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.

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.

One of the important problems in the environment is heavy metal pollution, and fluorescence is one of the best methods for their detection due to its sensitivity, selectivity, and relatively rapid and easy operation. In this study, 1,8-diaminonaphthalene functionalized super-stable mesoporous silica (DAN-LUS-1) was synthesized and used as a fluorescence probe to identify Hg2+ and Fe3+ in food samples. The TGA and FT-IR spectra illustrated that 1,8-diaminonaphthalene was grafted into LUS-1. XRD patterns verified that the LUS-1 and functionalized mesoporous silica have a hexagonal symmetrical array of nano-channels. SEM images showed that the rod-like morphology of LUS-1 was preserved in DAN-LUS-1. Also, surface area and pore diameter decreased from 824 m2 g⁻1 and 3.61 nm for the pure LUS-1 to 748 m2 g⁻1 and 3.43 nm for the DAN-LUS-1, as determined by N₂ adsorption-desorption isotherms. This reduction demonstrated that 1,8-diaminonaphthalene immobilized into the pore of LUS-1. The DAN-LUS-1 fluorescence properties as a chemical sensor were studied with a 340/407 nm excitation/emission wavelength that was quenched by Hg2+ and Fe3+ ions. Hg2+ and Fe3+ were quantified using the fluorescence response in the working range 8.25-13.79 × 10-6 and 3.84-10.71 × 10-6 mol/L, with detection limits of 8.5 × 10-8 M and 1.3 × 10-7 M, respectively. Hg2+ and Fe3+ were measured in vetiver grass and spinach. Since the Fe3+ quenching can move in the opposite direction with sodium hexametaphosphate (SHMP) as a hiding compound for Fe3+, consequently, the circuit logic system was established with Fe3+, Hg2+, and SHMP as inputs and the fluorescent quench as the output.

This study addressed the bioaccumulation and human health risk among the consumption of Spinacia oleracea grown in agricultural soil treated with humic acid (189-2310 ppm) and biochars (0.00-5.10%.wt). The biochars came from two local feedstocks of rice-husk (RH) and sugar-beet-pulp (SBP) pyrolyzed at temperatures 300 and 600 °C. Total concentrations of Cu, Cd, and Ni found in both the soil and biomass/biochar exceeded global safety thresholds. The bioaccumulation levels of HMs in spinach leaves varied, with Fe reaching the highest concentration at 765.27 mg kg-1 and Cd having the lowest concentration at 3.31 mg kg-1. Overall, the concentrations of Zn, Cd, Pb, and Ni in spinach leaves exceeded the safety threshold limits, so that its consumption is not recommended. The assessment of hazard quotient (HI) for the HMs indicated potential health hazards for humans (HI > 1) from consuming the edible parts of spinach. The biochar application rates of 4.35%wt and 0.00%.wt resulted in the highest (3.69) and lowest (3.15) HI values, respectively. The cumulative carcinogenic risk (TCR) ranged from 0.0085 to 0.0119, exceeding the cancer risk threshold. Introducing 5.10%wt biomass/biochar resulted in a 36% rise in TCR compared to the control. The utilization of humic acid alongside HMs-polluted biochars results in elevated levels of HMs bioaccumulation exceeding the allowable thresholds in crops (with a maximum increase of 49% at 2000 ppm humic acid in comparison to 189 ppm). Consequently, this raised the HI by 46% and the TCR by 22%. This study demonstrated that the utilization of HMs-polluted biochars could potentially pose supplementary health hazards. Moreover, it is evident that the utilization of HMs-polluted biochars in treating metal-contaminated soil does not effectively stabilize or reduce pollution.

Vacuum impregnation is a novel methodology for adding various substances to porous foods. This study aimed to develop a cost effective automate system for vacuum impregnation of food materials to enhance their nutritional, functional and sensory properties depending on the functionality of the impregnation solution. The developed vacuum impregnation system includes a vacuum chamber, vacuum pump and an automation setup for creating and maintaining vacuum conditions, feeding impregnated solutions to the samples and releasing vacuum. Fresh-cut spinach leaves were impregnated with ascorbic acid (AsA) and calcium chloride (Cacl2) (10% concentration) in the setup in order to test the effect of the process on some biochemical properties. Statistical analysis revealed significant effect of vacuum impregnation on the biochemical properties (total soluble solids, total phenolic content, flavonoid content and free radical scavenging activity) and color of spinach leaves during storage up to 4 days. Impregnation process showed significant increase in the total phenolic and flavonoid content of the spinach leaves. Increment up to 78% in antioxidant activity was seen for the uncoated impregnated leaves as compared to 59% activity in untreated samples. Thus, products with desired parameters can be produced with this process with minimal impact on their properties at a lower cost and in a shorter time period.

BACKGROUND: Optimization of a highly efficient transient expression system is critical for the study of gene function, particularly in those plants in which stable transformation methods are not widely available. Agrobacterium tumefaciens‑mediated transient transformation is a simple and low-cost method that has been developed and applied to a wide variety of plant species. However, the transient expression in spinach (Spinacia oleracea L.) is still not reported.
RESULTS: We developed a transient expression system in spinach leaves of the Sp75 and Sp73 varieties. Several factors influencing the transformation efficiency were optimized such as Agrobacterium strain, spinach seedling stage, leaf position, and the expression time after injection. Agrobacterium strain GV3101 (pSoup-p19) was more efficient than AGL1 in expressing recombinant protein in spinach leaves. In general, Sp75 leaves were more suitable than Sp73 leaves, regardless of grow stage. At four-leaf stage, higher intensity and efficiency of transient expression were observed in group 1 (G1) of Sp75 at 53 h after injection (HAI) and in G1 of Sp73 at 64 HAI. At six-leaf stage of Sp75, group 3 (G3) at 72 HAI were the most effective condition for transient expression. Using the optimized expression system, we detected the subcellular localization of a transcriptional co-activator SoMBF1c and a NADPH oxidase SoRbohF. We also detected the interaction of the protein kinase SoCRK10 and the NADPH oxidase SoRbohB.
CONCLUSIONS: This study established a method of highly efficient transient expression mediated by Agrobacterium in spinach leaves. The transient expression system will facilitate the analysis of gene function and lay a solid foundation for molecular design breeding of spinach.

Spinach (Spinacia oleracea) is one of the most famous vegetables worldwide, rich in essential metabolites for various health benefits. It is a valuable plant source that has the potential to be a nutraceutical. This study aimed to evaluate the single characteristic marker compound to establish the validation of HPLC-DAD methods applied to the development of a nutraceutical using spinach samples. Six metabolites (1-6) were identified from the spinach samples such as freeze-dried spinach (FDS) and spinach extract concentrate (SEC) by LC-Q-TOF/MS analysis. Among the six metabolites, 3\',4\',5-trihydroxy-3-methoxy-6,7-methylenedioxyflavone 4\'-glucuronide (TMG) was selected as a marker compound due to its highest abundance and high selectivity. The specificity, accuracy, linearity, precision, repeatability, limit of detection (LOD), and limit of quantification (LOQ) of TMG in the spinach samples (FDS and SEC) were validated according to AOAC international guideline. The specificity was confirmed by monitoring the well separation of the marker compound from other compounds of spinach samples in the base peak intensity (BPI) and ultraviolet (UV) chromatogram. The calibration curve of TMG (15.625~500 μg/mL) had reasonable linearity (R2 = 0.999) considered with LOD and LOQ values, respectively. Recovery rate of TMG was 93-101% for FDS and 90-95% for SEC. The precision was less than 3 and 6% in the intraday and interday. As a result, the HPLC-DAD validation method of TMG in the spinach samples (FDS and SEC) was first established with AOAC and KFDA regulations for approving functional ingredients in functional foods.

Insufficient disinfection of fresh-cut spinach poses significant health risks, along with potential issues like odor, color changes, and softening during short-term storage. To address these challenges, boric acid solutions were explored as an alternative to chlorine washes, which are known to produce toxic compounds. Among various concentrations, 1 % boric acid exhibited the most effective microbial inactivation, leading to substantial reductions in total mesophilic aerobic bacteria, total yeast and mold, and Enterobacteriaceae counts, with reductions of 1.64, 1.38, and 1.77 logs, respectively. Additionally, washing spinach leaves with this solution for 1 min maintained quality parameters, with enhanced antioxidant activity (55.26 mg kg-1 Trolox equivalent), increased total phenolic content (1214.06 mg kg-1 gallic acid equivalent), retention of chlorophyll a (839.16 mg kg-1), chlorophyll b (539.61 mg kg-1) and ascorbic acid content (264.72 mg kg-1). Mechanical properties such as puncture strength (1.81 N) and puncture distance (52.78 mm) also showed favorable outcomes, alongside optimal moisture content at 89.81 %. Notably, residual boric acid content was lowest in spinach leaves (1252.49 mg kg-1) and highest in the wash water (53.88 mg kg-1) after treatment. Scanning electron microscopy images demonstrated maintained tissue integrity, while Hunter Lab readings indicated minimal color changes post-washing. Additionally, sensory evaluations and various physicochemical analyses further supported the efficacy of boric acid washing. Consequently, washing spinach leaves with a 1 % boric acid solution for 1 min yielded favorable results across multiple quality parameters. These findings suggest the potential of boric acid as a safe and effective alternative disinfectant in the fresh-cut produce industry, highlighting its practical implications for food safety and quality. Future research should focus on exploring long-term effects and optimizing washing protocols for broader applications.

Rapid and accurate determination of pigment content is important for quality inspection of spinach leaves during storage. This study aimed to use hyperspectral imaging at two spectral ranges (visible/near-infrared, VNIR: 400-1000 nm; NIR: 900-1700 nm) to simultaneously determine the pigment (chlorophyll a, chlorophyll b, total chlorophyll, and carotenoids) content in spinach stored at different durations and conditions (unpackaged and packaged). Partial least squares (PLS), back propagation neural network (BPNN) and convolutional neural network (CNN) were used to establish single-task and multi-task regression models. Single-task CNN (STCNN) models and multi-task CNN (MTCNN) models obtained better performances than the other models. The models using VNIR spectra were superior to those using NIR spectra. The overall results indicated that hyperspectral imaging with multi-task learning could predict the quality attributes of spinach simultaneously for spinach quality inspection under various storage conditions. This research will guide food quality inspection by simultaneously inspecting multiple quality attributes.

BACKGROUND: The auxin/indole-3-acetic acid (Aux/IAA) gene family is a crucial element of the auxin signaling pathway, significantly influencing plant growth and development. Hence, we conducted a comprehensive investigation of Aux/IAAs gene family using the Sp75 and Monoe-Viroflay genomes in spinach.
RESULTS: A total of 24 definitive Aux/IAA genes were identified, exhibiting diverse attributes in terms of amino acid length, molecular weight, and isoelectric points. This diversity underscores potential specific roles within the family, such as growth regulation and stress response. Structural analysis revealed significant variations in gene length and molecular weight. These variations indicate distinct roles within the Aux/IAA gene family. Chromosomal distribution analysis exhibited a dispersed pattern, with chromosomes 4 and 1 hosting the highest and lowest numbers of Aux/IAA genes, respectively. Phylogenetic analysis grouped the identified genes into distinct clades, revealing potential evolutionary relationships. Notably, the phylogenetic tree highlighted specific gene clusters suggesting shared genetic ancestry and potential functional synergies within spinach. Expression analysis under NAA treatment unveiled gene-specific and time-dependent responses, with certain genes exhibiting distinct temporal expression patterns. Specifically, SpoIAA5 displayed a substantial increase at 2 h post-NAA treatment, while SpoIAA7 and SpoIAA9 demonstrated continuous rises, peaking at the 4-hour time point.
CONCLUSIONS: These observations indicate a complex interplay of gene-specific and temporal regulation in response to auxin. Moreover, the comparison with other plant species emphasized both shared characteristics and unique features in Aux/IAA gene numbers, providing insights into the evolutionary dynamics of this gene family. This comprehensive characterization of Aux/IAA genes in spinach not only establishes the foundation for understanding their specific functions in spinach development but also provides a valuable resource for experimental validation and further exploration of their roles in the intricate network of auxin signaling pathways.