3. Proline

Proline is one of the 20 standard amino acids that make up proteins. It is highly water soluble and acts as a potent osmoprotectant in bacteria and higher plants. Its concentrations in plant cells vary drastically depending upon both the exogenous and endogenous physiological conditions. Proline levels in plant cells can be increased up to 400-fold upon osmotic stress, and its concentrations can reach to over 200 mM in the cytoplasm of cultured tobacco cells (Binzel et al., 1987; Delauney and Verma, 1994). Accumulated proline can protect enzymes and other macromolecules from denaturation, serves as an energy reserve during stress, and acts as an antioxidant against reactive oxygen species (ROS) and other free radicals [Fig 1].

Accumulation of proline is due primarily to de novo proline biosynthesis. In E. coli and yeast, proline is synthesized from glutamate, which is catalyzed by three enzymes, GK (γ-glutamyl kinase), GPR (glutamyl-γ-phosphate reductase), also known as GSADH (glutamate-5-semialdehyde dehydrogenase, and P5CR (Δ1-pyrroline-5-carboxylate reductase) (Csonka and Hanson, 1991). In plants, proline can be synthesized from glutamate and ornithine. The biosynthesis of proline from glutamate is catalyzed by two enzymes, P5CS (Δ1-pyrroline-5-carboxylate synthetase) and P5CR [Fig 2] (Hu et al., 1992; Delauney and Verma, 1994). The biosynthesis of proline from ornithine can occur via two routes [Fig 3]. The first route involves the transamination of δ-NH2 group of ornithine, and is catalyzed by two enzymes, δ-OAT (ornithine δ-aminotransferase) and P5CR. The other route involves the transamination of α-NH2 group and is catalyzed by α-OAT (ornithine α-aminotransferase) and P2CR (Δ1-pyrroline-2-carboxylate reductase). Genes encoding P5CS, P5CR, and δ-OAT have been cloned (Delauney and Verma, 1994). However, genes for α-OAT and P2CR have yet to be cloned. Thus, proline could be synthesized from glutamate and ornithine via three pathways, i.e. the P5CS pathway, the δ-OAT1 pathway, and the α-OAT1 pathway.

Proline levels are reduced in plant cells in response to the removal of osmotic stress. Proline is converted to glutamate, which is catalyzed by PDH (proline dehydrogenase, also known as proline oxidase) and P5CDH (Δ1-pyrroline-5-carboxylate dehydrogenase). Proline recycling can provide both C- and N-sources for the needs of rebuilding of plants during recovery from stress. Proline recycling recovers only a portion of the energy used to synthesize it.

Our results have demonstrated that P5CS is the key enzyme that is responsible for the accumulation of proline under stress. Overexpression of P5CS leads to increased proline accumulation in transgenic plants. In bacteria and plants, amino acid biosynthesis is known to be regulated predominantely via feedback inhibition by the end product of the pathway [Fig 4]. Without exception, proline biosynthesis in bacteria and plants is also feedback-inhibited by the concentration of proline. In E. coli, a key residue, Asp-107 (D-107), of the GK enzyme (proB protein) is known to be critical for feedback inhibition by proline and an Asp-to-Asn (D-to-N) substitution position 107 removes the feedback inhibition by proline. In plants, the corresponding Asp residue (D-126) in P5CS plays no role in feedback inhibition. Two surrounding residues, Asp-126 (D-126) and Phe-129 (F-129), are critical for feedback regulation and a substitution of either D-126 or F-129 with Alanine (D126A or F129A) effectively abolishes the feedback inhibition by proline. Overexpression of this F129A mutant of P5CS has been demonstrated to confer the ability of high level proline accumulation and osmotolerance in transgenic plants [Fig 5-6].

Fig 1. Roles of proline in plants.

Proline (Pro) is one of the 20 amino acids that make proteins. Pro is a prevalent compatible solute (osmolyte) that accumulates in bacteria and plants when exposed to challenging environmental conditions. Osmotic stress (drought or high salinity) and oxidative stress could lead to Pro accomulation in plants. Pro serves as an ideal reservoir of carbon and nitrogen compounds, which could be easily recycled during recovery from stress. High concentrations of Pro does not cause toxicity to the cell, and, instead, it protects the cell via different mechanisms. There has been mount of evidence that suggests that Pro could help remove free dadicals, could stabilize enzymes, could help maintain cytosolic pH buffer and NAD+/NADH balance.

Fig 2. A bifunctional enzyme is key to proline synthesis from glutamate.

In bacteria, Pro is synthesized from glutamate (Glu) via the functions of three enzymes, known as ProB, ProA, and ProC. ProB and ProA form a protein complex to catalyze the first two reactions of this pathway.

In plants, a protein with two distinct domains exhibiting similarities to bacterial ProB and ProA, respectively, has been cloned and designated as Δ1-pyrroline-5-carboxylate synthetase (P5CS) (Hu et al., 1992; Delauney & Verma, 1994).

The last step is catalyzed by Δ1-pyrroline-5-carboxylate reductase (P5CR) in bacteria and in plants.

Fig 3. Proline can be synthesized from ornithine in plants.

Ornithine (Orn) is an amino acid, not used for protein synthesis. It has an α-amino group and a δ-amino group (B), which both could be used for amination of 2-ketoglutarate to produce glutamate. The remaining skeleton from Orn is either glutamic γ-semialdehyde (GSA) or α-keto-δ-aminovalerate (KAV), which could be reduced to proline.

(A) Genes encoding δ-ornithine transferase (δ-OAT1) has been cloned from plants (Hu et al., 1992; Delauney & Verma, 1994). This pathway plays important roles in proline synthesis under high N conditions (Delauney et al., 1993).

(B) In vivo tracer studies with labeled ornithine strongly suggest that the KAV to P2C pathway of ornithine metabolism is the main route of conversion of ornithine to proline in several plant species (Mestichelli et al, 1979). However, genes encoding α-OAT1 and P2CR have yet to be cloned in plants and its importance in proline synthesis remains to be demonstrated.

Fig 4. P5CS is feedback-inhibited by Pro and this inhibition can be eliminated by mutation

(A) Plant P5CS and bacterial the glutamyl kinase (GK or ProB) enzymes are feedback-inhibited by Pro, the end product of the Pro synthesis pathway from Glu.

(B) ProB and P5CS enzymes have a dedicated allosteric site for Pro binding, which is separate from their catalytic site, and mutations in this Pro binding site affect feedback inhibition without changing their catalytic activities. In bacteria, an Asp-to-Asn subistitution at position 107 (D107N) of ProB removes the feedback inhibition by Pro. The Asp residue, corresonding to the D107 of ProB is D128 in plant P5CS. Surprisingly, a D128A mutation of P5CS has no effect on feedback inhibition. However, a D126A mutation of P5CS has a greatly reduced level of feedback inhibition, and more drastically, an F129A mutation of P5CS has basically eliminated its feedback inhibition by Pro.

(C) The presence of 5 mM Pro could inhibit P5CS enzyme activity by 50%. For the D126A mutant of P5CS, it takes 85 mM Pro to inhibit its 50% activity. For the F129A mutant, it takes 960 mM to achieve 50% activity inhibition. The Pro concentration would never reach 960 mM in plant cells and thus, this F129A mutant is basically insensitive to Pro accumulation.

Fig 5. Overexpression of the P5CS and its Pro-insensitive mutant leads to accumulation of Pro

(A) Moth bean (Vigna aconitiforlia) P5CS and the F129A mutant were expressed in tobacco under the CaMV 35S promoter.

(B) The mRNA transcript and protein levels of the transgenes were high in transgenic tobacco plants as shown on Nothern blot and Western blot.

(C) Proline contents were high in transgenic tobacco plants under the normal growth condition and under salanity stress (200 mM NaCl). About 10-fold more Pro is detected in transgenic plants than in WT tobacco plants. About 2-fold increase in Pro is found in F129A plants than in P5CS-transgenic plants.

Fig 6. Improved osmotolerance in transgenic plants expressing P5CS and its F129A mutant.

(A) Transgenic plants expressing P5CS exhibited significantly improved growth at the seedling and adult stages, when treated with 200 mM NaCl.

(B). Transgenic plants expressing P5CS exhibited significantly improved root growth of adult stages, when treated with 200 mM NaCl.

(C-D). When germinated on agar-plate containing 200 mM NaCl, transgenic plants expressing P5CS had a higher germination rate and developed more seedling mass than the control plant (pBI121). Transgenic plants expressing the Pro-insensitive mutant F129A had even more improved osmotolerance than the P5CS plants.

References

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Csonka LN, Gelvin SB, Goodner BW, Siemieniak D, Slightom JL (1988) Nucleotide sequence of a mutation in the proB gene of Escherichia coli that confers proline overproduction and enhanced tolerance to osmotic stress. Gene 64: 199-205

Csonka LN, Hanson AD (1991) Prokaryotic osmoregulation: genetics and physiology. Annu Rev Microbiol 45: 569-606

Delauney AJ, Hu CA, Kavi Kishor PB, Verma DPS (1993) Trans-complementation of E. coli for ornithine aminotransferase and isolation of a cDNA clone from Vigna aconitifolia. J Biol Chem 268: 18673-18678

Delauney AJ, Verma DPS (1993) Proline biosynthesis and osmoregulation in plants. Plant J 4: 215-223

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Hu CA, Delauney A, Verma DPS (1992) A bifunctional enzyme (Δ1-pyrroline -5-carboxylate synthetase) catalyses the first two steps in proline biosynthesis. Proc Natl Acad Sci USA 89: 9354-9358

Kavi Kishor PB, Hong Z, Miao G-H and Verma DPS (1995) Overexpression of Δ1-pyrroline-5-carboxylate synthetase increases proline production and confers osmotolerance in transgenic plants. Plant Physiol. 108: 1387-1394

Mestichelli LJJ, Gupta RN, Spenser ID (1979) The biosynthetic route from ornithine to proline. J Biol Chem 254: 640-647

Szoke A, Miao G-H, Hong Z and Verma DPS (1992) Subcellular location of Δ1-pyrroline-5-carboxylate reductase in root/nodule and leaf of soybean. Plant Physiol 99:1642-1649

Zhang CS, Lu Q, Verma DPS (1995) Removal of feedback inhibition of Δ1-pyrroline-5-carboxylate synthetase, a bifunctional enzyme catalyzing the first two steps of proline biosynthesis in plants. J Biol Chem 270: 20491-20496

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