Evolution of unisexual flowers involves extreme changes in floral development. Spinach is one of the species to discern the formation and evolution of dioecy. MADS-box gene family is involved in regulation of floral organ identity and development and in many other plant developmental processes. However, there is no systematic analysis of MADS-box family genes in spinach. A comprehensive genome-wide analysis and transcriptome profiling of MADS-box genes were undertaken to understand their involvement in unisexual flower development at different stages in spinach. In total, 54 MADS-box genes found to be unevenly located across 6 chromosomes and can be divided into type I and type II genes. Twenty type I MADS-box genes are subdivided into Mα, Mβ and Mγ subgroups. While thirty-four type II SoMADSs consist of 3 MIKC*, and 31 MIKCC -type genes including sixteen floral homeotic MADS-box genes that are orthologous to the proposed Arabidopsis ABCDE model of floral organ identity determination, were identified in spinach. Gene structure, motif distribution, physiochemical properties, gene duplication and collinearity analyses for these genes are performed in detail. Promoters of both types of SoMADS genes contain mainly MeJA and ABA response elements. Expression profiling indicated that MIKCc genes exhibited more dynamic and intricate expression patterns compared to M-type genes and the majority of type-II genes AP1, SVP, and SOC1 sub-groups showed female flower-biased expression profiles, suggesting their role in carpel development, while PI showed male-biased expression throughout flower developmental stages, suggesting their role in stamen development. These results provide genomic resources and insights into spinach dioecious flower development and expedite spinach improvement.

Genotoxic substances widely exist in the environment and the food supply, posing serious health risks due to their potential to induce DNA damage and cancer. Traditional genotoxicity assays, while valuable, are limited by insufficient sensitivity, specificity, and efficiency, particularly when applied to complex food matrices. This study introduces a multiparametric high-content analysis (HCA) for the detection of genotoxic substances in complex food matrices. The developed assay measures three genotoxic biomarkers, including γ-H2AX, p-H3, and RAD51, which enhances the sensitivity and accuracy of genotoxicity screening. Moreover, the assay effectively distinguishes genotoxic compounds with different modes of action, which not only offers a more comprehensive assessment of DNA damage and the cellular response to genotoxic stress but also provides new insights into the exploration of genotoxicity mechanisms. Notably, the five tested food matrices, including coffee, tea, pak choi, spinach, and tomato, were found not to interfere with the detection of these biomarkers under proper dilution ratios, validating the robustness and reliability of the assay for the screening of genotoxic compounds in the food industry. The integration of multiple biomarkers with HCA provides an efficient method for detecting and assessing genotoxic substances in the food supply, with potential applications in toxicology research and food safety.

Cadmium (Cd) pollution poses significant threats to soil organisms and human health by contaminating the food chain. This study aimed to assess the impact of various concentrations (50, 250, and 500 mg·kg-1) of zinc oxide nanoparticles (ZnO NPs), bulk ZnO, and ZnSO4 on morphological changes and toxic effects of Cd in the presence of earthworms and spinach. The results showed that Zn application markedly improved spinach growth parameters (such as fresh weight, plant height, root length, and root-specific surface area) and root morphology while significantly reducing Cd concentration and Cd bioconcentration factors (BCF-Cd) in spinach and earthworms, with ZnO NPs exhibiting the most pronounced effects. Earthworm, spinach root, and shoot Cd concentration decreased by 82.3 %, 77.0 %, and 75.6 %, respectively, compared to CK. Sequential-step extraction (BCR) analysis revealed a shift in soil Cd from stable to available forms, consistent with the available Cd (DTPA-Cd) results. All Zn treatments significantly reduced Cd accumulation, alleviated Cd-induced stress, and promoted spinach growth, with ZnO NPs demonstrating the highest Cd reduction and Zn bioaugmentation efficiencies compared to bulk ZnO and ZnSO4 at equivalent concentrations. Therefore, ZnO NPs offer a safer and more effective option for agricultural production and soil heavy metal pollution management than other Zn fertilizers.

Soil salinity is a major nutritional challenge with poor agriculture production characterized by high sodium (Na+) ions in the soil. Zinc oxide nanoparticles (ZnO NPs) and biochar have received attention as a sustainable strategy to reduce biotic and abiotic stress. However, there is a lack of information regarding the incorporation of ZnO NPs with biochar to ameliorate the salinity stress (0, 50,100 mM). Therefore, the current study aimed to investigate the potentials of ZnO NPs application (priming and foliar) alone and with a combination of biochar on the growth and nutrient availability of spinach plants under salinity stress. Results demonstrated that salinity stress at a higher rate (100 mM) showed maximum growth retardation by inducing oxidative stress, resulted in reduced photosynthetic rate and nutrient availability. ZnO NPs (priming and foliar) alone enhanced growth, chlorophyll contents and gas exchange parameters by improving the antioxidant enzymes activity of spinach under salinity stress. While, a significant and more pronounced effect was observed at combined treatments of ZnO NPs with biochar amendment. More importantly, ZnO NPs foliar application with biochar significantly reduced the Na+ contents in root 57.69%, and leaves 61.27% of spinach as compared to the respective control. Furthermore, higher nutrient contents were also found at the combined treatment of ZnO NPs foliar application with biochar. Overall, ZnO NPs combined application with biochar proved to be an efficient and sustainable strategy to alleviate salinity stress and improve crop nutritional quality under salinity stress. We inferred that ZnO NPs foliar application with a combination of biochar is more effectual in improving crop nutritional status and salinity mitigation than priming treatments with a combination of biochar.

Methyl viologen (MV), also known as paraquat, is a widely used herbicide but has also been reported as highly toxic to different life forms. The mode of its operation is related to superoxide radical (O2.-) production and consequent oxidative damage. However, besides the damage to key macromolecules, reactive oxygen species (ROS; to which O2.- belongs) are also known as regulators of numerous ion transport systems located at cellular membranes. In this study, we used MV as a tool to probe the role of O2.- in regulating membrane-transport activity and systemic acquired tolerance in halophytic Chenopodium quinoa and glycophytic spinach plants. Both plant species showed growth reduction in terms of reduced shoot length, lower shoot fresh and dry weight, photosynthesis rate, and chlorophyll contents; however, quinoa showed less reduction in growth compared with spinach. This whole plant response was further examined by measuring the ion concentration, gene expression of ion transporters, activation of antioxidants, and osmolyte accumulation. We observed that at the mechanistic level, the differences in growth in response to MV were conferred by at least four complementary physiological mechanisms: (1) higher K+ loss from spinach leaves resulted from higher expression of MV-induced plasma membrane-based depolarization-activated K+ efflux GORK channel, (2) higher activation of high-affinity K+ uptake transporter HAK5 in quinoa, (3) higher antioxidant production and osmolyte accumulation in quinoa as compared with spinach, and (4) maintaining a higher rate of photosynthesis due to higher chlorophyll contents, and efficiency of photosystem II and reduced ROS and MDA contents. Obtained results also showed that MV induced O2.- significantly reduced N contents in both species but with more pronounced effects in glycophytic spinach. Taken together this study has shown the role of O2.- in regulating membrane ion transport and N metabolism in the leaves of halophyte vs. glycophyte in the context of oxidative stress tolerance.

Spinach (Spinacia oleracea L.) is a dioecious, diploid, wind-pollinated crop cultivated worldwide. Sex determination plays an important role in spinach breeding. Hence, this study aimed to understand the differences in sexual differentiation and floral organ development of dioecious flowers, as well as the differences in the regulatory mechanisms of floral organ development of dioecious and monoecious flowers. We compared transcriptional-level differences between different genders and identified differentially expressed genes (DEGs) related to spinach floral development, as well as sex-biased genes to investigate the flower development mechanisms in spinach. In this study, 9189 DEGs were identified among the different genders. DEG analysis showed the participation of four main transcription factor families, MIKC_MADS, MYB, NAC, and bHLH, in spinach flower development. In our key findings, abscisic acid (ABA) and gibberellic acid (GA) signal transduction pathways play major roles in male flower development, while auxin regulates both male and female flower development. By constructing a gene regulatory network (GRN) for floral organ development, core transcription factors (TFs) controlling organ initiation and growth were discovered. This analysis of the development of female, male, and monoecious flowers in spinach provides new insights into the molecular mechanisms of floral organ development and sexual differentiation in dioecious and monoecious plants in spinach.

Cultivated spinach (Spinacia oleracea) is a dioecious species. We report high-quality genome sequences for its two closest wild relatives, Spinacia turkestanica and Spinacia tetrandra, which are also dioecious, and are used to study the genetics of spinach domestication. Using a combination of genomic approaches, we assembled genomes of both these species and analyzed them in comparison with the previously assembled S. oleracea genome. These species diverged c. 6.3 million years ago (Ma), while cultivated spinach split from S. turkestanica 0.8 Ma. In all three species, all six chromosomes include very large gene-poor, repeat-rich regions, which, in S. oleracea, are pericentromeric regions with very low recombination rates in both male and female genetic maps. We describe population genomic evidence that the similar regions in the wild species also recombine rarely. We characterized 282 structural variants (SVs) that have been selected during domestication. These regions include genes associated with leaf margin type and flowering time. We also describe evidence that the downy mildew resistance loci of cultivated spinach are derived from introgression from both wild spinach species. Collectively, this study reveals the genome architecture of spinach assemblies and highlights the importance of SVs during the domestication of cultivated spinach.

Most vegetable crops are severely affected by the uptake of heavy metals from the soil. Heavy metals in vegetable bodies generate reactive oxygen species (ROS) that unbalance the antioxidant defense system. This study was initiated to determine the physiological and biochemical characteristics of spinach plants grown on soil contaminated with heavy metals and responding to Bacillus cereus and Bacillus aerius were isolated from soil contaminated with heavy metals. Heavy metal contamination led to a significant reduction in seed germination, seedling biomass, protein, and total nitrogen content of spinach plants grown in contaminated soils compared to control soils. In contrast, a significant increase in the content of metallothioneins and antioxidant enzymes was observed. Plants inoculated with B. cereus and B. aerius significantly reduced the oxidative stress induced by heavy metals by improving seed germination (%), seedling growth, nitrogen, and protein content. The content of metallothioneins and the activities of antioxidant enzymes were reduced in spinach plants grown from seeds inoculated with bacterial strains. In addition, plants inoculated with, B. cereus and B. aerius showed greater stomata opening than plants grown on soil contaminated with heavy metals, whose stomata were almost closed. These results suggested that both bacterial strains enhanced plant growth by reducing oxidative stress caused by metals.

The sex of dioecious plants is mainly determined by genetic factors, but it can also be converted by environmental cues such as exogenous phytohormones. Gibberellic acids (GAs) are well-known inducers of flowering and sexual development, yet the pathway of gibberellin-induced sex conversion in dioecious spinach (Spinacia oleracea L.) remains elusive. Based on sex detection before and after GA3 application using T11A and SSR19 molecular markers, we confirmed and elevated the masculinization effect of GA on a single female plant through exogenous applications of GA3, showing complete conversion and functional stamens. Silencing of GIBBERELLIC ACID INSENSITIVE (SpGAI), a single DELLA family protein that is a central GA signaling repressor, results in similar masculinization. We also show that SpGAI can physically interact with the spinach KNOX transcription factor SHOOT MERISTEMLESS (SpSTM), which is a homolog of the flower meristem identity regulator STM in Arabidopsis. The silencing of SpSTM also masculinized female flowers in spinach. Furthermore, SpSTM could directly bind the intron of SpPI to repress SpPI expression in developing female flowers. Overall, our results suggest that GA induces a female masculinization process through the SpGAI-SpSTM-SpPI regulatory module in spinach. These insights may help to clarify the molecular mechanism underlying the sex conversion system in dioecious plants while also elucidating the physiological basis for the generation of unisexual flowers so as to establish dioecy in plants.

Spinach is a significant source of vitamins, minerals, and antioxidants. These nutrients make it delicious and beneficial for human health. However, the genetic mechanism underlying the accumulation of nutrients in spinach remains unclear. In this study, we analyzed the content of chlorophyll a, chlorophyll b, oxalate, nitrate, crude fiber, soluble sugars, manganese, copper, and iron in 62 different spinach accessions. Additionally, 3,356,182 high-quality, single-nucleotide polymorphisms were found using resequencing and used in a genome-wide association study. A total of 2077 loci were discovered that significantly correlated with the concentrations of the nutritional elements. Data mining identified key genes in these intervals for four traits: chlorophyll, oxalate, soluble sugar, and Fe. Our study provides insights into the genetic architecture of nutrient variation and facilitates spinach breeding for good nutrition.