling functions defined by the abi2 and abi3 mutations have no effect on salt‐induction of the AtP5CS genes.

The two-step oxidation of proline in all eukaryotes is performed at the inner mitochondrial membrane by the consecutive action of proline dehydrogenase (ProDH) that produces ⌬ 1 -pyrroline-5-carboxylate (P5C) and P5C dehydrogenase (P5CDH) that oxidizes P5C to glutamate. This catabolic route is down-regulated in plants during osmotic stress, allowing free Pro accumulation. We show here that overexpression of MsProDH in tobacco and Arabidopsis or impairment of P5C oxidation in the Arabidopsis p5cdh mutant did not change the cellular Pro to P5C ratio under ambient and osmotic stress conditions, indicating that P5C excess was reduced to Pro in a mitochondrial-cytosolic cycle. This cycle, involving ProDH and P5C reductase, exists in animal cells and now demonstrated in plants. As a part of the cycle, Pro oxidation by the ProDH-FAD complex delivers electrons to the electron transport chain. Hyperactivity of the cycle, e.g. when an excess of exogenous L-Pro is provided, generates mitochondrial reactive oxygen species (ROS) by delivering electrons to O 2 , as demonstrated by the mitochondria-specific MitoSox staining of superoxide ions. Lack of P5CDH activity led to higher ROS production under dark and light conditions in the presence of Pro excess, as well as rendered plants hypersensitive to heat stress. Balancing mitochondrial ROS production during increased Pro oxidation is therefore critical for avoiding Pro-related toxic effects. Hence, normal oxidation of P5C to Glu by P5CDH is key to prevent P5C-Pro intensive cycling and avoid ROS production from electron run-off.Stress-related changes in Pro biosynthesis and degradation are conserved in different organisms and used to induce essential signaling pathways, such as p53-mediated apoptosis in mammalian cells (1) or osmo-protecting mechanisms in plants (2) and bacteria (3). Pro is one of the most common osmolytes that, together with glycine-betaine and soluble sugars, accumulates in plants in response to a wide array of abiotic and biotic stresses (2,4,5). Although the role of Pro as an osmo-protecting molecule in prokaryotes is well established (3, 6), the overall function of stress-accumulated free Pro in plants is still under debate (2, 7-9). In most plants, stress-induced free Pro accumulation results from enhanced Pro synthesis and concomitant silencing of Pro degradation (10 -13). Pro is produced in the cytosol and chloroplasts (14, 15) and degraded in the mitochondria by a short, strictly regulated cyclic pathway (Fig. 1). In the anabolic part of the pathway, glutamate is converted to ⌬ 1 -pyrroline-5-carboxylate (P5C) 3 by P5C synthetase (P5CS), and P5C-reductase (P5CR) reduces P5C to Pro. In an alternative pathway, P5C is generated from ornithine by mitochondrial ornithine-aminotransferase (16, 17) and is reduced to Pro by P5CR in the cytosol. Thus, a possible movement of P5C among organelles and cytosol is assumed. P5CS is considered the key enzyme in the synthesis route, and its expression is increased by osmotic stress in many plants (11, 16 -18). A singl...

Proline is not only an essential component of proteins but it also has important roles in adaptation to osmotic and dehydration stresses, redox control, and apoptosis. Here, we review pathways of proline biosynthesis in the three domains of life. Pathway reconstruction from genome data for hundreds of eubacterial and dozens of archaeal and eukaryotic organisms revealed evolutionary conservation and variations of this pathway across different taxa. In the most prevalent pathway of proline synthesis, glutamate is phosphorylated to γ -glutamyl phosphate by γ -glutamyl kinase, reduced to γ -glutamyl semialdehyde by γ -glutamyl phosphate reductase, cyclized spontaneously to 1 -pyrroline-5-carboxylate and reduced to proline by 1 -pyrroline-5-carboxylate reductase. In higher plants and animals the first two steps are catalysed by a bi-functional 1 -pyrroline-5-carboxylate synthase. Alternative pathways of proline formation use the initial steps of the arginine biosynthetic pathway to ornithine, which can be converted to 1 -pyrroline-5-carboxylate by ornithine aminotransferase and then reduced to proline or converted directly to proline by ornithine cyclodeaminase. In some organisms, the latter pathways contribute to or could be fully responsible for the synthesis of proline. The conservation of proline biosynthetic enzymes and significance of specific residues for catalytic activity and allosteric regulation are analysed on the basis of protein structural data, multiple sequence alignments, and mutant studies, providing novel insights into proline biosynthesis in organisms. We also discuss the transcriptional control of the proline biosynthetic genes in bacteria and plants.

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In an attempt to increase the insecticidal effect of the ␦-endotoxin crystal protein CryIC on the relatively Cry-insensitive larvae of Spodoptera littoralis, a combination of CryIC and endochitinase was used. CryIC comprising the first 756 amino acids from Bacillus thuringiensis K26-21 and endochitinase ChiAII encoded by Serratia marcescens were separately produced in Escherichia coli carrying the genes in overexpression vectors. The endochitinase on its own, even at very low concentrations (0.1 g/ml), perforated the larval midgut peritrophic membrane. When applied together with low concentrations of CryIC, a synergistic toxic effect was obtained. In the absence of chitinase, about 20 g of CryIC per ml was required to obtain maximal reduction in larval weight, while only 3.0 g of CryIC per ml caused a similar toxic effect in the presence of endochitinase. Thus, a combination of the Cry protein and an endochitinase could result in effective insect control in transgenic systems in which the Cry protein is not expressed in a crystalline form.