Pyrroline-5-carboxylate Synthetase Research Articles

Insertion of YFP at P5CS1 and AFL1 shows the potential, and potential complications, of gene tagging for functional analyses of stress-related proteins.

Crispr/CAS9-enabled homologous recombination to insert a tag in frame with an endogenous gene can circumvent difficulties such as context-dependent promoter activity that complicate analysis of gene expression and protein accumulation patterns. However, there have been few reports examining whether such gene targeting/gene tagging (GT) can alter expression of the target gene. The enzyme encoded by Δ1-pyrroline-5-carboxylate synthetase 1 (P5CS1) is key for stress-induced proline synthesis and drought resistance, yet its expression pattern and protein localisation have been difficult to assay. We used GT to insert YFP in frame with the 5' or 3' ends of the endogenous P5CS1 and At14a-Like 1 (AFL1) coding regions. Insertion at the 3' end of either gene generated homozygous lines with expression of the gene-YFP fusion indistinguishable from the wild type allele. However, for P5CS1 this occurred only after selfing and advancement to the T5 generation allowed initial homozygous lethality of the insertion to be overcome. Once this was done, the GT-generated P5CS1-YFP plants revealed new information about P5CS1 localisation and tissue-specific expression. In contrast, insertion of YFP at the 5' end of either gene blocked expression. The results demonstrate that GT can be useful for functional analyses of genes that are problematic to properly express by other means but also show that, in some cases, GT can disrupt expression of the target gene.

Evaluation of P5CS and ProDH activity in Paulownia tomentosa (Steud.) as an indicator of oxidative changes induced by drought stress.

The aim of the study was to investigate changes in proline metabolism in seedlings of tree species during drought stress. One month old Paulownia tomentosa seedlings were exposed to moisture conditions at various levels (irrigation at 100, 75, 50 and 25% of field capacity), and then the material (leaves and roots) was collected three times at 10-day intervals. The activity of enzymes involved in proline metabolism was closely related to drought severity; however, proline content was not directly impacted. The activity of pyrroline-5-carboxylate synthetase (P5CS), which catalyzes proline biosynthesis, increased in response to hydrogen peroxide accumulation, which was correlated with soil moisture. In contrast, the activity of proline dehydrogenase (ProDH), which catalyzes proline catabolism, decreased. Compared to proline, the activity of these enzymes may be a more reliable biochemical marker of stress-induced oxidative changes. The content of proline is dependent on numerous additional factors, i.e., its degradation is an important alternative energy source. Moreover, we noted tissue-specific differences in this species, in which roots appeared to be proline biosynthesis sites and leaves appeared to be proline catabolism sites. Further research is needed to examine a broader view of proline metabolism as a cycle regulated by multiple mechanisms and differences between species.

A complex array of factors regulate the activity of Arabidopsis thaliana δ1 -pyrroline-5-carboxylate synthetase isoenzymes to ensure their specific role in plant cell metabolism.

The first and committed step in proline synthesis from glutamate is catalyzed by δ1 -pyrroline-5-carboxylate synthetase (P5CS). Two P5CS genes have been found in most angiosperms, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate regulation at the transcriptional level, to date, the properties of the enzymes have been subjected to limited study. The isolation of Arabidopsis thaliana P5CS isoenzymes was achieved through heterologous expression and affinity purification. The two proteins were characterized with respect to kinetic and biochemical properties. AtP5CS2 showed KM values in the micro- to millimolar range, and its activity was inhibited by NADP+ , ADP and proline, and by glutamine and arginine at high levels. Mg2+ ions were required for activity, which was further stimulated by K+ and other cations. AtP5CS1 displayed positive cooperativity with glutamate and was almost insensitive to inhibition by proline. In the presence of physiological, nonsaturating concentrations of glutamate, proline was slightly stimulatory, and glutamine strongly increased the catalytic rate. Data suggest that the activity of AtP5CS isoenzymes is differentially regulated by a complex array of factors including the concentrations of proline, glutamate, glutamine, monovalent cationsand pyridine dinucleotides.

Lipid signaling and proline catabolism are activated in barley roots (Hordeum vulgare L.) during recovery from cold stress

Previous findings have shown that phospholipase D (PLD) contributes to the response to long-term chilling stress in barley by regulating the balance of proline (Pro) levels. Although Pro accumulation is one of the most prominent changes in barley roots exposed to this kind of stress, the regulation of its metabolism during recovery from stress remains unclear. Research has mostly focused on the responses to stress per se, and not much is known about the dynamics and mechanisms underlying the subsequent recovery. The present study aimed to evaluate how PLD, its product phosphatidic acid (PA), and diacylglycerol pyrophosphate (DGPP) modulate Pro accumulation in barley during recovery from long-term chilling stress. Pro metabolism involves different pathways and enzymes. The rate-limiting step is mediated by pyrroline-5-carboxylate synthetase (P5CS) in its biosynthesis, and by proline dehydrogenase (ProDH) in its catabolism. We observed that Pro levels decreased in recovering barley roots due to an increase in ProDH activity. The addition of 1-butanol, a PLD inhibitor, reverted this effect and altered the relative gene expression of ProDH. When barley tissues were treated with PA before recovery, the fresh weight of roots increased and ProDH activity was stimulated. These data contribute to our understanding of how acidic membrane phospholipids like PA help to control Pro degradation during recovery from stress.

L-Glutamate Seed Priming Enhances 2-Acetyl-1-pyrroline Formation in Fragrant Rice Seedlings in Response to Arsenite Stress.

2-Acetyl-1-pyrroline (2-AP) is a fragrance compound and flavor in fragrant rice whose precursors are generally glutamate (Glu) and proline (Pro). Our previous study revealed that exogenous Glu enhanced the arsenic (As) tolerance in fragrant rice by improving the ascorbic acid-glutathione cycle and the Pro content in roots. However, less is known about how Glu is involved in 2-AP biosynthesis in fragrant rice under As stress. Herein, a hydroponic experiment of L-Glu seed priming with 0, 100, and 500 μM l-glutamic acid solutions was conducted with two fragrant rice varieties. After that, the 10-day-old seedlings were cultured under 0 and 100 μM arsenite stress for 10 d. Results showed that the 2-AP and Pro contents were increased by 18-30% and 21-78% under As100 μM-Glu100 μM treatment in comparison to the control As100 μM to Glu0 μM, while the activities of pyrroline-5-carboxylate synthetase (P5CS) and proline dehydrogenase (ProDH) were increased by 19-46% and 3-19%, respectively. Furthermore, the 2-AP, Pro contents, and P5CS activity were correlated positively. Correspondingly, a significant abundance of differential expressed metabolites (18) and differential expressed genes (26) was observed in amino acid metabolism and glutathione metabolism pathways. In addition, several essential genes were verified and grouped into the pathways of glutathione metabolism, proline, and arginine metabolism with antioxidant defense system to comodulate 2-AP biosynthesis and stress detoxification. Therefore, the Glu seed priming treatment had a positive impact on the 2-AP biosynthesis of fragrant rice under 100 μM arsenite toxicity.

Arbuscular mycorrhizal fungi as an effective approach to enhance the growth and metabolism of soybean plants under thallium (TI) toxicity

Thallium (TI) is a toxic metal that can trigger harmful impacts on growth and metabolism of plants. Utilizing arbuscular mycorrhizal fungi (AMF) proves to be an effective strategy for alleviating heavy metal toxicity in plants. To this end, AMF were applied to mitigate TI toxic effects on the growth, primary and secondary metabolism of soybean plants. Here, TI stress inhibited the growth and photosynthetic parameters of soybean plants. It also increased the oxidative damage as demonstrated by increased levels of oxidative markers, (MDA and lipoxygenase (LOX) activity). However, AMF could mitigate the reduction in growth and photosynthesis induced by TI, as well as the induction of oxidative damage. To overcome TI toxicity, AMF increased the levels and metabolism of osmolytes such as proline in soybean plants. This was in line with the increased activities of key enzymes that involved in proline biosynthesis (e.g., P5CS (pyrroline-5-carboxylate synthetase), P5CR (pyrroline-5-carboxylate reductase) and OAT (ornithine aminotransferase) under the AMF and/or TI treatments. Furthermore, soybean plants could benefit from the synergism between AMF and TI to enhance the contents of individual (e.g., spermine and spermidine) and total polyamines as well as their metabolic enzymes (e.g., arginine decarboxylase and ornithine decarboxylase). Overall, the combined application of AMF emerges as a viable approach for alleviating TI toxicity in soybean plants.

Proline Metabolism Process and Antioxidant Potential of Lycium ruthenicum Murr. in Response to NaCl Treatments.

Salinity influences the level of antioxidants and proline content, which are both involved in the regulation of stress responses in plants. To examine the interplay between the antioxidant system and proline metabolism in plant stress acclimation, explants of Lycium ruthenicum were subjected to NaCl treatments, and the growth characteristics, antioxidant enzyme activities, proline accumulation, and metabolic enzyme content were analyzed. The results revealed that NaCl concentrations between 50 to 150 mM have a positive effect on the growth of L. ruthenicum explants. Increasing NaCl concentrations elevated the activities of superoxide dismutase (SOD) and catalase (CAT), while hydrogen peroxide (H2O2) content was inhibited, suggesting that the elevated antioxidants play a central protective role in superoxide anion (O2•-) and H2O2 scavenging processes in response to NaCl treatments. Also, high proline levels also protect antioxidant enzyme machinery, thus protecting the plants from oxidative damage and enhancing osmotic adjustment. Increasing levels of pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), and ornithine-δ-aminotransferase (δ-OAT) were observed, resulting in elevated level of proline. In addition, the expression levels of LrP5CS1, -2, -3, LrOAT-1, and -2 were promoted in NaCl treatments. According to the combined analysis of metabolic enzyme activities and their relative expression, it is confirmed that the glutamate (Glu) pathway is activated in L. ruthenicum faced with different levels of NaCl concentrations. However, Glu supplied by δ-OAT is fed back into the main pathway for proline metabolism.

Melatonin treatment alleviates chilling injury in mango fruit ‘Keitt’ by modulating proline metabolism under chilling stress

Mangoes often suffer from low temperature-induced chilling injury (CI) during postharvest cold storage. Therefore, advanced techniques are crucial and in high demand to solve the chilling stress of mango fruit for a higher value. This study addresses chilling stress modulation by investigating the effects of melatonin treatment on CI, proline metabolism, and related gene expressions of ‘Keitt’ mango during cold storage after dipped in 0 (control), 0.1 (MT1), and 0.2 mmol L-1 (MT2) melatonin solution for 30 min. The results revealed that melatonin treatment in MT1 significantly reduced CI development and increased proline content in mango fruit during cold storage compared to the control. These changes were along with increases in the activity of critical enzymes as well as the expression of encoding genes involved in proline biosynthesis, such as pyrroline-5-carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR), ornithine D-aminotransferase (OAT), P5CS2, P5CR2, and OAT3. Additionally, proline dehydrogenase (PDH) activity and the expression of the PDH3 gene associated with proline dehydrogenation were lower in MT1-treated mangoes than the controlled group. Thus, melatonin treatment has regulated proline metabolism resulting in the accumulation of proline, subsequently contributing to enhancing the chilling tolerance of ‘Keitt’ mango fruit.

Functional assessment of homozygous ALDH18A1 variants reveals alterations in amino acid and antioxidant metabolism.

Mono- and bi-allelic variants in ALDH18A1 cause a spectrum of human disorders associated with cutaneous and neurological findings that overlap with both cutis laxa and spastic paraplegia. ALDH18A1 encodes the bifunctional enzyme pyrroline-5-carboxylate synthetase (P5CS) that plays a role in the de novo biosynthesis of proline and ornithine. Here we characterize a previously unreported homozygous ALDH18A1 variant (p.Thr331Pro) in four affected probands from two unrelated families, and demonstrate broad-based alterations in amino acid and antioxidant metabolism. These four patients exhibit variable developmental delay, neurological deficits and loose skin. Functional characterization of the p.Thr331Pro variant demonstrated a lack of any impact on the steady-state level of the P5CS monomer or mitochondrial localization of the enzyme, but reduced incorporation of the monomer into P5CS oligomers. Using an unlabeled NMR-based metabolomics approach in patient fibroblasts and ALDH18A1-null human embryonic kidney cells expressing the variant P5CS, we identified reduced abundance of glutamate and several metabolites derived from glutamate, including proline and glutathione. Biosynthesis of the polyamine putrescine, derived from ornithine, was also decreased in patient fibroblasts, highlighting the functional consequence on another metabolic pathway involved in antioxidant responses in the cell. RNA sequencing of patient fibroblasts revealed transcript abundance changes in several metabolic and extracellular matrix-related genes, adding further insight into pathogenic processes associated with impaired P5CS function. Together these findings shed new light on amino acid and antioxidant pathways associated with ALDH18A1-related disorders, and underscore the value of metabolomic and transcriptomic profiling to discover new pathways that impact disease pathogenesis.

Selection of Newly Identified Growth-Promoting Archaea Haloferax Species With a Potential Action on Cobalt Resistance in Maize Plants.

Soil contamination with cobalt (Co) negatively impacts plant growth and production. To combat Co toxicity, plant growth-promoting microorganisms for improving plant growth are effectively applied. To this end, unclassified haloarchaeal species strain NRS_31 (OL912833), belonging to Haloferax genus, was isolated, identified for the first time, and applied to mitigate the Co phytotoxic effects on maize plants. This study found that high Co levels in soil lead to Co accumulation in maize leaves. Co accumulation in the leaves inhibited maize growth and photosynthetic efficiency, inducing oxidative damage in the tissue. Interestingly, pre-inoculation with haloarchaeal species significantly reduced Co uptake and mitigated the Co toxicity. Induced photosynthesis improved sugar metabolism, allocating more carbon to defend against Co stress. Concomitantly, the biosynthetic key enzymes involved in sucrose (sucrose-P-synthase and invertases) and proline (pyrroline-5- carboxylate synthetase (P5CS), pyrroline-5-carboxylate reductase (P5CR)) biosynthesis significantly increased to maintain plant osmotic potential. In addition to their osmoregulation potential, soluble sugars and proline can contribute to maintaining ROS hemostasis. Maize leaves managed their oxidative homeostasis by increasing the production of antioxidant metabolites (such as phenolics and tocopherols) and increasing the activity of ROS-scavenging enzymes (such as POX, CAT, SOD, and enzymes involved in the AsA/GSH cycle). Inside the plant tissue, to overcome heavy Co toxicity, maize plants increased the synthesis of heavy metal-binding ligands (metallothionein, phytochelatins) and the metal detoxifying enzymes (glutathione S transferase). Overall, the improved ROS homeostasis, osmoregulation, and Co detoxification systems were the basis underlying Co oxidative stress, mitigating haloarchaeal treatment's impact.

CO2 Enrichment Differentially Upregulated Sugar, Proline, and Polyamine Metabolism in Young and Old Leaves of Wheat and Sorghum to Mitigate Indium Oxide Nanoparticles Toxicity.

Soil contamination with indium oxide nanoparticles (In2O3-NPs) is a challenge for plant growth and productivity. Despite In2O3-NPs toxicity, their effects on plant growth and metabolism are largely unknown, particularly under future climate CO2 (eCO2). Therefore, the In2O3-NPs toxicity and stress mitigating impact of eCO2 in the young and old leaves of C3 (wheat) and C4 (sorghum) plants were investigated. Overall, In2O3-NPs significantly retard the biomass and photosynthetic machinery of all tested crops, particularly the young leaves of C3 plants. Consequently, In2O3-NPs altered C and N metabolism in C3 and C4 plants. On the other hand, eCO2 contrarily alleviated the hazardous effects of In2O3-NPs on growth and photosynthesis, especially in the young leaves of C4 plants. Increased photosynthesis consequently enhanced the soluble sugars’ accumulation and metabolism (e.g., sucrose P synthase, cytosolic, and vacuolar invertase) in all stressed plants, but to a greater extent in C4 young leaves. High sugar availability also induced TCA organic and fatty acids’ accumulation. This also provided a route for amino acids and polyamines biosynthesis, where a clear increase in proline biosynthetic enzymes [e.g., pyrroline-5-carboxylate synthetase (P5CS), ornithine aminotransferase (OAT), Pyrroline-5-carboxylate reductase (P5CR), pyrroline-5-carboxylate dehydrogenase (P5CDH), and proline dehydrogenase (PRODH)] and polyamine metabolic enzymes (e.g., spermine and spermidine synthases, ornithine decarboxylase, and adenosyl methionine decarboxylase) were mainly recorded in C4 young leaves. The observed increases in these metabolites involved in osmo- and redox-regulation to reduce In2O3-NPs induced oxidative damage. Overall, our study, for the first time, shed light on how eCO2 differentially mitigated In2O3-NPs stress in old and young leaves of different species groups under the threat of In2O3-NPs contamination.

Tamarix hispida ThEIL1 improves salt tolerance by adjusting osmotic potential and increasing reactive oxygen species scavenging capability

The ETHYLENE INSENSITIVE 3-LIKE (EIL) transcription factor (TF) family plays a key role in the ethylene signal transduction pathway. However, it is still unknown how EIL proteins mediate salt tolerance in Tamarix hispida. Here, we cloned and functionally characterized the TF ThEIL1 from T. hispida. ThEIL1 is a nuclear protein possessing transcriptional activation activity. In addition to binding to the PERE motif (GGATTCAA), a novel EIL protein-DNA interaction was identified, in which ThEIL1 binds to GCC-box (AGCCGCC). Gain- and loss-of-function analysis in T. hispida showed that ThEIL1 regulates the expression of trehalose-6-phosphate synthase (TPS), pyrroline-5-carboxylate synthetase (P5CS), and trehalose-6-phosphate phosphatase (TPP) to mediate the biosynthesis of proline and trehalose, respectively, thereby elevating osmotic potential. Additionally, ThEIL1 controls the activities of peroxidase (POD) and superoxide dismutase (SOD) by regulating the expression of genes encoding SODs and PODs, leading to increased reactive oxygen species scavenging capability. Moreover, overexpression of ThEIL1 in Arabidopsis significantly increased root length and fresh weight under salt treatment conditions. Furthermore, the physiological traits of T. hispida were consistent with those of Arabidopsis stably overexpressing ThEIL1. Thus, ThEIL1 serves as a TF that mediates salt tolerance by elevating osmotic potential and improving ROS scavenging capability.

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.

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.

Differential Contribution of P5CS Isoforms to Stress Tolerance in Arabidopsis.

Proline accumulation is a widespread response of plants to salt stress as well as drought and cold stress. In most plant species, two isoforms of pyrroline-5-carboxylate synthetase (P5CS) catalyze the first step in proline biosynthesis from glutamate. In Arabidopsis, these isoforms differ in their spatial and temporal expression patterns, suggesting sub-functionalization. P5CS1 has been identified as the major contributor to stress-induced proline accumulation, whereas P5CS2 has been considered important for embryo development and growth. In contrast to previous results, our analysis of P5CS1- and P5CS2-GFP fusion proteins indicates that both enzymes were exclusively localized in the cytosol. The comparison of the susceptibility of p5cs1 and p5cs2 mutants to infection with Pseudomonas syringae and salt stress provided novel information on the contribution of the two P5CS isoforms to proline accumulation and stress tolerance. In agreement with previous studies, salt-stressed p5cs1 mutants accumulated very little proline, indicating that P5CS1 contributed more to stress-induced proline accumulation, whereas its impact on stress tolerance was rather weak. Germination and establishment of p5cs2 mutants were impaired under ambient conditions, further supporting that P5CS2 is most important for growth and development, whereas its contribution to stress-induced proline accumulation was smaller than that of P5CS1. In contrast to p5cs1 mutants or wildtype plants, p5cs2 mutants were only weakly affected by sudden exposure to a high NaCl concentration. These findings show that proline content, which was intermediate in leaves of p5cs2 mutants, was not directly correlated with stress tolerance in our experiments. In rosettes of NaCl-exposed p5cs2 mutants, nearly no accumulation of Na+ was observed, and the plants showed neither chlorosis nor reduction of photosynthesis. Based on these data, we suggest a function of P5CS2 or P5CS2-mediated proline synthesis in regulating Na+ accumulation in leaves and thereby salt stress tolerance.

Δ1 -Pyrroline-5-carboxylate synthetase deficiency: An emergent multifaceted urea cycle-related disorder.

The bifunctional homooligomeric enzyme Δ1 -pyrroline-5-carboxylate synthetase (P5CS) and its encoding gene ALDH18A1 were associated with disease in 1998. Two siblings who presented paradoxical hyperammonemia (alleviated by protein), mental disability, short stature, cataracts, cutis laxa, and joint laxity, were found to carry biallelic ALDH18A1 mutations. They showed biochemical indications of decreased ornithine/proline synthesis, agreeing with the role of P5CS in the biosynthesis of these amino acids. Of 32 patients reported with this neurocutaneous syndrome, 21 familial ones hosted homozygous or compound heterozygous ALDH18A1 mutations, while 11 sporadic ones carried de novo heterozygous ALDH18A1 mutations. In 2015 to 2016, an upper motor neuron syndrome (spastic paraparesis/paraplegia SPG9) complicated with some traits of the neurocutaneous syndrome, although without report of cutis laxa, joint laxity, or herniae, was associated with monoallelic or biallelic ALDH18A1 mutations with, respectively, dominant and recessive inheritance. Of 50 SPG9 patients reported, 14 and 36 (34/2 familial/sporadic) carried, respectively, biallelic and monoallelic mutations. Thus, two neurocutaneous syndromes (recessive and dominant cutis laxa 3, abbreviated ARCL3A and ADCL3, respectively) and two SPG9 syndromes (recessive SPG9B and dominant SPG9A) are caused by essentially different spectra of ALDH18A1 mutations. On the bases of the clinical data (including our own prior patients' reports), the ALDH18A1 mutations spectra, and our knowledge on the P5CS protein, we conclude that the four syndromes share the same pathogenic mechanisms based on decreased P5CS function. Thus, these syndromes represent a continuum of increasing severity (SPG9A < SPG9B < ADCL3 ≤ ARCL3A) of the same disease, P5CS deficiency, in which the dominant mutations cause loss-of-function by dominant-negative mechanisms.

In Vitro Propagation, Isolation and Expression Studies of Suaeda edulis Genes Involved in the Osmoprotectants Biosynthesis

Halophytes are an excellent choice for the study of genes conferring salt tolerance to salt-sensitive plants and, they are suitable for reclamation and remediation of saline soil. We develop an <i>in vitro</i> plant propagation protocol and studies of genes involved with GB and Pro biosynthesis in Suaeda edulis. Axillary buds were used as explants and cultured in different treatments on Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of plant growth regulators. The highest number of multiple shoots was on MS medium containing 1 mg/L Benzyladenine (BA) and / or 2 g/L activated carbon with 5.5 ± 06 shoots per explant. The identification and expression analysis of genes involved in glycine betaine (GB) biosynthesis were S-adenosylmethionine synthetase (<i>SAMS</i>), choline monooxygenase (<i>CMO</i>) and betaine aldehyde dehydrogenase (<i>BADH</i>), and for proline (Pro) was pyrroline 5-carboxylate synthetase (<i>P5CS</i>). These sequences shared 90–95% of identity with others plant homologous in public databases. The amino acids sequence analysis showed that all these peptides contain some of the conserved motifs of those kinds of enzymes. The qRT-PCR analysis revealed a higher expression of <i>SeBADH</i>, <i>SeCMO</i>, and, <i>SeP5CS</i> genes in the roots and leaves from plants collected in the field in contrast with from <i>in vitro</i> plants. However, the expression level of <i>SeSAMS</i> was higher only in the leaves of plants collected in the field when compared to those cultivated <i>in vitro</i>.

Water and Salt Stress Metabolomics for Wheat Genotypes of India

Shoots of ten day old seedlings of nineteen wheat genotypes were evaluated for proline metabolism, H2O2, thiobarbituric acid reactive substances (TBARS) and 2,2 diphenyl-picrylhydrazyl (DPPH) radical scavenging activity under water deficit, water withholding and salinity stress conditions. Principle component analysis demarcated four groups: i.e. drought tolerant (Excalibar, Krichauff, Babax, Drysdale, Gladius and C306), salt tolerant (Kharchia, Type11, Krl 1-4 and Krl 19), low stress tolerant (C273, C518 and C591) and susceptible (HD2967, PBW621, WH1105, HD3086, PBW660 and PBW175). Salt stress treatment affected the length, fresh weight and dry weight of seedlings of all studied genotypes in comparison to water deficit and water withholding condition which may be due to higher contents of TBARS. Shoots of salt and drought tolerant genotypes possessed higher proline content and DPPH radical scavenging activity alongwith reduced content of TBARS in parallel with decreased H2O2 content under water stress conditions. The activities of proline synthesizing enzymes i.e. pyrroline-5-carboxylate synthetase (P5CS) and pyrroline-5-carboxylate reductase (P5CR) were significantly higher than proline degrading enzyme viz. proline dehydrogenase (PDH) under water stress as compared to salinity stress conditions. Overall, results indicated that P5CS, P5CR and PDH activities led to higher build up of proline under water stress, which might play a significant role in improving membrane stability by increasing radical scavenging activity and finally imparting stress tolerance in specific wheat genotypes.

Exogenous ethylene alleviates chilling injury of ‘Huangguan’ pear by enhancing the proline content and antioxidant activity

Peel browning spots (PBS) is a chilling injury (CI) symptom in cold-stored ‘Huangguan’ pear. In the present study, the effect of exogenous ethylene on CI and its correlation to proline and reactive oxygen species (ROS) metabolism was investigated. The results showed that ethylene treatment (100 μL L−1 for 24 h) could completely inhibit PBS appearance without significantly affecting fruit firmness, soluble solids content (SSC) and titratable acidity (TA), enhance ethylene production rate at the initiate stage of storage, and promote proline accumulation by increasing activities of pyrroline-5-carboxylate synthetase (P5CS), ornithine-delta-aminotransferase (OAT) and up-regulating expression of P5CS1 and OAT1 genes, decreasing proline dehydrogenase (PDH) activity and the expression of PDH1 gene in peel. Moreover, the ethylene treatment suppressed the accumulation of H2O2, O2- and malondialdehyde (MDA), maintained higher levels of ascorbic acid (AsA) and glutathione (GSH), and meanwhile enhanced activities of superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX), as well as expression of the genes such as SOD1, CAT1, and APX1 which could encode these enzymes in peel. These results suggested that ethylene alleviated the CI of ‘Huangguan’ pear by promoting the proline accumulation and antioxidant activity of peel.

Application of sucrose modulates the expressions of genes involved in proline and polyamine metabolism in maize seedlings exposed to drought

Sucrose, proline, and polyamines are compatible solutes accumulating in plant tissues and increasing cellular osmolarity under environmental stresses. These compatible solutes and hydrogen peroxide can function as signaling molecules in plants. There has been very little evidence how the supply of sucrose changes the biosynthesis of compatible solutes. This study aimed to assess the cross-talk among sucrose, H2O2, and compatible solutes on the expression of genes encoding key enzymes in the pathways of proline and polyamine metabolism in drought stressed maize seedlings. Drought stress (induced by polyethylene glycol solution) increased the expressions of genes encoding pyrroline-5-carboxylate synthetase (P5CS), arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (SAMDC), while decreased proline dehydrogenase (ProDH), diamine oxidase (DAO), and polyamine oxidase (PAO) expressions. Addition of sucrose to the stressed seedlings increased the P5CS, ADC and SAMDC expressions more than drought stress alone and reduced more the ProDH, DAO, and PAO expressions. Moreover, exogenous sucrose increased leaf water potential and the content of proline, polyamines, and total soluble sugars, whereas decreased H2O2 content and membrane damages under the drought stress conditions. Consequently, exogenous sucrose contributed to the preservation of water status and the amelioration of damage in maize seedlings under the drought stress.