Proline metabolism and its implications for plant-environment interaction

Abstract

Proline has long been known to accumulate in plants experiencing water limitation and this has driven studies of proline as a beneficial solute allowing plants to increase cellular osmolarity during water limitation. Proline metabolism also has roles in redox buffering and energy transfer and is involved in plant pathogen interaction and programmed cell death. Some of these unique roles of proline depend on the properties of proline itself, whereas others depend on the "proline cycle" of coordinated proline synthesis in the chloroplast and cytoplasm with proline catabolism in the mitochondria. The regulatory mechanisms controlling proline metabolism, intercellular and intracellular transport and connections of proline to other metabolic pathways are all important to the in vivo functions of proline metabolism. Connections of proline metabolism to the oxidative pentose phosphate pathway and glutamate-glutamine metabolism are of particular interest. The N-acetyl glutamate pathway can also produce ornithine and, potentially, proline but its role and activity are unclear. Use of model systems such as Arabidopsis thaliana to better understand both these long studied and newly emerging functions of proline can help in the design of next-generation experiments testing whether proline metabolism is a promising metabolic engineering target for improving stress resistance of economically important plants.

Figure 1.

Examples of stress-induced proline accumulation in Arabidopsis. (A) Proline content after transfer of 7-day-old seedlings to low water potential polyethylene glycol (PEG)-agar plates. Seedlings were held at -1.2 MPa for 4 days and then transferred back to high water potential media (-0.25 MPa). PEG-agar plates were used to mimic the decrease in water potential of a drying soil while allowing a constant and controlled severity of stress to be applied. Transfer to low water potential caused a nearly 100-fold increase in proline content over 4 days (circles). Proline levels declined back to control levels over a similar time period after return to high water potential (squares). Seedlings kept at -0.25 MPa for the entire experiment maintained proline content below 1 µmol g FW^-1 (triangles) Figure is modified from Sharma and Verslues (2010). Data are means ± standard error (n = 10–12) (B) Comparison of proline content after low water potential (PEG) or NaCl treatment. Seven-day-old seedlings were transferred to the indicated treatments and proline content measured 4 days later. Proline content increased with increasing severity of stress for low water potential and NaCl; however, low water potential imposed by PEG always elicited approximately ten-fold more proline accumulation than NaCl treatment of similar water potential. This is likely because proline is needed for osmotic adjustment in the low water potential treatment. In the salt treatment, ion uptake means there is less solute needed for osmotic adjustment and the proline that does accumulate may have other protective functions. Data are from Sharma and Verslues (2010) and are means ± standard error (n = 5–10).

Figure 2.

The core pathways of proline metabolism. Proline synthesis from glutamate occurs in the cytoplasm and/or chloroplast via two enzymatic steps. Proline catabolism to glutamate occurs in the mitochondria also by two enzymatic steps. Proline synthesis and catabolism both use the common intermediate P5C (formed by spontaneous cyclization of glutamic-5-semialdehyde produced by P5CS or ProDH). Solid lines indicate known metabolic or transport steps. Dashed lines indicate proposed but not demonstrated steps. Both mitochondrial proline Import carriers as well as a proline-glutamate exchanger have been demonstrated in transport studies of isolated mitochondria. Transport of P5C across the mitochondrial membrane to allow cyclic metabolism between ProDH and P5CR has been proposed but not demonstrated. The mitochondrial Basic Amino Acid Carher1 (BAC1) and BAC2 may also influence proline metabolism by movement of arginine or ornithine. OAT had been thought to be a cytoplasmic enzyme that functioned as an alterantive route to proline but more recent evidence has placed it in the mitochondria. Arabidopsis gene identification numbers and all gene name abbreviations are given in Table 1.

Figure 3.

Transcriptional regulation of core genes of proline metabolism by low water potential, salt stress or stress recovery. Consensus transcriptional regulation of proline metabolism from literature, our own work, and publically available microarray data (Genevestigator). Black lines indicate no change from basal level expression in unstressed conditions. Blue lines indicate up-regulation with the thickness of line approximately corresponding to the extent of up-regulation. Dashed red lines Indicate down regulation. Stress release refers to recovery immediately after either low water potential or salt stress.

Figure 4.

Connections of praline metabolism to other pathways. Connections of proline synthesis to nitrogen assimilation by GS and GOGAT; the N-acetyl-glutamate pathway (chloroplast); the oxidative pentose phosphate pathway (chloroplast); GABA metabolism (cytoplasm and mitochondria); and, ornithine-arginine metabolism (mitochondria). Note that in this illustration, proline synthesis is depicted solely in the chloroplast; however, cytoplasmic proline synthesis is also possible. Dashed lines indicate reactions that have not been experimentally observed in Arabidopsis. Transport steps are drawn merely as the most direct route as transporters for these steps remain mostly unidentified. Note that many of these reactions are catalyzed by multiple isozymes with different compartmentation; not all of the possible isozymes/localizations are shown. Arabidopsis gene identification numbers and gene name abbreviations are given in Table 1.

Figure 5.

Functions of proline and proline transport in growth and osmotic adjustment. (A) Scheme of possible long distance proline transport in plants experiencing water limitation. In this scenario, proline synthesized in the photo-synthetic tissue is transported to non-photosynthetic tissues (such as root) or other sinks and used to maintain metabolism and growth. A product of proline catabolism could then be transported back to the source tissue to sustain the cycle. Such a scenario would be consistent with high levels of proline observed in the phloem of drought stressed plants and may be one role of proline transporters that have been identified but whose physiological function is unclear. (B) Osmotic adjustment and proline compartmentation. Cell on the left is unstressed (high water potential, -0.3 MPa) and has relatively low levels of proline and other solutes. Cell on the right is exposed to a moderate low water potential stress (-1.0 MPa). The cell on the right has decreased osmotic potential (ψ_s) to maintain water potential (ψ_w) equilibrium with its surroundings. The solute accumulation reflected in the decreased ψs has allowed cell volume and turgor (ψ_p) to be maintained (as ψ_s and ψs are the dominant components of water potential in this example the cellular water status can be expressed as ψ_w = ψ_s + ψ_p). Note that the -0.6 MPa change of ψ_s in the cell at -1.0 MPa corresponds to an increased solute concentration of approximately 240 mM. Accumulation of proline (and other compatible solutes) in the relatively small volume of cytoplasm is matched by accumulation of potassium and other solutes in the larger vacuole volume. Thus, an increase in proline that may not seem significant when expressed on a bulk tissue basis can cause larger changes in osmotic potential by eliciting matching accumulation of other solutes in the larger vacuole. Note that the figure also depicts the close association of chloroplasts (green), mitochondria (red) and peroxisomes (gray) which is likely to be beneficial In stressed plants (Rivero et al., 2009).