Oxalate as a potent and selective inhibitor of spinach (Spinacia oleracea) leaf NADPH-dependent hydroxypyruvate reductase.
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ABSTRACT:
SUBMITTER: Kleczkowski LA
PROVIDER: S-EPMC1151153 | biostudies-other | 1991 May
REPOSITORIES: biostudies-other
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Project description:BackgroundSpinach (Spinacia oleracea L.) is an important leafy vegetable crop, and leaf-related traits including leaf length, leaf width, and petiole length, are important commercial traits. However, the underlying genes remain unclear. The objective of the study was to conduct QTL mapping of leaf-related traits in spinach.ResultsA BC1 population was used to construct the linkage map and for QTL mapping of leaf length, leaf width, petiole length, and the ratio of leaf length to width in 2015 and 2019. Two genetic linkage maps were constructed by specific locus amplified fragment sequencing (SLAF-seq), and kompetitive allele specific PCR (KASP) technology, respectively using BC1 population in 2015. Based on the results of 2015, the specific linkage groups (LG) detected QTLs were generated using BC1 population in 2019. A total of 13 QTLs were detected for leaf-related traits, only five QTLs being repeatedly detected in multiple years or linkage maps. Interestingly, the major QTLs of leaf length, petiole length, and the ratio of leaf length to width were highly associated with the same SNP markers (KM3102838, KM1360385 and KM2191098). A major QTL of leaf width was mapped on chromosome 1 from 41.470-42.045 Mb. And 44 genes were identified within the region. Based on the GO analysis, these genes were significantly enriched on ribonuclease, lyase activity, phosphodiester bond hydrolysis process, and cell wall component, thus it might change cell size to determine leaves shape.ConclusionsFive QTLs for leaf-related traits were repeatedly detected at least two years or linkage maps. The major QTLs of leaf length, petiole length, and the ratio of leaf length to width were mapped on the same loci. And three genes (Spo10792, Spo21018, and Spo21019) were identified as important candidate genes for leaf width.
| S-EPMC8223354 | biostudies-literature
Project description:A novel hydroxypyruvate reductase preferring NADPH to NADH as a cofactor was purified over 1500-fold from spinach leaf extracts. The enzyme was an oligomer of about 70 kDa, composed of two subunits of 38 kDa each. The Km for hydroxypyruvate (with NADPH) was about 0.8 mM in the pH range 5.5-6.5, and 0.3 mM at pH 8.2. The Vmax. was highest in the pH range 5.5-6.5 and decreased by about 65% at pH 8.2. Above pH 6.0, the enzyme was prone to a strong substrate inhibition by hydroxypyruvate. The reductase could use glyoxylate as an alternative substrate, with rates up to one-quarter of those with hydroxypyruvate. This glyoxylate-dependent activity preferred NADPH to NADH as a cofactor. Rabbit antibodies prepared against NADPH(NADH)-hydroxypyruvate reductase were highly specific for this enzyme and did not cross-react with peroxisomal NADH(NADPH)-dependent hydroxypyruvate reductase, as found by Western immunoblots of proteins from leaf extracts of spinach, pea and wheat. Antibodies raised against purified NADH(NADPH)-hydroxypyruvate reductase were also highly specific, recognizing only their own antigen. To our knowledge, this is the first report in the literature of the occurrence of NADPH(NADH)-hydroxypyruvate reductase in leaves, and the first to provide immunological comparison of leaf hydroxypyruvate reductases. Because of the relatively high rates of the novel reductase in leaf extracts (at least 20 mumol/h per mg of chlorophyll), this enzyme might be an important side-component of the glycollate pathway (photorespiration), possibly utilizing hydroxypyruvate 'leaked' from peroxisomes, and thus contributing to the glycerate pool derived from glycollate. Because of the glyoxylate-dependent activity, the enzyme may also contribute to glycollate formation in leaves.
| S-EPMC1148826 | biostudies-other
Project description:Spinacia oleracea strain:Spinach Raw sequence reads
| PRJNA576552 | ENA
Project description:Spinach (Spinacia oleracea L.) is an economically important vegetable crop. Here we describe the complete mitochondrial DNA sequence of spinach, which has a length of 329,613 bp and a GC content of 43.4%. It is separated by a pair of directly repeated elements of 7286 bp, to form a large single copy region of 229,375 bp and a small single copy region of 85,666 bp. The genome contains 29 protein-coding genes, 4 pseudogenes, 24 tRNA genes, and 3 rRNA genes. A phylogenetic analysis revealed that spinach was closely related to Beta vulgaris (sugar beet), both belonging to the Amaranthaceae family.
| S-EPMC7799700 | biostudies-literature
Project description:A novel reductase displaying high specificity for glyoxylate and NADPH was purified 3343-fold from spinach leaves. The enzyme was found to be an oligomer of about 125 kDa, composed of four equal subunits of 33 kDa each. A Km for glyoxylate was about 14-fold lower with NADPH than with NADH (0.085 and 1.10 mM respectively), but the maximal activity, 210 mumol/min per mg of protein, was similar with either cofactor. Km values for NADPH and NADH were 3 and 150 microM respectively. Optimal rates with either NADPH or NADH were found in the pH range 6.5-7.4. The enzyme also showed some reactivity towards hydroxypyruvate with rates less than 2% of those observed for glyoxylate. Results of immunological studies, using antibodies prepared against either glyoxylate reductase or spinach peroxisomal hydroxypyruvate reductase, suggested substantial differences in molecular structure of the two proteins. The high rates of NADPH(NADH)-glyoxylate reductase in crude leaf extracts of spinach, wheat and soya bean (30-45 mumol/h per mg of chlorophyll) and its strong affinity for glyoxylate suggest that the enzyme may be an important side component of photorespiration in vivo. In leaves of nitrogen-fixing legumes, this reductase may also be involved in ureide breakdown, utilizing the glyoxylate produced during allantoate metabolism.
| S-EPMC1147336 | biostudies-other
Project description:Spinach (Spinacia oleracea L.) is an economically important and globally consumed popular leafy vegetable that is heat-sensitive. Heat stress caused by global climate change is one of the primary deleterious elements limiting spinach production worldwide. Little work has been done to explore the heat-responsive mechanisms of spinach under high temperature-induced stress. In the present study, we used iTRAQ-based proteomic and transcriptomic approaches to investigate physiological, metabolic, and proteomic responses of spinach in response to day / night temperature of 35°C / 25°C compared to 20°C / 15°C for 4 days. A total of 3,543 differentially expressed genes (DEGs) were detected using transcriptome sequencing, of which 2,086 DEGs were downregulated and 1,457 were upregulated. The DEGs were mainly involved in superoxide dismutase activity, catalase, and peroxidase activity. A total of 3,246 differentially abundant proteins were detected using iTAQ-based quantitative proteomic approach, from which 567 differentially expressed proteins (DEPs) (277 upregulated and 290 downregulated) were identified. DEPs were mainly assigned to pathways related to metabolism, signal transduction, protein degradation, defense, and antioxidant. Four genes - superoxide dismutase (SOD, LOC110788339), catalase (CAT, LOC110790286), peroxidase (POD, LOC110775253), and heat shock protein (HSP, LOC110799288) - were validated using quantitative real-time PCR (qRT-PCR) to verify the proteomic and transcriptomic analyses, showing different transcriptional and translational expression levels. The findings of this study provide a fundamental understanding of the metabolic pathways and biological processes that control adaptation to heat stress in spinach, and provide novel insight into the development of heat-tolerant spinach.
2020-06-24 | GSE153070 | GEO
Project description:Minor alleles (MA) have been associated with disease incidence in human studies, enabling the identification of diagnostic risk factors for various diseases. However, allelic mapping has rarely been performed in plant systems. The goal of this study was to determine whether a difference in MA prevalence is a strong enough risk factor to indicate a likely significant difference in disease resistance against white rust (WR; Albugo occidentalis) in spinach (Spinacia oleracea). We used WR disease severity ratings (WR-DSRs) in a diversity panel of 267 spinach accessions to define resistant- and susceptibility-associated groups within the distribution scores and then tested the single-nucleotide polymorphism (SNP) variants to interrogate the MA prevalence in the most susceptible (MS) vs. most resistant (MR) individuals using permutation-based allelic association tests. A total of 448 minor alleles associated with WR severity were identified in the comparison between the 25% MS and the 25% MR accessions, while the MA were generally similar between the two halves of the interquartile range. The minor alleles in the MS group were distributed across all six chromosomes and made up ~71% of the markers that were also strongly associated with WR in parallel performed genome-wide association study. These results indicate that susceptibility may be highly determined by the disproportionate overrepresentation of minor alleles, which could be used to select for resistant plants. Furthermore, by focusing on the distribution tails, allelic mapping could be used to identify plant markers associated with quantitative traits on the most informative segments of the phenotypic distribution.
| S-EPMC6885047 | biostudies-literature
Project description:Nitrogen (N) is critical to the growth and productivity of crops. To understand the molecular mechanisms influenced by N stress, we used RNA-Sequencing (RNA-Seq) to analyze differentially expressed genes (DEGs) in root and leaf tissues of spinach. N stress negatively influenced photosynthesis, biomass accumulation, amino acid profiles, and partitioning of N across tissues. RNA-seq analysis revealed that N stress caused most transcriptomic changes in roots, identifying 1,346 DEGs. High-affinity nitrate transporters (NRT2.1, NRT2.5) and glutamine amidotransferase (GAT1) genes were strongly induced in roots in response to N deplete and replete conditions, respectively. GO and KEGG analyses revealed that the functions associated with metabolic pathways and nutrient reservoir activity were enriched due to N stress. Whereas KEGG pathway enrichment analysis indicated the upregulation of DEGs associated with DNA replication, pyrimidine, and purine metabolism in the presence of high N in leaf tissue. A subset of transcription factors comprising bHLH, MYB, WRKY, and AP2/ERF family members was over-represented in both tissues in response to N perturbation. Interesting DEGs associated with N uptake, amino acid metabolism, hormonal pathway, carbon metabolism, along with transcription factors, were highlighted. The results provide valuable information about the underlying molecular processes in response to N stress in spinach and; could serve as a resource for functional analysis of candidate genes/pathways and enhancement of nitrogen use efficiency.
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Project description:When intact spinach chloroplasts were supplied with [32P]Pi, stromal protein phosphorylation was found to occur in the dark. On illumination the thylakoid protein kinase was activated and the amount of label found in thylakoid proteins quickly exceeded that incorporated into stromal protein, such that the latter was found to account for only 10-15% of the total radioactivity bound to chloroplast proteins after 5 min illumination. The rate of phosphorylation of stromal polypeptides was unchanged by light. After SDS/polyacrylamide-gel electrophoresis, more than 15 labelled polypeptides of stromal origin were observed. A polypeptide with an Mr of approx. 70 000 had the highest specific activity of labelling. Both the large and small subunits of the ribulose-1,5-bisphosphate carboxylase were phosphorylated. The level of phosphorylation of stromal protein was increased by CO2 fixation in intact chloroplasts. This increase was not observed in the absence of NaHCO3 or in the presence of the phosphoribulokinase inhibitor DL-glyceraldehyde. These effects appeared to be largely due to changes in the phosphorylation state of the large and small subunits of ribulose-1,5-bisphosphate carboxylase. Studies with the reconstituted chloroplast system showed that the thylakoid protein kinase(s) played no part in the phosphorylation of stromal protein. The rate and level of phosphorylation of stromal protein was unaffected by the activation state of the thylakoid protein kinase and was unchanged when thylakoids were omitted from the reaction medium. The phosphorylation of stromal proteins is therefore catalysed by a discrete soluble protein kinase.
| S-EPMC1152708 | biostudies-other
Project description:Betacyanins have been reported as water-soluble, nitrogenous pigments found in the order Caryophyllales, and they are known for powerful natural antioxidant. The biofortification of secondary metabolites such as anthocyanins and betacyanins has recently been performed in food crops by metabolic engineering through genetic modification. However, the distribution of genetically modified foods is strictly regulated. Therefore, we aimed to develop a new method for biofortifying natural antioxidants, betacyanins, without genetic modification. We first detected the presence of betacyanins in red-tube spinach (Spinacia oleracea) through ultraviolet-visible spectroscopy and mass spectrometry. We then hydroponically cultivated plants in the presence of three candidate compounds for betacyanin biofortification: dopamine, Ca2+, and sucrose. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) and antioxidant activity analyses showed that sucrose was most successful in biofortifying betacyanins, and reverse transcription polymerase chain reaction (RT-PCR) indicated that several genes involved in betacyanin biosynthesis were induced by sucrose. Therefore, strategic hydroponics represents a new approach for cultivating betacyanin-enriched vegetables.
| S-EPMC6128657 | biostudies-literature