Metabolic pathway engineering of high-salinity-induced overproduction of L-proline improves high-salinity stress tolerance of an ectoine-deficient Halomonas elongata.

Halophilic bacteria have adapted to survive in high-salinity environments by accumulating amino acids and their derivatives as organic osmolytes. L-Proline (Pro) is one such osmolyte that is also being used as a feed stimulant in the aquaculture industry. Halomonas elongata OUT30018 is a moderately halophilic bacterium that accumulates ectoine (Ect), but not Pro, as an osmolyte. Due to its ability to utilize diverse biomass-derived carbon and nitrogen sources for growth, H. elongata OUT30018 is used in this work to create a strain that overproduces Pro, which could be used as a sustainable Pro-rich feed additive. To achieve this, we replaced the coding region of H. elongata OUT30018's Ect biosynthetic operon with the artificial self-cloned proBm1AC gene cluster that encodes the Pro biosynthetic enzymes: feedback-inhibition insensitive mutant γ-glutamate kinase (γ-GKD118N/D119N), γ-glutamyl phosphate reductase, and pyrroline-5-carboxylate reductase. Additionally, the putA gene, which encodes the key enzyme of Pro catabolism, was deleted from the genome to generate H. elongata HN6. While the Ect-deficient H. elongata KA1 could not grow in minimal media containing more than 4% NaCl, H. elongata HN6 thrived in the medium containing 8% NaCl by accumulating Pro in the cell instead of Ect, reaching a concentration of 353.1 ± 40.5 µmol/g cell fresh weight, comparable to the Ect accumulated in H. elongata OUT30018 in response to salt stress. With its genetic background, H. elongata HN6 has the potential to be developed into a Pro-rich cell factory for upcycling biomass waste into single-cell feed additives, contributing to a more sustainable aquaculture industry.IMPORTANCEWe report here the evidence for de novo biosynthesis of Pro to be used as a major osmolyte in an ectoine-deficient Halomonas elongata. Remarkably, the concentration of Pro accumulated in H. elongata HN6 (∆ectABC::mCherry-proBm1AC ∆putA) is comparable to that of ectoine accumulated in H. elongata OUT30018 in response to high-salinity stress. We also found that among the two γ-glutamate kinase mutants (γ-GKD118N/D119N and γ-GKD154A/E155A) designed to resemble the two known Escherichia coli feedback-inhibition insensitive γ-GKD107N and γ-GKE143A, the γ-GKD118N/D119N mutant is the only one that became insensitive to feedback inhibition by Pro in H. elongata. As Pro is one of the essential feed additives for the poultry and aquaculture industries, the genetic makeup of the engineered H. elongata HN6 would allow for the sustainable upcycling of high-salinity waste biomass into a Pro-rich single-cell eco-feed.

Screening cotton genotypes for their drought tolerance ability based on the expression level of dehydration-responsive element-binding protein and proline biosynthesis-related genes and morpho-physio-biochemical responses.

Drought stress adversely affects growth, development, productivity, and fiber quality of cotton (Gossypium hirsutum L). Breeding strategies to enhance drought tolerance require an improved knowledge of plant drought responses necessitating proper identification of drought-tolerant genotypes of crops, including cotton. The objective of this study was to classify the selected cotton genotypes for their drought tolerance ability based on morpho-physio-biochemical traits using Hierarchical Ward's cluster analysis. Five genotypes of cotton (Takfa 3, Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5) were selected as plant materials, and were grown under well-watered (WW; 98 ± 2% field capacity) and water-deficit (WD; 50 ± 2% field capacity) conditions for 16 days during the flower initiation stage. Data on morpho-physio-biochemical parameters and gene expression levels for these parameters were collected, and subsequently genotypes were classified either as a drought tolerant or drought susceptible one. Upregulation of GhPRP (proline-rich protein), GhP5CS (Δ1-pyrroline-5-carboxylate synthetase), and GhP5CR (Δ1-pyrroline-5-carboxylate reductase) in relation to free proline enrichment was observed in Takfa 3 genotype under WD condition. An accumulation of free proline, total soluble sugar, and potassium in plants under WD conditions was detected, which played a key role as major osmolytes controlling cellular osmotic potential. Magnesium and calcium concentrations were also enriched in leaves under WD conditions, functioning as essential elements and regulating photosynthetic abilities. Leaf greenness, net photosynthetic rate, stomatal conductance, and transpiration rate were also declined under WD conditions, leading to growth retardation, especially aboveground traits of Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5 genotypes. An increase in leaf temperature (1.1 - 4.0 °C) and crop water stress index (CWSI > 0.75) in relation to stomatal closure and reduced transpiration rate was recorded in cotton genotypes under WD conditions compared with WW conditions. Based on the increase of free proline, soluble sugar, leaf temperature, and CWSI, as well as the decrease of aboveground growth traits and physiological attributes, five genotypes were categorized into two cluster groups: drought tolerant (Takfa 3) and drought susceptible (Takfa 6, Takfa 7, Takfa 84-4, and Takfa 86-5). The identified drought-tolerant cotton genotype, namely, Takfa 3, may be grown in areas experiencing drought conditions. It is recommended to further validate the yield traits of Takfa 3 under rainfed field conditions in drought-prone environments.

Strigolactone and nitric oxide collaborate synergistically to boost tomato seedling resilience to arsenic toxicity via modulating physiology and antioxidant system.

Arsenic (As) poses a significant environmental threat as a metalloid toxin, adversely affecting the health of both plants and animals. Strigolactones (SL) and nitric oxide (NO) are known to play crucial roles in plant physiology. Therefore, the present experiment was designed to investigate the potential cumulative role of SL (GR24-0.20 µM) and NO (100 µM) in mitigating the adverse effect of AsV (53 µM) by modulating physiological mechanisms in two genotypes of tomato (Riogrand and Super Strain 8). A sample randomized design with four replicates was used to arrange the experimental pots in the growth chamber. 45-d old both tomato cultivars under AsV toxicity exhibited reduced morphological attributes (root and shoot length, root and shoot fresh weight, and root and shoot dry weight) and physiological and biochemical characteristics [chlorophyll (Chl) a and b content, activity of δ-aminolevulinic acid dehydratase activity (an enzyme responsible for Chl biosynthesis), and carbonic anhydrase activity (an enzyme responsible for photosynthesis), and enhanced Chl degradation, overproduction of reactive oxygen species (ROS) and lipid peroxidation due to enhanced malondialdehyde (MDA) content. However, the combined application of SL and NO was more effective in enhancing the tolerance of both varieties to AsV toxicity compared to individual application. The combined application of SL and NO improved growth parameters, biosynthesis of Chls, NO and proline. However, the combined application significantly suppressed cellular damage by inhibiting MDA and overproduction of ROS in leaves and roots, as confirmed by the fluorescent microscopy study and markedly upregulated the antioxidant enzymes (catalase, peroxidase, superoxide dismutase, ascorbate dismutase and glutathione reductase) activity. This study provides clear evidence that the combined application of SL and NO supplementation significantly improves the resilience of tomato seedlings against AsV toxicity. The synergistic effect of SL and NO was confirmed by the application of cPTIO (an NO scavenger) with SL and NO. However, further molecular studies could be imperative to conclusively validate the simultaneous role of SL and NO in enhancing plant tolerance to abiotic stress.

Regulation of proline metabolism, AsA-GSH cycle, cadmium uptake and subcellular distribution in Brassica napus L. under the effect of nano-silicon.

Cadmium (Cd) is known to have detrimental effects on plant growth and human health. Recent studies showed that silicon nanoparticles (SNPs) can decrease Cd toxicity in plants. Therefore, a study was conducted using 50 µM Cd and 1.50 mM SNPs to investigate Cd uptake, subcellular distribution, proline (Pro) metabolism, and the antioxidant defense system in rapeseed seedlings. In this study, results indicated that Cd stress negatively affected rapeseed growth, and high Cd contents accumulated in both shoots and roots. However, SNPs significantly decreased Cd contents in shoots and roots. Moreover, substantial increases were found in root fresh weight by 40.6% and dry weight by 46.6%, as well as shoot fresh weight by 60.1% and dry weight by 113.7% with the addition of SNPs. Furthermore, the addition of SNPs alleviated oxidative injury by maintaining the ascorbate-glutathione (AsA-GSH) cycle and increased Pro biosynthesis which could be due to high activities of Δ1-pyrroline-5-carboxylate synthase (P5CS) and reductase (P5CR) and decreased proline dehydrogenase (ProDH) activity. Furthermore, the addition of SNPs accumulated Cd in the soluble fraction (42%) and cell wall (45%). Results indicate that SNPs effectively reduce Cd toxicity in rapeseed seedlings which may be effective in promoting both rapeseed productivity and human health preservation.

Autophagy loss impedes cancer-associated fibroblast activation via downregulating proline biosynthesis.

Cancer-associated fibroblasts (CAFs) are considered one of the most critical stromal cells that interact with pancreatic ductal adenocarcinoma (PDAC) and promote tumor growth, metastasis, and treatment resistance. Previous studies illustrated macroautophagy/autophagy contributes to CAF activation during tumor progression. Here in our study, we found that autophagy deficiency in CAFs impedes CAF activation by inhibiting proline biosynthesis and collagen production. Furthermore, we uncovered that autophagy promotes proline biosynthesis through mitophagy-mediated regulation of NADK2 (NAD kinase 2, mitochondrial), an enzyme responsible for production of mitochondrial NADP(H). Using an orthotopic mouse model of PDAC, we found that inhibiting mitophagy by targeting PRKN (parkin RBR E3 ubiquitin protein ligase) in the stroma reduced tumor weight. Thus, inhibition of CAFs mitophagy might be an attractive strategy for stroma-focused anti-cancer intervention. Abbreviations: ACTA2/α-SMA: actin alpha 2, smooth muscle, aorta; ACTB/ß-actin: actin, beta; ALDH18A1/P5CS: aldehyde dehydrogenase 18 family, member A1; ATG3: autophagy related 3; ATG5: autophagy related 5; BNIP3L: BCL2/adenovirus E1B interacting protein 3-like; CAFs:cancer-associated fibroblasts; COL1A1: collagen, type I, alpha 1; DES: desmin; ECM: extracellular matrix; FABP4: fatty acid binding protein 4, adipocyte; FAP/FAPα: fibroblast activation protein; IHC: immunohistochemical staining; LAMP1: lysosomal-associated membrane protein 1; NADK2: NAD kinase 2, mitochondrial; PC1: pro-collagen 1; PDAC: pancreatic ductal adenocarcinoma; PDGFR: platelet derived growth factor receptor; PDPN: podoplanin; PRKN: parkin RBR E3 ubiquitin protein ligase; PSCs: pancreatic stellate cells; VIM: vimentin; WT: wild-type.

Expression and kinetic characterization of PYCR3.

PYCRs are proline biosynthetic enzymes that catalyze the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate (P5C) to proline in humans. PYCRs - especially PYCR1 - are upregulated in many types of cancers and have been implicated in the altered metabolism of cancer cells. Of the three isoforms of PYCR, PYCR3 remains the least studied due in part to the lack of a robust recombinant expression. Herein, we describe a procedure for the expression of soluble SUMO-PYCR3 in Escherichia coli, purification of the fusion protein, and removal of the SUMO tag. PYCR3 is active with either NADPH or NADH as the coenzyme. Bi-substrate kinetic measurements obtained by varying the concentrations of both L-P5C and NADH, along with product inhibition data for l-proline, suggest a random ordered bi bi mechanism. A panel of 19 proline analogs was screened for inhibition, and the kinetics of competitive inhibition (with L-P5C) were measured for five of the compounds screened, including N-formyl-l-proline, a validated inhibitor of PYCR1. N-formyl-l-proline was found to be ten times more selective for PYCR1 over PYCR3. The SUMO-PYCR3 expression system should be useful for testing the isoform specificity of PYCR1 inhibitors.

TaERF87 and TaAKS1 synergistically regulate TaP5CS1/TaP5CR1-mediated proline biosynthesis to enhance drought tolerance in wheat.

Drought stress limits wheat production and threatens food security world-wide. While ethylene-responsive factors (ERFs) are known to regulate plant response to drought stress, the regulatory mechanisms responsible for a tolerant phenotype remain unclear. Here, we describe the positive regulatory role of TaERF87 in mediating wheat tolerance to drought stress. TaERF87 overexpression (OE) enhances drought tolerance, while silencing leads to drought sensitivity in wheat. RNA sequencing with biochemical assays revealed that TaERF87 activates the expression of the proline biosynthesis genes TaP5CS1 and TaP5CR1 via direct binding to GCC-box elements. Furthermore, proline accumulates to higher levels in TaERF87- and TaP5CS1-OE lines than that in wild-type plants under well-watered and drought stress conditions concomitantly with enhanced drought tolerance in these transgenic lines. Moreover, the interaction between TaERF87 and the bHLH transcription factor TaAKS1 synergistically enhances TaP5CS1 and TaP5CR1 transcriptional activation. TaAKS1 OE also increases wheat drought tolerance by promoting proline accumulation. Additionally, our findings verified that TaERF87 and TaAKS1 are targets of abscisic acid-responsive element binding factor 2 (TaABF2). Together, our study elucidates the mechanisms underlying a positive response to drought stress mediated by the TaABF2-TaERF87/TaAKS1-TaP5CS1/TaP5CR1 module, and identifies candidate genes for the development of elite drought-tolerant wheat varieties.

Kinetics of human pyrroline-5-carboxylate reductase in L-thioproline metabolism.

L-Thioproline (L-thiazolidine-4-carboxylate, L-T4C) is a cyclic sulfur-containing analog of L-proline found in multiple kingdoms of life. The oxidation of L-T4C leads to L-cysteine formation in bacteria, plants, mammals, and protozoa. The conversion of L-T4C to L-Cys in bacterial cell lysates has been attributed to proline dehydrogenase and L-Δ1-pyrroline-5-carboxylate (P5C) reductase (PYCR) enzymes but detailed kinetic studies have not been conducted. Here, we characterize the dehydrogenase activity of human PYCR isozymes 1 and 2 with L-T4C using NAD(P)+ as the hydride acceptor. Both PYCRs exhibit significant L-T4C dehydrogenase activity; however, PYCR2 displays nearly tenfold higher catalytic efficiency (136 M-1 s-1) than PYCR1 (13.7 M-1 s-1). Interestingly, no activity was observed with either L-Pro or the analog DL-thiazolidine-2-carboxylate, indicating that the sulfur at the 4-position is critical for PYCRs to utilize L-T4C as a substrate. Inhibition kinetics show that L-Pro is a competitive inhibitor of PYCR1 [Formula: see text] with respect to L-T4C, consistent with these ligands occupying the same binding site. We also confirm by mass spectrometry that L-T4C oxidation by PYCRs leads to cysteine product formation. Our results suggest a new enzyme function for human PYCRs in the metabolism of L-T4C.

Enzymology and Regulation of δ1-Pyrroline-5-Carboxylate Synthetase 2 From Rice.

Under several stress conditions, such as excess salt and drought, many plants accumulate proline inside the cell, which is believed to help counteracting the adverse effects of low water potential. This increase mainly relies upon transcriptional induction of δ1-pyrroline-5-carboxylate synthetase (P5CS), the enzyme that catalyzes the first two steps in proline biosynthesis from glutamate. P5CS mediates both the phosphorylation of glutamate and the reduction of γ-glutamylphosphate to glutamate-5-semialdehyde, which spontaneously cyclizes to δ1-pyrroline-5-carboxylate (P5C). In most higher plants, two isoforms of P5CS have been found, one constitutively expressed to satisfy proline demand for protein synthesis, the other stress-induced. Despite the number of papers to investigate the regulation of P5CS at the transcriptional level, to date, the properties of the enzyme have been only poorly studied. As a consequence, the descriptions of post-translational regulatory mechanisms have largely been limited to feedback-inhibition by proline. Here, we report cloning and heterologous expression of P5CS2 from Oryza sativa. The protein has been fully characterized from a functional point of view, using an assay method that allows following the physiological reaction of the enzyme. Kinetic analyses show that the activity is subjected to a wide array of regulatory mechanisms, ranging from product inhibition to feedback inhibition by proline and other amino acids. These findings confirm long-hypothesized influences of both, the redox status of the cell and nitrogen availability, on proline biosynthesis.

Peculiar substrate specificity of δ1-pyrroline-5-carboxylate reductase in the obligately fermentative bacterium Zymomonas mobilis.

BACKGROUND: The enzyme that catalyzes the last step in proline synthesis, δ1-pyrroline-5-carboxylate reductase, showed in most cases a distinct preference in vitro for NADPH as the electron donor. METHODS AND RESULTS: A Zymomonas mobilis gene coding for a δ1-pyrroline-5-carboxylate reductase was cloned and heterologously expressed, and the recombinant protein was purified and characterized. The enzyme showed higher affinity to, and higher catalytic rate with NADH, with a specific activity of about 600 nkat (mg protein)-1. The molecular basis of this feature was investigated by analysis of the dinucleotide binding domain in silico. CONCLUSIONS: We postulate that the main determinants of coenzyme preference for P5C reductases are the length and the sequence of the motif A, whereas the overall sequence identity is insufficient to predict it a priori. Results are discussed in view of the obligately fermentative metabolism of this bacterium.

How signaling pathways link extracellular mechano-environment to proline biosynthesis: A hypothesis: PINCH-1 and kindlin-2 sense mechanical signals from extracellular matrix and link them to proline biosynthesis.

We propose a signaling pathway in which cell-extracellular matrix (ECM) adhesion components PINCH-1 and kindlin-2 sense mechanical signals from ECM and link them to proline biosynthesis, a vital metabolic pathway for macromolecule synthesis, redox balance, and ECM remodeling. ECM stiffening promotes PINCH-1 expression via integrin signaling, which suppresses dynamin-related protein 1 (DRP1) expression and mitochondrial fission, resulting in increased kindlin-2 translocation into mitochondria and interaction with Δ1 -pyrroline-5-carboxylate (P5C) reductase 1 (PYCR1). Kindlin-2 interaction with PYCR1 protects the latter from proteolytic degradation, leading to elevated PYCR1 level. Additionally, PINCH-1 promotes P5C synthase (P5CS) expression and P5C synthesis, which, together with increased PYCR1 level, support augmented proline biosynthesis. This signaling pathway is frequently activated in fibrosis and cancer, resulting in increased proline biosynthesis and excessive collagen matrix production, which in turn further promotes ECM stiffening. Targeting this signaling pathway, therefore, may provide an effective strategy for alleviating fibrosis and cancer progression.

Expression of Arabidopsis Ornithine Aminotransferase (AtOAT) encoded gene enhances multiple abiotic stress tolerances in wheat.

KEY MESSAGE: The drought and salt tolerances of wheat were enhanced by ectopic expression of the Arabidopsis ornithine aminotransferase (AtOAT) encoded gene. The OAT was confirmed to play a role in proline biosynthesis in wheat. Proline (Pro) accumulation is a common response to both abiotic and biotic stresses in plants. Ornithine aminotransferase (OAT) is pyridoxal-5-phosphate dependent enzyme involved in plant proline biosynthesis. During stress condition, proline is synthesized via glutamate and ornithine pathways. The OAT is the key enzyme in ornithine pathway. In this study, an OAT gene AtOAT from Arabidopsis was expressed in wheat for its functional characterization under drought, salinity, and heat stress conditions. We found that the expression of AtOAT enhanced the drought and salt stress tolerances of wheat by increasing the proline content and peroxidase activity. In addition, it was confirmed that the expression of AtOAT also played a partial tolerance to heat stress in the transgenic wheat plants. Moreover, quantitative real-time PCR (qRT-PCR) analysis showed that the transformation of AtOAT up-regulated the expression of the proline biosynthesis associated genes TaOAT, TaP5CS, and TaP5CR, and down-regulated that of the proline catabolism related gene TaP5CDH in the transgenic plants under stress conditions. Moreover, the genes involved in ornithine pathway (Orn-OAT-P5C/GSA-P5CR-Pro) were up-regulated along with the up-regulation of those genes involved in glutamate pathway (Glu-P5CS-P5C/GSA-P5CR-Pro). Therefore, we concluded that the expression of AtOAT enhanced wheat abiotic tolerance via modifying the proline biosynthesis by up-regulating the expression of the proline biosynthesis-associated genes and down-regulating that of the proline catabolic gene under stresses condition.

Structure, biochemistry, and gene expression patterns of the proline biosynthetic enzyme pyrroline-5-carboxylate reductase (PYCR), an emerging cancer therapy target.

Proline metabolism features prominently in the unique metabolism of cancer cells. Proline biosynthetic genes are consistently upregulated in multiple cancers, while the proline catabolic enzyme proline dehydrogenase has dual, context-dependent pro-cancer and pro-apoptotic functions. Furthermore, the cycling of proline and Δ1-pyrroline-5-carboxylate through the proline cycle impacts cellular growth and death pathways by maintaining redox homeostasis between the cytosol and mitochondria. Here we focus on the last enzyme of proline biosynthesis, Δ1-pyrroline-5-carboxylate reductase, known as PYCR in humans. PYCR catalyzes the NAD(P)H-dependent reduction of Δ1-pyrroline-5-carboxylate to proline and forms the reductive half of the proline metabolic cycle. We review the research on the three-dimensional structure, biochemistry, inhibition, and cancer biology of PYCR. To provide a global view of PYCR gene upregulation in cancer, we mined RNA transcript databases to analyze differential gene expression in 28 cancer types. This analysis revealed strong, widespread upregulation of PYCR genes, especially PYCR1. Altogether, the research over the past 20 years makes a compelling case for PYCR as a cancer therapy target. We conclude with a discussion of some of the major challenges for the field, including developing isoform-specific inhibitors, elucidating the function of the long C-terminus of PYCR1/2, and characterizing the interactome of PYCR.

Disease variants of human Δ1-pyrroline-5-carboxylate reductase 2 (PYCR2).

Pyrroline-5-carboxylate reductase (PYCR in humans) catalyzes the final step of l-proline biosynthesis by catalyzing the reduction of L-Δ1-pyrroline-5-carboxylate (L-P5C) to l-proline using NAD(P)H as the hydride donor. In humans, three isoforms PYCR1, PYCR2, and PYCR3 are known. Recent genome-wide association and clinical studies have revealed that homozygous mutations in human PYCR2 lead to postnatal microcephaly and hypomyelination, including hypomyelinating leukodystrophy type 10. To uncover biochemical and structural insights into human PYCR2, we characterized the steady-state kinetics of the wild-type enzyme along with two protein variants, Arg119Cys and Arg251Cys, that were previously identified in patients with microcephaly and hypomyelination. Kinetic measurements with PYCR2 suggest a sequential binding mechanism with L-P5C binding before NAD(P)H and NAD(P)+ releasing before L-Pro. Both disease-related variants are catalytically impaired. Depending on whether NADPH or NADH was used, the catalytic efficiency of the R119C protein variant was 40 or 366 times lower than that of the wild-type enzyme, while the catalytic efficiency of the R251C protein variant was 7 or 26 times lower than that of the wild-type enzyme. In addition, thermostability and circular dichroism measurements suggest that the R251C protein variant has a pronounced folding defect. These results are consistent with the involvement of Arg119Cys and Arg251Cys in disease pathology.

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.

Proteomic patterns associated with response to breast cancer neoadjuvant treatment.

Tumor relapse as a consequence of chemotherapy resistance is a major clinical challenge in advanced stage breast tumors. To identify processes associated with poor clinical outcome, we took a mass spectrometry-based proteomic approach and analyzed a breast cancer cohort of 113 formalin-fixed paraffin-embedded samples. Proteomic profiling of matched tumors before and after chemotherapy, and tumor-adjacent normal tissue, all from the same patients, allowed us to define eight patterns of protein level changes, two of which correlate to better chemotherapy response. Supervised analysis identified two proteins of proline biosynthesis pathway, PYCR1 and ALDH18A1, that were significantly associated with resistance to treatment based on pattern dominance. Weighted gene correlation network analysis of post-treatment samples revealed that these proteins are associated with tumor relapse and affect patient survival. Functional analysis showed that knockdown of PYCR1 reduced invasion and migration capabilities of breast cancer cell lines. PYCR1 knockout significantly reduced tumor burden and increased drug sensitivity of orthotopically injected ER-positive tumor in vivo, thus emphasizing the role of PYCR1 in resistance to chemotherapy.

Trypanosoma cruzi synthesizes proline via a Δ1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools.

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. Δ1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from Δ1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is an NADPH-dependent cytosolic enzyme with a Kmapp for P5C of 27.7 µM and with a higher expression in the insect-resident form of the parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Kiapp=45±0.7µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model, cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite, and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase.

Trypanosoma cruzi synthesizes proline via a delta1-pyrroline-5-carboxylate reductase whose activity is fine-tuned by NADPH cytosolic pools

In Trypanosoma cruzi, the etiological agent of Chagas disease, the amino acid proline participates in processes related to T. cruzi survival and infection, such as ATP production, cell differentiation, host-cell invasion, and in protection against osmotic, nutritional, and thermal stresses and oxidative imbalance. However, little is known about proline biosynthesis in this parasite. delta1-Pyrroline-5-carboxylate reductase (P5CR, EC 1.5.1.2) catalyzes the biosynthesis of proline from delta1-pyrroline-5-carboxylate (P5C) with concomitant NADPH oxidation. Herein, we show that unlike other eukaryotes, T. cruzi biosynthesizes proline from P5C, which is produced exclusively from glutamate. We found that TcP5CR is a NADPH-dependent cytosolic enzyme with a Km app for P5C of 23.9 mM and with a higher expression in the insect-resident form of parasite. High concentrations of the co-substrate NADPH partially inhibited TcP5CR activity, prompting us to analyze multiple kinetic inhibition models. The model that best explained the obtained data included a non-competitive substrate inhibition mechanism (Ki app = 45 ± 0.7 µM). Therefore, TcP5CR is a candidate as a regulatory factor of this pathway. Finally, we show that P5C can exit trypanosomatid mitochondria in conditions that do not compromise organelle integrity. These observations, together with previously reported results, lead us to propose that in T. cruzi TcP5CR participates in a redox shuttle between the mitochondria and the cytoplasm. In this model cytoplasmic redox equivalents from NADPH pools are transferred to the mitochondria using proline as a reduced metabolite and shuttling to fuel electrons to the respiratory chain through proline oxidation by its cognate dehydrogenase

Novel Maize NAC Transcriptional Repressor ZmNAC071 Confers Enhanced Sensitivity to ABA and Osmotic Stress by Downregulating Stress-Responsive Genes in Transgenic Arabidopsis.

NAC TFs play crucial roles in response to abiotic stresses in plants. Here, ZmNAC071 was identified as a nuclear located transcriptional repressor. Overexpression of ZmNAC071 in Arabidopsis enhanced sensitivity of transgenic plants to ABA and osmotic stress. The expression levels of SODs, PODs, P5CSs, and AtMYB61 were inhibited by ZmNAC071, which results in reduced ROS scavenging and proline content, increased ROS level, and water loss. Besides, the expression levels of some ABA or abiotic stress-related genes, like ABIs, RD29A, DREBs, and LEAs were also significantly inhibited by ZmNAC071. Yeast one-hybrid assay demonstrated that ZmNAC071 specifically bound to the cis-acting elements containing CGT[G/A] core sequences in the promoter of stress-related genes, suggesting that ZmNAC071 may participate in the regulation of transcription of these genes through recognizing the core sequences CGT[G/A]. These results will facilitate further studies concerning the cis-elements and downstream genes targeted by ZmNAC071 in maize.

Potential roles of melatonin and sulfur in alleviation of lanthanum toxicity in tomato seedlings.

Owing to the active use of rare-earth elements in many areas, it is necessary to study their behavior in the environment and their biological impact on plants. Despite the role of melatonin and sulfur in plant growth, development and abiotic stress tolerance; it is still not clear how they have a strong regulatory influence and synergistic effect on growth, physiological and biochemical characteristics of plants under different environmental stresses. Therefore, this study highlights how melatonin and sulfur together potentially involved in a reversal of lanthanum-inhibited photosynthetic and growth responses in tomato seedlings. Here, we reported that seedlings grown in a medium containing 150 µM lanthanum exhibited increased overproduction of reactive oxygen species (ROS) and lipid peroxidation together with increased Chlorophyll degradation, and activity of chlorophyllase, proline dehydrogenase and glycolate oxidase (GOx), and decreased photosynthesis and growth. However, the application of melatonin and sulfur showed significant responses on tomato seedlings, although the response of their combined treatment was more effective by further increasing photosynthesis and growth under lanthanum toxicity. Melatonin supplied with sulfur suppressed ROS formation, lipid peroxidation and activity of GOx, and increased photosynthesis by upregulating activities of carbonic anhydrase and ribulose-1,5-bisphosphate carboxylase/oxygenase. Also, sulfur supplementation with melatonin to seedlings resulted in an elevation in the accumulation of Chl and proline by increasing δ-aminolevulinic acid and activity of δ-aminolevulinic acid dehydratase and Δ1-pyrroline-5-carboxylate synthetase activity. The administration of melatonin with sulfur substantially induced upregulation of enzymes (superoxide dismutase, ascorbate peroxidase, monodehydroascorbate reductase, dehydroascorbate reductase and glutathione reductase) activities involved in the antioxidant system, thereby mitigating ROS-induced oxidative damage. Thus, this study provides strong evidence that melatonin and sulfur have strong regulatory influence and synergistic role in alleviating the adverse effect of lanthanum-toxicity by increasing photosynthesis and growth.