Pyrroline-5-carboxylate Research Articles

Regulation of 2-Acetyl-1-pyrroline Content in Fragrant Rice under Different Temperatures at the Grain-Filling Stage.

2-Acetyl-1-pyrroline (2-AP) is a key volatile organic compound in fragrant rice aroma. However, the effects of temperature on 2-AP biosynthesis in fragrant rice and its regulation mechanism have been rarely reported. In the present study, three fragrant rice varieties were used as plant materials, and four temperature treatments during the grain-filling stage, i.e., (T1) 22/17 °C, (T2) 27/22 °C, (T3) 32/27 °C, and (T4) 37/32 °C, were adopted. The results showed that grain contents of 2-AP, proline, and γ-aminobutyric acid (GABA) significantly (P < 0.05) increased with decreased temperature, while the lowest and highest 2-AP contents were recorded in the T4 and T1 treatments, respectively. Higher pyrroline-5-carboxylic acid (P5C) content was recorded in low-temperature treatments (T1 and T2) than in high-temperature treatments (T3 and T4). The transcript levels of genes BADH2, PRODH, and OAT significantly (P < 0.05) decreased with decreased temperature. Lower transcript levels of genes P5CR, P5CS2, DAO2, DAO4, and DAO5 were recorded in low-temperature treatments (T1 and T2) than in high-temperature treatments (T3 and T4). In conclusion, low temperature increased 2-AP content and high temperature decreased 2-AP content in fragrant rice. We deduced that temperature regulated 2-AP biosynthesis through the metabolism of proline and GABA.

Radical SAM Enzyme PylB Generates a Lysyl Radical Intermediate in the Biosynthesis of Pyrrolysine by Using SAM as a Cofactor.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S-adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-Nε-methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

Proline catabolism is a key factor facilitating Candida albicans pathogenicity.

Candida albicans, the primary etiology of human mycoses, is well-adapted to catabolize proline to obtain energy to initiate morphological switching (yeast to hyphal) and for growth. We report that put1-/- and put2-/- strains, carrying defective Proline UTilization genes, display remarkable proline sensitivity with put2-/- mutants being hypersensitive due to the accumulation of the toxic intermediate pyrroline-5-carboxylate (P5C), which inhibits mitochondrial respiration. The put1-/- and put2-/- mutations attenuate virulence in Drosophila and murine candidemia models and decrease survival in human neutrophils and whole blood. Using intravital 2-photon microscopy and label-free non-linear imaging, we visualized the initial stages of C. albicans cells infecting a kidney in real-time, directly deep in the tissue of a living mouse, and observed morphological switching of wildtype but not of put2-/- cells. Multiple members of the Candida species complex, including C. auris, are capable of using proline as a sole energy source. Our results indicate that a tailored proline metabolic network tuned to the mammalian host environment is a key feature of opportunistic fungal pathogens.

Pyrroline-5-carboxylate metabolism protein complex detected in Arabidopsis thaliana leaf mitochondria.

Proline dehydrogenase (ProDH) and pyrroline-5-carboxylate (P5C) dehydrogenase (P5CDH) catalyse the oxidation of proline into glutamate via the intermediates P5C and glutamate-semialdehyde (GSA), which spontaneously interconvert. P5C and GSA are also intermediates in the production of glutamate from ornithine and α-ketoglutarate catalysed by ornithine δ-aminotransferase (OAT). ProDH and P5CDH form a fused bifunctional PutA enzyme in Gram-negative bacteria and are associated in a bifunctional substrate-channelling complex in Thermus thermophilus; however, the physical proximity of ProDH and P5CDH in eukaryotes has not been described. Here, we report evidence of physical proximity and interactions between Arabidopsis ProDH, P5CDH, and OAT in the mitochondria of plants during dark-induced leaf senescence when all three enzymes are expressed. Pairwise interactions and localization of the three enzymes were investigated using bimolecular fluorescence complementation with confocal microscopy in tobacco and sub-mitochondrial fractionation in Arabidopsis. Evidence for a complex composed of ProDH, P5CDH, and OAT was revealed by co-migration of the proteins in native conditions upon gel electrophoresis. Co-immunoprecipitation coupled with mass spectrometry analysis confirmed the presence of the P5C metabolism complex in Arabidopsis. Pull-down assays further demonstrated a direct interaction between ProDH1 and P5CDH. P5C metabolism complexes might channel P5C among the constituent enzymes and directly provide electrons to the respiratory electron chain via ProDH.

Zinc supplementation and light intensity affect 2-acetyl-1-pyrroline (2AP) formation in fragrant rice

BackgroundImproving the yield and aroma content of fragrant rice is the focus of fragrant rice research. Light and Zinc (Zn) management generally cause regulations in the 2-acetyl-1-pyrroline (2AP) accumulation in fragrant rice. In addition, Zn promotes rice growth and improves rice yield, which has the potential to compensate for the negative impact of low light on fragrant rice yield. However, the potential of Zn to improve fragrant rice yield and 2AP content under shading conditions has not been verified.MethodsField experiments were conducted in the rice season (May–September) in 2019 to 2021. Two light i.e., normal light (NL) and low light (LL) and four Zn levels i.e., 0 kg Zn ha− 1 (N0), 1 kg Zn ha− 1 (Zn1), 2 kg Zn ha− 1(Zn2), and 3 kg Zn ha− 1 (Zn3), which applied at booting stage was set up. The grain yield, 2AP contents, Zn content in polished rice, photosynthesis related indicators, MDA content, antioxidant enzyme activity and the biochemical parameters related to 2AP formation were investigated.ResultsShading reduced yield by 8.74% and increased 2AP content by 24.37%. In addition, shading reduced net photosynthetic rate (Pn), superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), and increased proline, γ-aminobutyric acid (GABA), and pyrroline-5-carboxylic acid (P5C), proline dehydrogenase (PDH), △1-pyrroline-5-carboxylic acid synthetase (P5CS), malondialdehyde (MDA). With increasing Zn application levels, yield, 2AP, Zn content in polished rice, Pn, proline, P5C, GABA, PDH, P5CS, SOD, CAT and POD increased, and MDA decreased. Significant Light and Zn interaction effect on 2AP content was detected, and both shading and increasing Zn application increased the 2AP content.ConclusionShading can increase the 2AP content but reduce the yield of fragrant rice. Increasing Zn application under shading conditions can further promote the biosynthesis of 2AP, but the effect of improving yield is limited.

Pyrroline-5-carboxylate dehydrogenase is an essential enzyme for proline dehydrogenase function during dark-induced senescence in Arabidopsis thaliana.

During leaf senescence, nitrogen is remobilized and carbon backbones are replenished by amino acid catabolism, with many of the key reactions occurring in mitochondria. The intermediate Δ1 -pyrroline-5-carboxylate (P5C) is common to some catabolic pathways, thus linking the metabolism of several amino acids, including proline and arginine. Specifically, mitochondrial proline catabolism involves sequential action of proline dehydrogenase (ProDH) and P5C dehydrogenase (P5CDH) to produce P5C and then glutamate. Arginine catabolism produces urea and ornithine, the latter in the presence of α-ketoglutarate being converted by ornithine δ-aminotransferase (OAT) into P5C and glutamate. Metabolic changes during dark-induced leaf senescence (DIS) were studied in Arabidopsis thaliana leaves of Col-0 and in prodh1prodh2, p5cdh, and oat mutants. Progression of DIS was followed by measuring chlorophyll and proline contents for 5 days. Metabolomic profiling of 116 compounds revealed similar profiles of Col-0 and oat metabolism, distinct from prodh1prodh2 and p5cdh metabolism. Metabolic dynamics were accelerated in p5cdh by one day. Notably, more P5C and proline accumulated in p5cdh than in prodh1prodh2. ProDH1 enzymatic activity and protein amount were significantly down-regulated in p5cdh mutant at day 4 of DIS. Mitochondrial P5C levels appeared critical in determining the flow through interconnected amino acid remobilisation pathways to sustain senescence. This article is protected by copyright. All rights reserved.

Effects of Sowing Date and Nitrogen (N) Application Rate on Grain Yield, Nitrogen Use Efficiency and 2-Acetyl-1-Pyrroline Formation in Fragrant Rice

Purpose: This study aimed to assess the effects of the sowing date and nitrogen application rate on the grain yield, nitrogen use efficiency (NUE), 2-acetyl-△1-pyrroline (2-AP) contents and biochemical parameters related to 2-AP formation in fragrant rice. Methods: A factorial split-plot arrangement of treatments was set up in a split-zone experiment with two factors and three levels carried out for 3 years. The main plots included two sowing dates: April 1st and May 1st. Subplots contained three different nitrogen levels i.e., 0 kg N ha−1 (N0), 120 kg N ha−1 (N1) and 180 kg N ha−1 (N2). Results: The results indicated that compared with April 1st, the delay in the sowing date (May 1st) decreased the number of grains per panicle, 1000-grain weight, grain yield, NUE and contents of 2-AP, △1-pyrroline, proline and pyrroline-5-carboxylic acid (P5C), as well as the proline dehydrogenase (ProDH) activity. Furthermore, compared with N0, higher-N (N1 and N2) applications increased the panicle number, the number of grains per panicle, grain yield and contents of 2-AP, △1-pyrroline, proline and P5C, as well as the activities of ProDH and ornithine aminotransferase (OAT). The seed-setting rate, 1000-grain weight and NUE were decreased under N2 compared to N1, but the 2-AP content and yield were higher under the high-N application N2 (180 kg ha−1) compared to N1 (120 kg ha−1). Conclusions: Sowing on April 1st and the N2 (180 kg ha−1) application improved the yield and promoted 2-AP biosynthesis, while sowing on May 1st reduced the yield and 2-AP content. Therefore, sowing on April 1st with 180 kg ha−1 of nitrogen is the best, which can not only ensure the yield but also improve the fragrant quality of rice.

Intriguing Role of Proline in Redox Potential Conferring High Temperature Stress Tolerance.

Proline is a proteinogenic amino acid synthesized from glutamate and ornithine. Pyrroline-5-carboxylate synthetase and pyrroline-5-carboxylate reductase are the two key enzymes involved in proline synthesis from glutamate. On the other hand, ornithine-δ-aminotransferase converts ornithine to pyrroline 5-carboxylate (P5C), an intermediate in the synthesis of proline as well as glutamate. Both proline dehydrogenase and P5C dehydrogenase convert proline back to glutamate. Proline accumulation is widespread in response to environmental challenges such as high temperatures, and it is known to defend plants against unpropitious situations promoting plant growth and flowering. While proline accumulation is positively correlated with heat stress tolerance in some crops, it has detrimental consequences in others. Although it has been established that proline is a key osmolyte, its exact physiological function during heat stress and plant ontogeny remains unknown. Emerging evidence pointed out its role as an overriding molecule in alleviating high temperature stress (HTS) by quenching singlet oxygen and superoxide radicals. Proline cycle acts as a shuttle and the redox couple (NAD+/NADH, NADP+/NADPH) appears to be highly crucial for energy transfer among different cellular compartments during plant development, exposure to HTS conditions and also during the recovery of stress. In this review, the progress made in recent years regarding its involvement in heat stress tolerance is highlighted.

Staphylococcus aureus Does Not Synthesize Arginine from Proline under Physiological Conditions.

The Gram-positive pathogen Staphylococcus aureus is the only bacterium known to synthesize arginine from proline via the arginine-proline interconversion pathway despite having genes for the well-conserved glutamate pathway. Since the proline-arginine interconversion pathway is repressed by CcpA-mediated carbon catabolite repression (CCR), CCR has been attributed to the arginine auxotrophy of S. aureus. Using ribose as a secondary carbon source, here, we demonstrate that S. aureus arginine auxotrophy is not due to CCR but due to the inadequate concentration of proline degradation product. Proline is degraded by proline dehydrogenase (PutA) into pyrroline-5-carboxylate (P5C). Although the PutA expression was fully induced by ribose, the P5C concentration remained insufficient to support arginine synthesis because P5C was constantly consumed by the P5C reductase ProC. When the P5C concentration was artificially increased by either PutA overexpression or proC deletion, S. aureus could synthesize arginine from proline regardless of carbon source. In contrast, when the P5C concentration was reduced by overexpression of proC, it inhibited the growth of the ccpA deletion mutant without arginine. Intriguingly, the ectopic expression of the glutamate pathway enzymes converted S. aureus into arginine prototroph. In an animal experiment, the arginine-proline interconversion pathway was not required for the survival of S. aureus. Based on these results, we concluded that S. aureus does not synthesize arginine from proline under physiological conditions. We also propose that arginine auxotrophy of S. aureus is not due to the CcpA-mediated CCR but due to the inactivity of the conserved glutamate pathway. IMPORTANCE Staphylococcus aureus is a versatile Gram-positive human pathogen infecting various human organs. The bacterium's versatility is partly due to efficient metabolic regulation via the carbon catabolite repression system (CCR). S. aureus is known to interconvert proline and arginine, and CCR represses the synthesis of both amino acids. However, when CCR is released by a nonpreferred carbon source, S. aureus can synthesize proline but not arginine. In this study, we show that, in S. aureus, the intracellular concentration of pyrroline-5-carboxylate (P5C), the degradation product of proline and the substrate of proline synthesis, is too low to synthesize arginine from proline. These results call into question the notion that S. aureus synthesizes arginine from proline.

Proline Oxidation Supports Mitochondrial ATP Production When Complex I Is Inhibited.

The oxidation of proline to pyrroline-5-carboxylate (P5C) leads to the transfer of electrons to ubiquinone in mitochondria that express proline dehydrogenase (ProDH). This electron transfer supports Complexes CIII and CIV, thus generating the protonmotive force. Further catabolism of P5C forms glutamate, which fuels the citric acid cycle that yields the reducing equivalents that sustain oxidative phosphorylation. However, P5C and glutamate catabolism depend on CI activity due to NAD+ requirements. NextGen-O2k (Oroboros Instruments) was used to measure proline oxidation in isolated mitochondria of various mouse tissues. Simultaneous measurements of oxygen consumption, membrane potential, NADH, and the ubiquinone redox state were correlated to ProDH activity and F1FO-ATPase directionality. Proline catabolism generated a sufficiently high membrane potential that was able to maintain the F1FO-ATPase operation in the forward mode. This was observed in CI-inhibited mouse liver and kidney mitochondria that exhibited high levels of proline oxidation and ProDH activity. This action was not observed under anoxia or when either CIII or CIV were inhibited. The duroquinone fueling of CIII and CIV partially reproduced the effects of proline. Excess glutamate, however, could not reproduce the proline effect, suggesting that processes upstream of the glutamate conversion from proline were involved. The ProDH inhibitors tetrahydro-2-furoic acid and, to a lesser extent, S-5-oxo-2-tetrahydrofurancarboxylic acid abolished all proline effects. The data show that ProDH-directed proline catabolism could generate sufficient CIII and CIV proton pumping, thus supporting ATP production by the F1FO-ATPase even under CI inhibition.

Effect of foliar copper application on grain yield, 2-acetyl-1-Pyrroline and copper content in fragrant rice

Literature reporting the effects of application of exogenous copper (Cu) on the grain yield, 2-acetyl-1-pyrroline (2-AP) and Cu content of fragrant rice is scarce. Pot experiments were conducted in early and later season in 2020 with two fragrant rice cultivars, Meixiangzhan and Yuxiangyouzhan, at heading stage using four treatments, i.e.: 0 (CK), 20 (T1), 40 (T2), and 60 mM (T3) CuSO4·5H2O to assess the effects of foliar copper application on the yield, 2-AP and Cu content. Results showed that T2 treatment improved the yield of Meixiangzhan and Yuxiangyouzhan by 6.88% and 11.35% respectively, by increased seed-setting rate. Meanwhile, T2 treatment greatly increased the grains 2-AP content by 21.95% and 10.15% in early season, and by 5.40% and 27.25% in later season, respectively, which was significant associated with the contents of proline, pyrroline-5-carboxylic acid (P5C) and pyrroline, and the activities of proline dehydrogenase (PDH), ornithine aminotransferase (OAT) and pyrroline-5-carboxylic acid synthetase (P5CS). Foliar copper application increased the grain Cu content and showed the highest values in T3 treatment. Overall, foliar copper application at heading stage had a positive effect on yield and grain 2-AP and Cu content of fragrant rice. The results from this study show that T2 treatment (moderate CuSO4 application) had the most desirable outcomes, synergistically improving the yield and aroma of fragrant rice as well as enriching the grain copper content.

Proline synthesis through PYCR1 is required to support cancer cell proliferation and survival in oxygen-limiting conditions.

SummaryThe demands of cancer cell proliferation alongside an inadequate angiogenic response lead to insufficient oxygen availability in the tumor microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing potential produced through tricarboxylic acid (TCA) cycle activity and mitochondrial respiration are functionally linked. As the oxidizing activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidizing NADH with the synthesis of proline as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting continued TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Understanding the Role of Estrogen Receptor Status in PRODH/POX-Dependent Apoptosis/Survival in Breast Cancer Cells.

Simple SummaryThe estrogen receptor (ER) status and the availability of agonists or antagonists of these receptors determine the processes of growth, differentiation, and proliferation of breast cancer cells. Estrogens and anti-estrogenic compounds have been shown to influence breast cancer cell survival/apoptosis via action through the mitochondrial enzyme proline dehydrogenase/proline oxidase (PRODH/POX). In this review, we highlight the molecular effects of ER stimulation/inhibition in signaling pathways.It has been suggested that activation of estrogen receptor α (ER α) stimulates cell proliferation. In contrast, estrogen receptor β (ER β) has anti-proliferative and pro-apoptotic activity. Although the role of estrogens in estrogen receptor-positive breast cancer progression has been well established, the mechanism of their effect on apoptosis is not fully understood. It has been considered that ER status of breast cancer cells and estrogen availability might determine proline dehydrogenase/proline oxidase (PRODH/POX)-dependent apoptosis. PRODH/POX is a mitochondrial enzyme that converts proline into pyrroline-5-carboxylate (P5C). During this process, ATP (adenosine triphosphate) or ROS (reactive oxygen species) are produced, facilitating cell survival or death, respectively. However, the critical factor in driving PRODH/POX-dependent functions is proline availability. The amount of this amino acid is regulated at the level of prolidase (proline releasing enzyme), collagen biosynthesis (proline utilizing process), and glutamine, glutamate, α-ketoglutarate, and ornithine metabolism. Estrogens were found to upregulate prolidase activity and collagen biosynthesis. It seems that in estrogen receptor-positive breast cancer cells, prolidase supports proline for collagen biosynthesis, limiting its availability for PRODH/POX-dependent apoptosis. Moreover, lack of free proline (known to upregulate the transcriptional activity of hypoxia-inducible factor 1, HIF-1) contributes to downregulation of HIF-1-dependent pro-survival activity. The complex regulatory mechanism also involves PRODH/POX expression and activity. It is induced transcriptionally by p53 and post-transcriptionally by AMPK (AMP-activated protein kinase), which is regulated by ERs. The review also discusses the role of interconversion of proline/glutamate/ornithine in supporting proline to PRODH/POX-dependent functions. The data suggest that PRODH/POX-induced apoptosis is dependent on ER status in breast cancer cells.

Proline metabolism and biosynthesis behave differently in response to boron-deficiency and toxicity in Brassica napus

Proline biosynthesis and accumulation is a common response to unfavorable environment in many plants. This work aimed to elucidate the effects of boron (B)-deficiency and toxicity on proline metabolism and biosynthesis in Brassica napus in a hydroponic experiment. The results showed that B-deficiency and toxicity exert injurious impact on plant growth, accumulated high malondialdehyde (MDA) content, and caused the destruction of subcellular structure. Proline accumulated in both B deprivation and B toxicity plants, except B toxicity-treated root. In roots, B-deficiency increased ornithine content and pyrroline-5-carboxylate reductase (P5CR) activity, with the higher expression of BnaC03.P5CR, whilst decreased glutamate, glutamate-1-semialdehyde (GSA), pyrroline-5-carboxylate (P5C) contents and ornithine-δ-aminotransferase (δ-OAT), pyrroline-5-carboxylate synthetase (P5CS), proline dehydrogenase (ProDH) activities in terms of down-regulated the BnaC04.P5CS2, BnaA04.P5CS2, and BnaAnn.ProDH expression. The glutamate and GSA contents were decreased while P5C, arginine, and ornithine contents were enhanced in leaves under B-deficient and toxicity conditions. Lower glutamate pathway-related substance contents, P5CR, and δ-OAT activities while higher ProDH activity along with the same trend of related-gene expression were observed in B-toxicity-treated roots. Importantly, principal component analysis (PCA) in conjunction with correlation analysis indicated that ornithine pathway-related substances and enzymes contributed more to proline accumulation in B-deficient plant and B toxicity-treated leaves. Collectively, proline accumulation is caused by increased synthesis and decreased decomposition, and positively contributed, at least partly, by regulated ornithine pathway.

FSMP-12. A ROLE FOR PROLINE BIOSYNTHESIS IN HYPOXIC GLIOBLASTOMA

Hypoxia is a common feature of glioblastoma, and a known driver of therapy resistance in brain tumours. Understanding the metabolic adaptations to hypoxia is key to develop new effective treatments for patients. A recent screening study highlighted Pyrroline-5-carboxylate reductase-like (PYCRL) as one of the top three genes that allowed tumour survival in hypoxia. PYCRL is one of the three enzymes involved in proline biosynthesis along with the mitochondrial pyrroline-5-carboxylate reductase 1 and 2 (PYCR1/2). The latter use glutamine as the carbon source to fuel the pyrroline-5-carboxylate (P5C)-to-proline reaction, whereas the cytosolic PYCRL is known to use ornithine to produce proline. Our investigations have shown that PYCRL differs from PYCR1 and 2 in the impact on cellular redox, which is a critical factor in hypoxic survival. Our data suggest that PYCRL activity is required for normal regulation of glioblastoma cell growth and the ability to deal with cellular stress, and that this enzyme may therefore represent a novel target in the treatment of these devastating tumours. Importantly, our study also begins to provide much-needed clarity over the network surrounding proline metabolism and redox maintenance.

Genetic analysis of Pycr1 and Pycr2 in mice.

The final step in proline biosynthesis is catalyzed by three pyrroline-5-carboxylate reductases, PYCR1, PYCR2, and PYCR3, which convert pyrroline-5-carboxylate (P5C) to proline. Mutations in human PYCR1 and ALDH18A1 (P5C Synthetase) cause Cutis Laxa (CL), whereas mutations in PYCR2 cause hypomyelinating leukodystrophy 10 (HLD10). Here, we investigated the genetics of Pycr1 and Pycr2 in mice. A null allele of Pycr1 did not show integument or CL-related phenotypes. We also studied a novel chemically-induced mutation in Pycr2. Mice with recessive loss-of-function mutations in Pycr2 showed phenotypes consistent with neurological and neuromuscular disorders, including weight loss, kyphosis, and hind-limb clasping. The peripheral nervous system was largely unaffected, with only mild axonal atrophy in peripheral nerves. A severe loss of subcutaneous fat in Pycr2 mutant mice is reminiscent of a CL-like phenotype, but primary features such as elastin abnormalities were not observed. Aged Pycr2 mutant mice had reduced white blood cell counts and altered lipid metabolism, suggesting a generalized metabolic disorder. PYCR1 and -2 have similar enzymatic and cellular activities, and consistent with previous studies, both were localized in the mitochondria in fibroblasts. Both PYCR1 and -2 were able to complement the loss of Pro3, the yeast enzyme that converts P5C to proline, confirming their activity as P5C reductases. In mice, Pycr1; Pycr2 double mutants were sub-viable and unhealthy compared to either single mutant, indicating the genes are largely functionally redundant. Proline levels were not reduced, and precursors were not increased in serum from Pycr2 mutant mice or in lysates from skin fibroblast cultures, but placing Pycr2 mutant mice on a proline-free diet worsened the phenotype. Thus, Pycr1 and -2 have redundant functions in proline biosynthesis, and their loss makes proline a semi-essential amino acid. These findings have implications for understanding the genetics of CL and HLD10, and for modeling these disorders in mice.

Harvest Time Effects on Yield, Quality and Aroma of Fragrant Rice

Fragrant rice is popular among the public because of its distinctive fragrant smell. To explore the effects of different harvest time on yield, quality, and aroma-related substances of fragrant rice in the after full heading stage. Under field conditions, we used a completely randomized block design. Using fragrant rice: Xiangyaxiangzhan (XY), Meixiangzhan-2 (MXZ), 19Xiang (19X) as materials, sampling was conducted within one week before maturity and the determination of fragrant rice yield and grain quality at different harvest times. 2-Acetyl-1-Pyrroline (2-AP), Pyrroline-5-carboxylic acid synthetase (P5CS), Pyrroline-5-carboxylic acid (P5C), Proline, Proline dehydrogenase (PDH), Ornithine aminotransferase (OTC) are the related aspects on which we worked on. The results showed that the head rice rate increased significantly with harvest time extension. However, the protein and amylose content in fragrant rice was significantly reduced. In addition, the activity of P5CS showed a decreasing trend, and the content of P5C in the grains showed an increasing trend, the activity of OTC, 2-AP and proline in the grains showed a parabolic trend. Overall, the harvest time within a week had no significant effect on the yield of fragrant rice, but it did affect the aroma accumulation and quality of fragrant rice. The aroma accumulation of different varieties is different, the timely harvest of rice should be considered a variety.

Proline Synthesis Through PYCR1 is Required to Support Cancer Cell Proliferation and Survival in Oxygen-Limiting Conditions

The demands of cancer cell proliferation alongside an inadequate angiogenic response leads to insufficient oxygen availability in the tumour microenvironment. Within the mitochondria, oxygen is the major electron acceptor for NADH, with the result that the reducing activity of the tricarboxylic acid (TCA) cycle and mitochondrial respiration are functionally linked. As the oxidising activity of the TCA cycle is required for efficient synthesis of anabolic precursors, tumoral hypoxia could lead to a cessation of proliferation without another means of correcting the redox imbalance. We show here that in hypoxic conditions, mitochondrial pyrroline 5-carboxylate reductase 1 (PYCR1) activity is increased, oxidising NADH with proline synthesised as a by-product. We further show that PYCR1 activity is required for the successful maintenance of hypoxic regions by permitting oxygen-sparing TCA cycle activity, and that its loss leads to significantly increased hypoxia in vivo and in 3D culture, resulting in widespread cell death.

Appropriate Activity Assays Are Crucial for the Specific Determination of Proline Dehydrogenase and Pyrroline-5-Carboxylate Reductase Activities.

Accumulation of proline is a widespread plant response to a broad range of environmental stress conditions including salt and osmotic stress. Proline accumulation is achieved mainly by upregulation of proline biosynthesis in the cytosol and by inhibition of proline degradation in mitochondria. Changes in gene expression or activity levels of the two enzymes catalyzing the first reactions in these two pathways, namely pyrroline-5-carboxylate (P5C) synthetase and proline dehydrogenase (ProDH), are often used to assess the stress response of plants. The difficulty to isolate ProDH in active form has led several researchers to erroneously report proline-dependent NAD+ reduction at pH 10 as ProDH activity. We demonstrate that this activity is due to P5C reductase (P5CR), the second and last enzyme in proline biosynthesis, which works in the reverse direction at unphysiologically high pH. ProDH does not use NAD+ as electron acceptor but can be assayed with the artificial electron acceptor 2,6-dichlorophenolindophenol (DCPIP) after detergent-mediated solubilization or enrichment of mitochondria. Seemingly counter-intuitive results from previous publications can be explained in this way and our data highlight the importance of appropriate and specific assays for the detection of ProDH and P5CR activities in crude plant extracts.

Pyrroline-5-carboxylate synthase senses cellular stress and modulates metabolism by regulating mitochondrial respiration.

Pyrroline-5-carboxylate synthase (P5CS) catalyzes the synthesis of pyrroline-5-carboxylate (P5C), a key precursor for the synthesis of proline and ornithine. P5CS malfunction leads to multiple human diseases; however, the molecular mechanism underlying these diseases is unknown. We found that P5CS localizes in mitochondria in rod- and ring-like patterns but diffuses inside the mitochondria upon cellular starvation or exposure to oxidizing agents. Some of the human disease-related mutant forms of P5CS also exhibit diffused distribution. Multimerization (but not the catalytic activity) of P5CS regulates its localization. P5CS mutant cells have a reduced proliferation rate and are sensitive to cellular stresses. Flies lacking P5CS have reduced eclosion rates. Lipid droplets accumulate in the eyes of the newly eclosed P5CS mutant flies, which degenerate with aging. The loss of P5CS in cells leads to abnormal purine metabolism and lipid-droplet accumulation. The reduced lipid-droplet consumption is likely due to decreased expression of the fatty acid transporter, CPT1, and few β-oxidation-related genes following P5CS knockdown. Surprisingly, we found that P5CS is required for mitochondrial respiratory complex organization and that the respiration defects in P5CS knockout cells likely contribute to the metabolic defects in purine synthesis and lipid consumption. This study links amino acid synthesis with mitochondrial respiration and other key metabolic processes, whose imbalance might contribute to P5CS-related disease conditions.