Regulation of Oxalate Metabolism in Spinach Revealed by RNA-Seq-Based Transcriptomic Analysis.

Although spinach (Spinacia oleracea L.) is considered to be one of the most nutrient-rich leafy vegetables, it is also a potent accumulator of anti-nutritional oxalate. Reducing oxalate content would increase the nutritional value of spinach by enhancing the dietary bioavailability of calcium and other minerals. This study aimed to investigate the proposed hypothesis that a complex network of genes associated with intrinsic metabolic and physiological processes regulates oxalate homeostasis in spinach. Transcriptomic (RNA-Seq) analysis of the leaf and root tissues of two spinach genotypes with contrasting oxalate phenotypes was performed under normal physiological conditions. A total of 2308 leaf- and 1686 root-specific differentially expressed genes (DEGs) were identified in the high-oxalate spinach genotype. Gene Ontology (GO) analysis of DEGs identified molecular functions associated with various enzymatic activities, while KEGG pathway analysis revealed enrichment of the metabolic and secondary metabolite pathways. The expression profiles of genes associated with distinct physiological processes suggested that the glyoxylate cycle, ascorbate degradation, and photorespiratory pathway may collectively regulate oxalate in spinach. The data support the idea that isocitrate lyase (ICL), ascorbate catabolism-related genes, and acyl-activating enzyme 3 (AAE3) all play roles in oxalate homeostasis in spinach. The findings from this study provide the foundation for novel insights into oxalate metabolism in spinach.

Comparative analysis of tissue-specific transcriptomic responses to nitrogen stress in spinach (Spinacia oleracea).

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.

Systematized biosynthesis and catabolism regulate citrulline accumulation in watermelon.

Citrulline, a non-protein amino acid, is present in large amounts in watermelon (Citrullus lanatus (Thunb.) Matsum. & Nakai Cucurbitaceae) fruits. Amino acid profiling of various tissues of cv. Charleston Gray during plant development confirmed progressive accumulation of citrulline only in the fruit flesh and rind tissues. Citrulline content was positively correlated with precursor (ornithine) and by-product (arginine) amino acids during fruit ripening. Genetic variation in the partitioning of citrulline and related amino acids in the flesh and rind tissues was confirmed in a sub-set of watermelon cultivars. No correlation was established between morphological fruit traits (size and rind properties) and citrulline content. To understand the regulation of citrulline accumulation, we investigated the expression of genes associated with its biosynthesis and catabolism in flesh and rind tissues during fruit development. The expression of ornithine carbamoyltransferase (OTC) involved in the ultimate step of citrulline synthesis remained steady in both tissues. The expression of N-acetylornithine aminotransferase (N-AOA) involved in the production of N-acetylornithine and N-acetylornithine deacetylase (AOD-3) involved in ornithine synthesis coincided with increasing accumulation of citrulline in flesh and rind tissues during fruit development. Down-regulation N-acetylornithine-glutamate acetyltransferase (N-AOGA) suggests the subordinate role of the non-cyclic pathway in citrulline synthesis. Eccentricity between citrulline accumulation and expression of carbamoyl phosphate synthases (CPS-1, CPS-2) during fruit development suggest that the localized synthesis of carbamoyl phosphates may not be required for citrulline synthesis. Most genes involved in citrulline break-down (Argininosuccinate synthases - ASS-1, ASS-2, and ASS-3, Argininosuccinate lyases - ASL-1, Ornithine decarboxylase - ODC, Arginine decarboxylase - ADC) were consistently down-regulated during fruit development.