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The emergence of unisexual flower is an important event during plant evolution. The molecular mechanism underlying the formation of unisexual flowers remains unclear in dioecious spinach. In this study, we identified the spinach MALE STERILITY1 gene, SpMS1, which serves as a masculine factor to regulate male fertility and sex reversion. Silencing SpMS1 led to stamen sterility in male flowers and the development of masculine traits in female flowers. Overexpression of SpMS1 in wild-type Arabidopsis resulted in sterile stamens and irregular pollen exine. Notably, ectopic expression of SpMS1 in Arabidopsis ms1 mutants restored pollen viability and flower fertility. Furthermore, our findings demonstrate that SpMS1 interacts with MADS-box transcription factor SpAP1 to regulate unisexual flower development. Thus, SpMS1 exhibits a conserved function in pollen fertility akin to bisexual flowers, while also acting as a key regulator of unisexual flower development in spinach. This study sheds light on the mechanism of sex differentiation in dioecious plants and also provides valuable insights for manipulating male sterility in plant breeding programs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site–for further information please contact journals.permissions@oup.com.)

Background: Consumption of leafy vegetables is a primary route of cadmium (Cd) exposure in the human body. Salicylic acid (SA) is a major stress signaling molecule that alleviates Cd toxicity in various plants. Our study aimed to investigate the effects of different SA concentrations on spinach growth, cadmium accumulation, and stress resistance physiology under cadmium stress (50 µmol/L)., Results: Cd stress significantly markedly decreased spinach growth and biomass, reduced its photosynthetic efficiency, increased activities of antioxidative enzymes, and upregulated the relative expression of several genes involved in cadmium absorption and transport compared to the control. The exogenous application of SA mitigated the harmful effects of Cd in spinach. 0.8 and 1.6 mmol/L SA significantly increased spinach root length, plant height, and biomass and decreased the Cd content in shoots by 30.03 and 17.35% compared to the Cd-treated group. Moreover, SA alleviated the yellowing of leaves caused by Cd stress. Exogenous SA ameliorated Cd toxicity in spinach by reducing reactive oxygen species, malondialdehyde, proline, and soluble protein levels. Exogenous SA application reduced Cd absorption in spinach leaves by downregulating the expression of genes involved in Cd transport, such as SoHMA4-like, SoNramp3.1-like, SoNramp6-like, and SoNramp7.2-like. Principal component analysis and correlation analysis showed that exogenous SA application under Cd stress was correlated with plant Cd content, photosynthetic pigment content, and relative expression of Cd absorption and transportation-related genes., Conclusions: To summarize, these findings indicate that SA mitigates Cd toxicity in spinach by reversing the adverse effects of Cd stress on plant growth and reducing Cd accumulation in the shoots., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

In the region of slightly acidic pH (рН 5.7), the manganese cluster in oxygen-evolving complex of photosystem II (PSII) is more resistant to exogenous reductants. The effect of such pH on the heat inactivation efficiency of the electron transport chain (O 2 evolution and 2,6-dichlorophenolindophenol reduction) in PSII membranes and thylakoid membranes was investigated. Under thylakoid membranes illumination accompanied by lumen acidification, their resistance to heat inactivation increases. In the presence of protonophores, the rate of heat inactivation increases, which seems to be associated not with the protonophore mechanism, but with structural and/or functional changes in membranes. In PSII membrane preparations, the efficiency of the oxygen evolution inhibition at pH 5.7 is also lower than at pH 6.5. The role of reactive oxygen species in thermal inactivation of photosynthetic membranes was investigated using a lipophilic cyclic hydroxylamine ESR spin probe., (© 2024. The Author(s) under exclusive licence to The Botanical Society of Japan.)

The spectral composition of some light-emitting diodes (LEDs) reportedly results in higher crop yield, prevents wilting, and reduces thermal damage to plants. The use of LEDs for postharvest storage and shelf-life extension has been limited, but the potential of this technology will allow for greater applications in horticulture and the food industry. In this experiment, 'Winterbor' kale (Brassica oleracea) and 'Melody' spinach (Spinacia oleracea) plants were measured for the light compensation point and stomatal response under 14 different wavelengths of light ranging from 405 to 661 nm. Data collected from these measurements were used to select two different wavelengths of LEDs and determine the proper irradiance levels for an LED irradiance storage test on spinach and kale. Treatments comprising blue, red, and amber lights were effective at increasing the stomatal opening, while the green light resulted in reduced stomatal opening. For spinach, the light response curve showed that light compensation points at 500 nm and 560 nm were 65.3 and 64.7 μmol m -2  s -1 , respectively. For kale, the light compensation points at 500 nm and 560 nm were 50.8 and 44.1 μmol m -2  s -1 , respectively. For the storage test experiment at room temperature, kale and spinach were stored under four different treatments: dark treatment (control), standard white fluorescent light, 500 nm, and 560 nm LED wavelengths. For spinach, the moisture content was 70.1% at 560 nm and 53.7% for dark, moisture losses of 41.5% under the 560-nm treatment and 52.0% for the dark treatment. The fresh basis moisture content was 74.6% at 560 nm and 59.3% in the dark. Moisture loss under the 560 nm treatment was 39.6% while the dark treatment had a 54.0% moisture loss. A visual assessment scale was monitored, 560 nm resulted in the top visual quality for kale compared to the other treatments with the lowest visual quality under the dark treatment at day 4. For spinach, the visual quality for 560 nm treatment was statistically the standard white fluorescent light and 500 nm, with poor-quality product occurring by day 4 and the lowest-quality product occurring at day 5. The LED treatments improved the shelf life of spinach and kale, likely as a result of stomatal aperture closure, photosynthetic rate near the light compensation point and stability of the atmospheric moisture content. This study provides valuable information on the extension of the shelf life of leafy greens during storage. Reducing fresh produce waste in grocery stores will increase revenue, thereby benefiting the Canadian economy while providing social and environmental benefits that entail increased food security and reduced food waste., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

This study unveils the detoxification potential of insecticide-tolerant plant beneficial bacteria (PBB), i.e., Ciceribacter azotifigens SF1 and Serratia marcescens SRB1, in spinach treated with fipronil (FIP), profenofos (PF) and chlorantraniliprole (CLP) insecticides. Increasing insecticide doses (25-400 μg kg -1 soil) significantly curtailed germination attributes and growth of spinach cultivated at both bench-scale and in greenhouse experiments. Profenofos at 400 μg kg -1 exhibited maximum inhibitory effects and reduced germination by 55%; root and shoot length by 78% and 81%, respectively; dry matter accumulation in roots and shoots by 79% and 62%, respectively; leaf number by 87% and leaf area by 56%. Insecticide application caused morphological distortion in root tips/surfaces, increased levels of oxidative stress, and cell death in spinach. Application of insecticide-tolerant SF1 and SRB1 strains relieved insecticide pressure resulting in overall improvement in growth and physiology of spinach grown under insecticide stress. Ciceribacter azotifigens improved germination rate (10%); root biomass (53%); shoot biomass (25%); leaf area (10%); Chl-a (45%), Chl-b (36%) and carotenoid (48%) contents of spinach at 25 μg CLP kg -1 soil. PBB inoculation reinvigorated the stressed spinach and modulated the synthesis of phytochemicals, proline, malondialdehyde (MDA), superoxide anions (O 2 • - ), and hydrogen peroxide (H 2 O 2 ). Scanning electron microscopy (SEM) revealed recovery in root tip morphology and stomatal openings on abaxial leaf surfaces of PBB-inoculated spinach grown with insecticides. Ciceribacter azotifigens inoculation significantly increased intrinsic water use efficiency, transpiration rate, vapor pressure deficit, intracellular CO 2 concentration, photosynthetic rate, and stomatal conductance in spinach exposed to 25 μg FIP kg -1 . Also, C. azotifigens and S. marcescens modulated the antioxidant defense systems of insecticide-treated spinach. Bacterial strains were strongly colonized to root surfaces of insecticide-stressed spinach seedlings as revealed under SEM. The identification of insecticide-tolerant PBBs such as C. azotifigens and S. marcescens hold the potential for alleviating abiotic stress to spinach, thereby fostering enhanced and safe production within polluted agroecosystems., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

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 GA 3 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 GA 3 , 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., (© 2024 Society for Experimental Biology and John Wiley & Sons Ltd.)

Microplastics, a type of new environmental pollutant, have received much attention for their negative effects on organisms and environment. We examined the effects of microplastics on seed germination and seedling physiological characteristics of spinach ( Spinacia oleracea ) under alkali stress, taking polystyrene microspheres with a diameter of 100 nm (200, 400, 800, 1600 mg·L -1 ) as the microplastic treatment, and mixed NaHCO 3 and Na 2 CO 3 as alkaline salt solution (5, 10, 20, 40 mmol·L -1 ) according to the molar ratio of 1:1. The results showed that the presence of MPs (≥400 mg·L -1 ) inhibited seed germination, and that the length of roots and shoots increased at low while decreased at high concentration of MPs. Different concentrations of alkali alone could inhibit seed germination, root and bud elongation. With the increases of MPs concentration, SOD activity of spinach seedlings gradually decreased, while POD activity firstly increased and then decreased, and chlorophyll content increased at low concentration (200 mg·L -1 ) and decreased significantly at medium and high concentration (≥400 mg·L -1 ). Different alkali stresses reduced chlorophyll content of spinach seedlings, and the effects on SOD and POD were 'promotion at low concentration and inhibition at high'. In the treatments of microplastics (200, 800 mg·L -1 ) and alkali (5, 20 mmol·L -1 ) combined exposure, germination of spinach seeds was inhibited, and chlorophyll content decreased. The activities of SOD and POD in spinach seedlings were reduced under the combined exposure except the treatment of 200 mg·L -1 MPs and 5 mmol·L -1 alkali. Compared to the alkali stress, the combination of low concentration of MPs (200 mg·L -1 ) and alkali could improve germination rate, germination index, germination vigor and vigor index of seeds, and significantly promoted the elongation of roots and shoots, while the addition of high concentration of MPs (800 mg·L -1 ) reduced the germination rate, germination index, germination vigor and vigor index of seeds and inhibited the growth of roots and buds. The different concentrations of combined exposure inhibited the activities of SOD and POD and decreased the content of chlorophyll in spinach seedlings.

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.

Background: Bolting refers to the early flowering stem production on agricultural and horticultural crops before harvesting. Indeed, bolting is an event induced by the coordinated effects of various environmental factors and endogenous genetic components, which cause a large reduction in the quality and productivity of vegetable crops like spinach. However, little is known about the signaling pathways and molecular functions involved in bolting mechanisms in spinach. The genetic information regarding the transition from vegetative growth to the reproductive stage in spinach would represent an advantage to regulate bolting time and improvement of resistant cultivars to minimize performance loss., Results: To investigate the key genes and their genetic networks controlling spinach bolting, we performed RNA-seq analysis on early bolting accession Kashan and late-bolting accession Viroflay at both vegetative and reproductive stages and found a significant number of differentially expressed genes (DEGs) ranging from 195 to 1230 in different comparisons. These genes were mainly associated with the signaling pathways of vernalization, photoperiod/circadian clock, gibberellin, autonomous, and aging pathways. Gene ontology analysis uncovered terms associated with carbohydrate metabolism, and detailed analysis of expression patterns for genes of Fructose-1, 6-bisphosphate aldolase, TREHALOSE-6-PHOSPHATE SYNTHASE 1, FLOWERING PROMOTING FACTOR 1, EARLY FLOWERING, GIGANTEA, and MADS-box proteins revealed their potential roles in the initiating or delaying of bolting., Conclusion: This study is the first report on identifying bolting and flowering-related genes based on transcriptome sequencing in spinach, which provides insight into bolting control and can be useful for molecular breeding programs and further study in the regulation of the genetic mechanisms related to bolting in other vegetable crops.

The visual perception of freshness is an important factor considered by consumers in the purchase of fruits and vegetables. However, panel testing when evaluating food products is time consuming and expensive. Herein, the ability of an image processing-based, nondestructive technique to classify spinach freshness was evaluated. Images of spinach leaves were taken using a smartphone camera after different storage periods. Twelve sensory panels ranked spinach freshness into one of four levels using these images. The rounded value of the average from all twelve panel evaluations was set as the true label. The spinach image was removed from the background, and then converted into a gray scale and CIE-Lab color space (L*a*b*) and Hue, Saturation and Value (HSV). The mean value, minimum value, and standard deviation of each component of color in spinach leaf were extracted as color features. Local features were extracted using the bag-of-words of key points from Oriented FAST (Features from Accelerated Segment Test) and Rotated BRIEF (Binary Robust Independent Elementary Features). The feature combinations selected from the spinach images were used to train machine learning models to recognize freshness levels. Correlation analysis between the extracted features and the sensory evaluation score showed a positive correlation (0.5 < r < 0.6) for four color features, and a negative correlation (‒0.6 < r < ‒0.5) for six clusters in the local features. The support vector machine classifier and artificial neural network algorithm successfully classified spinach samples with overall accuracy 70% in four-class, 77% in three-class and 84% in two-class, which was similar to that of the individual panel evaluations. Our findings indicate that a model using support vector machine classifiers and artificial neural networks has the potential to replace freshness evaluations currently performed by non-trained panels., Competing Interests: The authors have declared that no competing interests exist.

Fe(II) cations bind with high efficiency and specificity at the high-affinity (HA), Mn-binding site (termed the "blocking effect" since Fe blocks further electron donation to the site) of the oxygen-evolving complex (OEC) in Mn-depleted, photosystem II (PSII) membrane fragments (Semin et al. in Biochemistry 41:5854, 2002). Furthermore, Fe(II) cations can substitute for 1 or 2Mn cations (pH dependent) in Ca-depleted PSII membranes (Semin et al. in Journal of Bioenergetics and Biomembranes 48:227, 2016; Semin et al. in Journal of Photochemistry and Photobiology B 178:192, 2018). In the current study, we examined the effect of Ca 2+ cations on the interaction of Fe(II) ions with Mn-depleted [PSII(-Mn)] and Ca-depleted [PSII(-Ca)] photosystem II membranes. We found that Ca 2+ cations (about 50 mM) inhibit the light-dependent oxidation of Fe(II) (5 µM) by about 25% in PSII(-Mn) membranes, whereas inhibition of the blocking process is greater at about 40%. Blocking of the HA site by Fe cations also decreases the rate of charge recombination between Q A - and Y Z •+ from t 1/2  = 30 ms to 46 ms. However, Ca 2+ does not affect the rate during the blocking process. An Fe(II) cation (20 µM) replaces 1Mn cation in the Mn 4 CaO 5 catalytic cluster of PSII(-Ca) membranes at pH 5.7 but 2 Mn cations at pH 6.5. In the presence of Ca 2+ (10 mM) during the substitution process, Fe(II) is not able to extract Mn at pH 5.7 and extracts only 1Mn at pH 6.5 (instead of two without Ca 2+ ). Measurements of fluorescence induction kinetics support these observations. Inhibition of Mn substitution with Fe(II) cations in the OEC only occurs with Ca 2+ and Sr 2+ cations, which are also able to restore oxygen evolution in PSII(-Ca) samples. Nonactive cations like La 3+ , Ni 2+ , Cd 2+ , and Mg 2+ have no influence on the replacement of Mn with Fe. These results show that the location and/or ligand composition of one Mn cation in the Mn 4 CaO 5 cluster is strongly affected by calcium depletion or rebinding and that bound calcium affects the redox potential of the extractable Mn4 cation in the OEC, making it resistant to reduction.

Heavy metal like cadmium (Cd) is inessential and highly toxic and is posing serious environmental problems for agriculture worldwide. Presence of Cd gives rise to several physiological and structural disorders that leads to reduction in growth and performance of agricultural plants. Evidence related to subcellular distribution and accumulation of Cd is still enigmatic. Experiment was conducted using hydroponic culture to examine the subcellular accumulation of Cd in Spinacia oleracea L. leaves under Cd stress (50 μM and 100 μM); moreover, the Cd toxicity alleviation using 5 mM silicon (Si) was investigated. Our findings suggest that fresh and dry biomass, shoot and root length, leaf area and length of leaf declined when exposed to Cd stress (50 μM and 100 μM); however, an increase was noticed when Cd treated plants were supplied with Si (5 mM). The content of Ca 2+ , Mg 2+ and Fe 2+ in apoplastic washing fluid and symplasm were found to be lower in plants treated with alone Cd, when compared to control. Higher Cd 2+ :Ca 2+ , Cd 2+ :Fe 2+ and Cd 2+ :Mg 2+ ratios were detected under cadmium stress in both apoplast and symplast of leaves which were lowered by the addition of 5 mM Si. The novelty of the current study is the detection of increased apoplastic and symplastic Cd concentration in aerial part (i.e., spinach leaves) under alone Cd treatment which was considerably reduced when supplied with Si. Moreover, a noticeable increase in spinach growth and beneficial ionic concentrations suggest that Si can ameliorate the Cd stress in crop plants., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Light harvesting in photosynthesis is currently an issue on-debate and studied widely in all over the world. Studies on light harvesting mainly focus on enlightening molecular mechanism of the process and enhancing absorption capacity of light harvesting complexes (LHCs). Enhancement of absorption capacity of LHCs can be done either by natural methods or by synthetic methods. Quantum dots (QDs), fluorescent semiconductor nanocrystals, are important constituents of inorganic-organic hybrid structures which are built to enhance absorption capacity of LHCs through synthetic methods. In this study, we synthesized carbon and heteroatom doped carbon QDs through a microwave assisted synthesis method. Each QD had unique photophysical and structural properties. Photosynthetic pigments (PP) (isolated from spinach leaves) were mixed with each QD separately to build a QD-PP hybrid structure. Our results revealed that significant amount of energy is transferred from carbon QDs to PPs and therefore chlorophyll fluorescence capacity of PPs enhanced significantly in 360-420 nm excitation wavelength interval. Our results suggested that non-toxic, inexpensive and easily synthesized carbon QDs can be an important constituent for hybrid structures to enhance absorption capacity of LHCs in highly energetic region of visible spectrum.

In this work, the effect of the inoculation of silver-incorporated titanium dioxide nanoparticles (Ag-TiO2 NPs) in spinach seeds was evaluated on certain growth, physiology and phytotoxicity parameters of the plants. This is an important crop for human consumption with high nutritional value due to their low calorie and fat content, providing various vitamins and minerals, especially iron. These NPs were obtained by means of the sol-gel method and heat treatment; the resulting powder material was characterized using X-ray diffraction and scanning electron microscopy and the influence of these NPs on plants was measured by estimating the germination rate, monitoring morphological parameters and evaluating phytotoxicity. The photosynthetic activity of the spinach plants was estimated through the quantification of the Ratio of Oxygen Evolution (ROE) by the photoacoustic technique. Samples of TiO2 powder with particle size between 9 and 43 nm were used to quantify the germination rate, which served to determine a narrower size range between 7 and 26 nm in the experiments with Ag-TiO2 NPs; the presence of Ag in TiO2 powder samples was confirmed by energy-dispersive X-ray spectroscopy. The analysis of variance showed that the dependent variable (plant growth) could be affected by the evaluated factors (concentration and size) with significant differences. The statistical trend indicated that the application of the Ag-TiO2 NPs suspension of lowest concentration and smallest particle size could be a promoting agent of the growth and development of these plants. The inoculation with NPs of 8.3 nm size and lowest concentration was related to the highest average ROE value, 24.6 ± 0.2%, while the control group was 20.2 ± 0.2%. The positive effect of the Ag-TiO2 NPs treatment could be associated to the generation of reactive oxygen species, antimicrobial activity, increased biochemical attributes, enzymatic activity or improvements in water absorption., Competing Interests: The authors have declared that no competing interests exist.

Soil pollution with cadmium (Cd) has posed a threat to our food safety. And rice consumption is the main source of Cd intake in China. Rice intercropping with water spinach is an efficient way for crop production and phytoremediation in Cd-contaminated soil. However, few people work on the Cd remediation by a combination of the passivation and intercropping. In this study, two passivators (the Si-Ca-Mg ameliorant and the Fe-modified biochar with microbial inoculants) were used in the monoculture and intercropping systems to evaluate the potential of co-effect of passivators and cropping systems on the plant growth and Cd phytoremediation. Results showed that the highest rice biomass and rice yield were presented in the intercropping system with the passivator additions, however, relatively lower biomass was showed in water spinach due to the competition with rice. And more Cd accumulated in water spinach while lower Cd in that of different rice parts. The intercropping system with the addition of the Si-Ca-Mg ameliorant and the microbial Fe-modified biochar significantly reduced the Cd contents in brown rice by 58.86% and 63.83%, while notably enhanced the Cd accumulation of water spinach by 32.0% and 22.0%, compared with the monoculture without passivation, respectively. This probably due to the increased pH, the lowered Cd availability in soil, and the reduced TF and BCF values in rice plants with passivator applications. Collectively, this study indicated that rice-water spinach intercropping, especially with the passivator additions, may function as an effective way for Cd remediation and guarantee rice grain safety., (Copyright © 2020 Elsevier Inc. All rights reserved.)

High variability in somatic embryo (SE)-forming capacity has previously been observed in several spinach cultivars. Such variability frequently accounted for more variation in embryogenic response of the explants than the factor being investigated. Hence, the variability in embryogenic capacity was examined in the present study at both the population and the single-seedling level, using seeds of spinach cultivar Matador obtained from nine European seed companies. Seed population obtained from Slovenia (Sl) was superior to others, with the highest regeneration frequency (100%) and the highest mean SE number (14.4). A total of 82% of these seedlings had 80-100% of regenerating explants, while in populations with intermediate embryogenic capacity approximately 40% of seedlings had 20-60% of regenerating explants. The explants from the majority of seedlings (52-100%) in the least responsive populations were irresponsive. Furthermore, the explants from Sl seedlings regenerated from 10-20 (43.5%) up to > 20 (27.6%) SEs on average, while the explants from the majority of seedlings belonging to other populations regenerated 1-10 SEs. The present study strongly indicates that the variability of plant material must not be overlooked, because choosing more responsive individuals for one treatment and less responsive ones for another may lead to misinterpretation of the data.

Microorganism technologies can provide a potential alternative to traditional methods of removing heavy metals to conserve agricultural soils. This study aimed to identify and characterize heavy metals-resistant bacteria (HM-RB) isolated from industry-affected soil and their desired impact as bioremediators of heavy metals-stressed spinach plants. Three of 135 isolates were selected based on a high level of resistance to heavy metals. Based on morphological and biochemical characteristics, the selected isolates were identified as Bacillus subtilis subsp. spizizenii DSM 15029 T DSM (MA3), Paenibacillus jamilae DSM 13815 T DSM (LA22), or Pseudomonas aeruginosa DSM 1117 DSM (SN36). Experiments were implemented to investigate the three isolated HM-RB ability on improving attributes of growth, physio-biochemistry, and components of the antioxidant defense system of spinach plant exposed to the stress of cadmium (Cd 2+ ; 2 mM), lead (Pb 2+ ; 2 mM) or 2 mM Cd 2+ +2 mM Pb 2+ . Compared to control, Cd 2+ or Pb 2+ stress markedly lowered plant fresh and dry weights, leaf contents of chlorophylls and carotenoids, rates of transpiration (Tr), net photosynthesis (Pn) and stomatal conductance (gs), relative water content (RWC), and membrane stability index (MSI). In contrast, contents of α.tochopherol (α.TOC), ascorbic acid (AsA), glutathione (GSH), proline, soluble sugars, Cd 2+ , and Pb 2+ , as well as activities of enzymatic and non-enzymatic antioxidants were markedly elevated. The application of HM-RB promoted the tolerance to heavy metal stress in spinach plants by improving Tr, Pn, gs, RWC, and MSI, while activities of enzymatic and non-enzymatic antioxidants were suppressed. These results reflected positively in promoting plant growth under heavy metal stress. Therefore, the application of HM-RB as potential bioremediators may be a promising strategy for promoting plant growth and productivity under heavy metal stress., Competing Interests: Declaration of competing interest The authors have no conflict of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Plant tissues subjected to short or prolonged freezing to a fixed sub-freezing temperature are expected to undergo similar freeze-desiccation but the former causes substantially less injury than the latter. To gain metabolic insight into this differential response, metabolome changes in spinach (Spinacia oleracea L.) leaves were determined following short-term (0.5 and 3.0 h) vs. prolonged freezing (5.5 and 10.5 h) at -4.5°C resulting in reversible or irreversible injury, respectively. LD 50 , the freezing duration causing 50% injury, was estimated to be ∼3.1 h and defined as the threshold beyond which tissues were irreversibly injured. From 39 identified metabolites, 19 were selected and clustered into 3 groups: (1) signaling-related (salicylic acid, aliphatic and aromatic amino acids), (2) injury-related (GABA, lactic acid, maltose, fatty acids, policosanols, TCA intermediates) and (3) recovery-related (ascorbic acid, α-tocopherol). Initial accumulation of salicylic acid during short-term freezing followed by a decline may be involved in triggering tolerance mechanisms in moderately injured tissues, while its resurgence during prolonged freezing may signal programmed cell death. GABA accumulated with increasing freezing duration, possibly to serve as a 'pH-stat' against cytoplasmic acidification resulting from lactic acid accumulation. Mitochondria seem to be more sensitive to prolonged freezing than chloroplasts since TCA intermediates decreased after LD 50 while salicylic acid and maltose, produced in chloroplasts, accumulate even at 10.5-h freezing. Fatty acids and policosanols accumulation with increasing freezing duration indicates greater injury to membrane lipids and epicuticular waxes. Ascorbic acid and α-tocopherol accumulated after short-term freezing, supposedly facilitating recovery while their levels decreased in irreversibly injured tissues., (© 2019 Scandinavian Plant Physiology Society.)

Irrigation of crop plants with microcystins (MCs) contaminated water could be a threat to human health via bioaccumulation. Despite the fact MCs bioaccumulation in crop plants is well documented, MCs depuration, as well as the mechanism involved remains unclear. The objectives of the present study were to investigate the bioaccumulation and depuration of microcystin-LR (MC-LR) in lettuce (Lactuca sativa L.) and spinach (Spinacia oleracea L.), as well as to explore the role of glutathione (GSH) biosynthesis in MC-LR depuration. The tested plants were irrigated with deionized water containing 10 μg L -1 MC-LR for 12 days (bioaccumulation), and subsequently, with either deionized water only or deionized water containing 0.5 mM buthionine sulfoximine (BSO, a specific inhibitor of GSH biosynthesis) for 12 days (depuration). After bioaccumulation period, highest concentrations of MC-LR found in lettuce and spinach were 114.4 and 138.5 μg kg -1 dry weight (DW) respectively. Depuration rates of MC-LR in lettuce and spinach were 9.5 and 8.1 μg kg -1 DW d -1 , which deceased to 3.7 and 4.6 μg kg -1 DW d -1 in treatments with BSO application. GSH content in both lettuce and spinach were not significantly affected during depuration without BSO; whereas after treatment with BSO, GSH content significantly decreased by 36.0% and 24.7% in lettuce and spinach on 15 d, and the decrease remained on 18 d and 21 d in lettuce. Moreover, during the bioaccumulation period, activities of glutathione reductase (GR) and glutathione S-transferase (GST) were enhanced in both plants. Our results suggested that GSH biosynthesis played an important role in MC-LR depuration in the tested plants. Concerning human health risk, most of the estimated daily intake (EDI) values during the bioaccumulation period exceeded the tolerable daily intake (TDI) guideline. However, the risk could be alleviated by irrigating with MCs-free water for a certain amount of time before harvest., (Copyright © 2019. Published by Elsevier Ltd.)

Photosynthetic induction, a gradual increase in photosynthetic rate on a transition from darkness or low light to high light, has ecological significance, impact on biomass accumulation in fluctuating light and relevance to photoprotection in strong light. However, the experimental quantification of the component electron fluxes in and around both photosystems during induction has been rare. Combining optimized chlorophyll fluorescence, the redox kinetics of P700 [primary electron donor in Photosystem I (PSI)] and membrane inlet mass spectrometry in the absence/presence of inhibitors/mediator, we partially estimated the components of electron fluxes in spinach leaf disks on transition from darkness to 1,000 �mol photons�m-2�s-1 for up to 10 min, obtaining the following findings: (i) the partitioning of energy between both photosystems did not change noticeably; (ii) in Photosystem II (PSII), the combined cyclic electron flow (CEF2) and charge recombination (CR2) to the ground state decreased gradually toward 0 in steady state; (iii) oxygen reduction by electrons from PSII, partly bypassing PSI, was small but measurable; (iv) cyclic electron flow around PSI (CEF1) peaked before becoming somewhat steady; (v) peak magnitudes of some of the electron fluxes, all probably photoprotective, were in the descending order: CEF1 > CEF2 + CR2 > chloroplast O2 uptake; and (vi) the chloroplast NADH dehydrogenase-like complex appeared to aid the antimycin A-sensitive CEF1. The results are important for fine-tuning in silico simulation of in vivo photosynthetic electron transport processes; such simulation is, in turn, necessary to probe partial processes in a complex network of interactions in response to environmental changes., (� The Author(s) 2019. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Spinach (Spinacia oleracea L.) is a vegetable plant with high nutritional properties. In the present work, we studied responses of in vitro shoot cultures to salt stress (0 (control), 100, 200 and 300 mM NaCl) and salt stress-induced accumulation of 20-hydroxyecdysone (20E). Our results revealed that effect of low to moderate level of salinity stress (100-200 mM) was less pronounced on growth and tissue water content (TWC) of shoot cultures compared to higher salinity level (300 mM). The salt treated shoot cultures showed better osmotic adjustment in terms of significant accumulation of compatible solutes and total soluble sugars and also higher antioxidant enzyme activity. As the NaCl stress was increased, there was a corresponding linear raise in the Na + accumulation while the contents of both K + and Ca 2+ decreased significantly. We also studied salt-stress induced accumulation of a bioactive compound; 20E and results showed that 200 mM salt treated shoot cultures accumulated significantly 2.9 fold higher 20E as compared to untreated shoot cultures. The results suggest that Spinacia oleracea exhibits considerable salt tolerance with better osmotic adjustment and can be considered a suitable candidate for the production of bioactive secondary metabolite.

High temperatures seriously limit plant growth and productivity. Investigating heat-responsive molecular mechanisms is important for breeding heat-tolerant crops. In this study, heat-responsive mechanisms in leaves from a heat-sensitive spinach ( Spinacia oleracea L.) variety Sp73 were investigated using two-dimensional gel electrophoresis (2DE)-based and isobaric tags for relative and absolute quantification (iTRAQ)-based proteomics approaches. In total, 257 heat-responsive proteins were identified in the spinach leaves. The abundance patterns of these proteins indicated that the photosynthesis process was inhibited, reactive oxygen species (ROS) scavenging pathways were initiated, and protein synthesis and turnover, carbohydrate and amino acid metabolism were promoted in the spinach Sp73 in response to high temperature. By comparing this with our previous results in the heat-tolerant spinach variety Sp75, we found that heat inhibited photosynthesis, as well as heat-enhanced ROS scavenging, stress defense pathways, carbohydrate and energy metabolism, and protein folding and turnover constituting a conservative strategy for spinach in response to heat stress. However, the heat-decreased biosynthesis of chlorophyll and carotenoid as well as soluble sugar content in the variety Sp73 was quite different from that in the variety Sp75, leading to a lower capability for photosynthetic adaptation and osmotic homeostasis in Sp73 under heat stress. Moreover, the heat-reduced activities of SOD and other heat-activated antioxidant enzymes in the heat-sensitive variety Sp73 were also different from the heat-tolerant variety Sp75, implying that the ROS scavenging strategy is critical for heat tolerance.

In the present study, we have investigated the effect of hydroxyectoine (Ect-OH), a heterocyclic amino acid, on oxygen evolution in photosystem II (PS II) membrane fragments and on photoinhibition of Mn-depleted PS II (apo-WOC-PS II) preparations. The degree of photoinhibition of apo-WOC-PS II preparations was estimated by the loss of the capability of exogenous electron donor (sodium ascorbate) to restore the amplitude of light-induced changes of chlorophyll fluorescence yield (∆F). It was found that Ect-OH (i) stimulates the oxygen-evolving activity of PS II, (ii) accelerates the electron transfer from exogenous electron donors (K 4 [Fe(CN) 6 ], DPC, TMPD, Fe 2+ , and Mn 2+ ) to the reaction center of apo-WOC-PS II, (iii) enhances the protective effect of exogenous electron donors against donor-side photoinhibition of apo-WOC-PS II preparations. It is assumed that Ect-OH can serve as an artificial electron donor for apo-WOC-PS II, which does not directly interact with either the donor or acceptor side of the reaction center. We suggest that the protein conformation in the presence of Ect-OH, which affects the extent of hydration, becomes favorable for accepting electrons from exogenous donors. To our knowledge, this is the first study dealing with redox activity of Ect-OH towards photosynthetic pigment-protein complexes.

Understanding the effects of many essential non-steroidal anti-inflammatory drugs (NSAIDs) on plants is still limited, especially at environmentally realistic concentrations. This paper presents the influence of three of the most frequently used NSAIDs (diclofenac, ibuprofen, and naproxen) at environmentally realistic concentrations on the autochthonous green leafy vegetables: orache (Atriplex patula L.), spinach (Spinacia oleracea L.) and lettuce (Lactuca sativa L.). Our research was focused on the determination of the photosynthetic parameters, the emission rate of volatile organic compounds, and the evaluation of the ultrastructure of leaves of studied vegetables after exposure to abiotic stress induced by environmental pollutants, namely NSAIDs. The data obtained indicate a moderate reduction of foliage physiological activity as a response to the stress induced by NSAIDs to the selected green leafy vegetables. The increase of the 3-hexenal and monoterpene emission rates with increasing NSAIDs concentration could be used as a sensitive and a rapid indicator to assess the toxicity of the NSAIDs. Microscopic analysis showed that the green leafy vegetables were affected by the selected NSAIDs. In comparison to the controls, the green leafy vegetables treated with NSAIDs presented irregular growth of glandular trichomes on the surface of the adaxial side of the leaves, less stomata, cells with less cytoplasm, irregular cell walls and randomly distributed chloroplasts. Of the three NSAIDs investigated in this study, ibuprofen presented the highest influence. The results obtained in this study can be used to better estimate the impact of drugs on the environment and to improve awareness on the importance of the responsible use of drugs.

High temperature is a major environmental factor affecting plant growth. Heat shock proteins (Hsps) are molecular chaperones that play important roles in improving plant thermotolerance during heat stress. Spinach (Spinacia oleracea) is very sensitive to high temperature; however, the specific function of Hsps in spinach is unclear. In this study, cytosolic heat shock 70 protein (SoHSC70), which was induced by heat stress, was cloned from spinach. Overexpressing SoHSC70 in spinach calli and Arabidopsis enhanced their thermotolerance. In contrast, spinach seedlings with silenced SoHSC70 by virus-induced gene silencing (VIGS) showed more sensitivity to heat stress. Further analysis revealed that overexpressing SoHSC70 altered relative electrical conductivity (REC), malondialdehyde (MDA) content, photosynthetic rate, reactive oxygen species (ROS) accumulation and the activities of antioxidant enzymes, such as superoxide dismutase (SOD), peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) after the heat treatment. Taken together, our results suggest that overexpressing SoHSC70 positively affects heat tolerance by reducing membrane damage and ROS accumulation and improving activities of antioxidant enzymes., (Copyright © 2019. Published by Elsevier B.V.)

Photosynthetic phenotyping requires quick characterization of dynamic traits when measuring large plant numbers in a fluctuating environment. Here, we evaluated the light-induced fluorescence transient (LIFT) method for its capacity to yield rapidly fluorometric parameters from 0.6 m distance. The close approximation of LIFT to conventional chlorophyll fluorescence (ChlF) parameters is shown under controlled conditions in spinach leaves and isolated thylakoids when electron transport was impaired by anoxic conditions or chemical inhibitors. The ChlF rise from minimum fluorescence (F o ) to maximum fluorescence induced by fast repetition rate (F m-FRR ) flashes was dominated by reduction of the primary electron acceptor in photosystem II (Q A ). The subsequent reoxidation of Q A - was quantified using the relaxation of ChlF in 0.65 ms (F r1 ) and 120 ms (F r2 ) phases. Reoxidation efficiency of Q A - (F r1 /F v , where F v  = F m-FRR  - F o ) decreased when electron transport was impaired, while quantum efficiency of photosystem II (F v /F m ) showed often no significant effect. ChlF relaxations of the LIFT were similar to an independent other method. Under increasing light intensities, F r2 '/F q ' (where F r2 ' and F q ' represent F r2 and F v in the light-adapted state, respectively) was hardly affected, whereas the operating efficiency of photosystem II (F q '/F m ') decreased due to non-photochemical quenching. F m-FRR was significantly lower than the ChlF maximum induced by multiple turnover (F m-MT ) flashes. However, the resulting F v /F m and F q '/F m ' from both flashes were highly correlated. The LIFT method complements F v /F m with information about efficiency of electron transport. Measurements in situ and from a distance facilitate application in high-throughput and automated phenotyping.

Examination of plants with strong Cu tolerance and an understanding of their Cu-tolerance mechanisms are of considerable significance for the remediation of Cu-contaminated soil. Although spinach may be a plant with strong Cu tolerance, the threshold of Cu tolerance in this plant and its physiological response mechanisms to Cu are still unclear. In this study, we examined that the effects of different Cu concentrations on the growth parameters, antioxidant enzyme activities, and photosynthesis of spinach seedlings. The results showed that when treated with a low Cu concentration (100 mg L -1 CuSO 4 ), the biomass of spinach seedlings increased, whereas the MDA content, the activities of antioxidant enzymes, P n , g s and T r were not significantly different from those in the control (P > 0.05), and Y(II), qP reached their maximum values, indicating that a low Cu concentration (100 mg L -1 CuSO 4 ) had minimal negative effects on the life activities of spinach seedlings. In contrast, when treated with high Cu concentrations (800-1000 mg L -1 CuSO 4 ), the total biomass of spinach seedlings was markedly decreased, the MDA contents increased, antioxidant enzyme activities initially increased and then decreased to varying degrees, the contents of chlorophyll, P n , T r , Fv/Fm, qP , NPQ, and Y(II) were all decreased. However the growth of spinach did not terminate, implying that the lethal threshold concentration of Cu for spinach is greater than 1000 mg L -1 CuSO 4 used in this study. In summary, spinach exhibits a high tolerance to Cu and can be considered as an alternative plant for the remediation of Cu-contaminated soils., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The effects of factors such as weather conditions, leaf age and irrigation water disinfection on the main bacterial genera (total bacterial, Enterobacteriaceae and Pseudomonas) of baby spinach were studied. Culture-dependent and independent quantification techniques were compared. Cultivation was carried out over two consecutive trials in commercial open field divided in two plots: 1) baby spinach irrigated with untreated surface water and 2) baby spinach irrigated with chlorine dioxide (ClO 2 ) treated water. In all the cases, higher concentrations of bacteria were detected using molecular quantification in comparison with culture dependent techniques. Based on the obtained results, wind speed, solar radiation and relative humidity seem to have an impact on the levels of total bacterial, Enterobacteriaceae and Pseudomonas during cultivation of baby spinach. However, further studies would be needed to confirm this tendency. Water disinfection treatments (ClO 2 ), when applied to irrigation water, impacted differently the bacterial genera evaluated in the present study. Thus, although no significant effects were observed in total bacterial enumerations of baby spinach irrigated with ClO 2 treated water; significant reductions were detected in Enterobacteriaceae (19%) and Pseudomonas spp. (14%) levels. These results were also confirmed using specific culture-dependent methods. On the other hand, leaf age did not influence the levels of the main bacterial genera of baby spinach. Considering that, a large proportion of foodborne and pathogenic bacteria associated to fresh produce belong to the Enterobacteriaceae family and Pseudomonas genera, reductions in these bacterial groups could be beneficial. However, these groups are very diverse, making difficult to link the measurement of Enterobacteriaceae and Pseudomonas levels with the presence/abundance of potential pathogenic and spoilage microorganisms., (Copyright © 2018. Published by Elsevier Ltd.)

Photoprotective non-photochemical quenching (NPQ) represents an effective way to dissipate the light energy absorbed in excess by most phototrophs. It is often claimed that NPQ formation/relaxation kinetics are determined by xanthophyll composition. We, however, found that, for the alveolate alga Chromera velia, this is not the case. In the present paper, we investigated the reasons for the constitutive high rate of quenching displayed by the alga by comparing its light harvesting strategies with those of a model phototroph, the land plant Spinacia oleracea. Experimental results and in silico studies support the idea that fast quenching is due not to xanthophylls, but to intrinsic properties of the Chromera light harvesting complex (CLH) protein, related to amino acid composition and protein folding. The pKa for CLH quenching was shifted by 0.5 units to a higher pH compared with higher plant antennas (light harvesting complex II; LHCII). We conclude that, whilst higher plant LHCIIs are better suited for light harvesting, CLHs are 'natural quenchers' ready to switch into a dissipative state. We propose that organisms with antenna proteins intrinsically more sensitive to protons, such as C. velia, carry a relatively high concentration of violaxanthin to improve their light harvesting. In contrast, higher plants need less violaxanthin per chlorophyll because LHCII proteins are more efficient light harvesters and instead require co-factors such as zeaxanthin and PsbS to accelerate and enhance quenching.

The steroid 20-hydroxyecdysone (20E) is a major component of phytoecdysteroid in plants and may play a defensive role against insect pests in higher plants. In spinach, the biosynthesis and accumulation of 20E have been investigated during the vegetative stage; however, these processes have not been clearly studied during the reproductive stage, particularly in male and female individuals. In this study, we analyzed the level and distribution of 20E in individual male and female spinach plants during the reproductive stage via high performance liquid chromatography (HPLC). We found that 20E biosynthesis and accumulation were markedly different between male and female spinach during the late flowering stage. Compared with the male plant, biosynthesis of 20E in the leaves was more active and its accumulation in the floral parts was higher in female plants during the late flowering stage. These results indicate that the female reproductive organs at least in PE-positive plants could be effectively protected against harmful insects via active biosynthesis and accumulation of PE during the late flowering stage to protect floral parts from harmful insects for seed formation and store the available 20E in seeds for the next generation., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

A field experiment was conducted to assess the impact of pesticides viz., chlorpyrifos, dimethoate and dieldrin alone and in combination with different soil amendments like chemical fertilizer, farmyard manure and combination of 50% chemical fertilizer and 50% farmyard manure on the growth, photosynthetic pigments, oxidative stress markers: superoxide radical (SOR) and hydrogen peroxide (H 2 O 2 ) and their consequent damage on lipids in terms of malondialdehyde equivalents and electrolyte leakage, and different antioxidant enzymes activity like superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), glutathione-s-transferase (GST), ascorbate peroxidase (APX) and glutathione reductase (GR) in palak (Spinacia oleracea L.) grown widely in tropical croplands. The pesticides effect was analyzed at its recommended dose in agriculture for growing plants when applied two times after germination. Our results showed distinct increase in SOR and H 2 O 2 which further manifested in marked oxidative damages in case of pesticides treatment individually that lead to decline in growth characteristics and yield. Further the application of different types of soil amendments led to consistent increase in levels of antioxidant system along with the amelioration in oxidative damage indices which was comparable across the combined treatments. The photosynthetic pigments, yield, antioxidants were maximum in case of pesticides applied in combination with 50% chemical fertilizer and 50% farmyard manure (pesticides+NF). It was apparent from the results that the application of pesticides+NF might be recommended in tropics as it ameliorates oxidative damages and maintains high quality of plant along yield which in turn will lead to no negative consequences on the global population., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Upon transition of plants from darkness to light the initiation of photosynthetic linear electron transfer (LET) from H 2 O to NADP + precedes the activation of CO 2 fixation, creating a lag period where cyclic electron transfer (CET) around photosystem I (PSI) has an important protective role. CET generates ΔpH without net reduced NADPH formation, preventing overreduction of PSI via regulation of the cytochrome b 6 f (cytb 6 f) complex and protecting PSII from overexcitation by inducing non-photochemical quenching. The dark-to-light transition also provokes increased phosphorylation of light-harvesting complex II (LHCII). However, the relationship between LHCII phosphorylation and regulation of the LET/CET balance is not understood. Here, we show that the dark-to-light changes in LHCII phosphorylation profoundly alter thylakoid membrane architecture and the macromolecular organization of the photosynthetic complexes, without significantly affecting the antenna size of either photosystem. The grana diameter and number of membrane layers per grana are decreased in the light while the number of grana per chloroplast is increased, creating a larger contact area between grana and stromal lamellae. We show that these changes in thylakoid stacking regulate the balance between LET and CET pathways. Smaller grana promote more efficient LET by reducing the diffusion distance for the mobile electron carriers plastoquinone and plastocyanin, whereas larger grana enhance the partition of the granal and stromal lamellae plastoquinone pools, enhancing the efficiency of CET and thus photoprotection by non-photochemical quenching.

Plant nanobionics aims to embed non-native functions to plants by interfacing them with specifically designed nanoparticles. Here, we demonstrate that living spinach plants (Spinacia oleracea) can be engineered to serve as self-powered pre-concentrators and autosamplers of analytes in ambient groundwater and as infrared communication platforms that can send information to a smartphone. The plants employ a pair of near-infrared fluorescent nanosensors-single-walled carbon nanotubes (SWCNTs) conjugated to the peptide Bombolitin II to recognize nitroaromatics via infrared fluorescent emission, and polyvinyl-alcohol functionalized SWCNTs that act as an invariant reference signal-embedded within the plant leaf mesophyll. As contaminant nitroaromatics are transported up the roots and stem into leaf tissues, they accumulate in the mesophyll, resulting in relative changes in emission intensity. The real-time monitoring of embedded SWCNT sensors also allows residence times in the roots, stems and leaves to be estimated, calculated to be 8.3 min (combined residence times of root and stem) and 1.9 min mm -1 leaf, respectively. These results demonstrate the ability of living, wild-type plants to function as chemical monitors of groundwater and communication devices to external electronics at standoff distances.

Salinity is a common problem under irrigated agriculture, especially in low rainfall and high evaporative demand areas of southwestern United States and other semi-arid regions around the world. However, studies on salinity effects on soil microbial communities are relatively few while the effects of irrigation-induced salinity on soil chemical and physical properties and plant growth are well documented. In this study, we examined the effects of salinity, temperature, and temporal variability on soil and rhizosphere microbial communities in sand tanks irrigated with prepared solutions designed to simulate saline wastewater. Three sets of experiments with spinach (Spinacia oleracea L., cv. Racoon) were conducted under saline water during different time periods (early winter, late spring, and early summer). Bacterial 16S V4 rDNA region was amplified utilizing fusion primers designed against the surrounding conserved regions using MiSeq® Illumina sequencing platform. Across the two sample types, bacteria were relatively dominant among three phyla-the Proteobacteria, Cyanobacteria, and Bacteroidetes-accounted for 77.1% of taxa detected in the rhizosphere, while Proteobacteria, Bacteroidetes, and Actinobacteria accounted for 55.1% of taxa detected in soil. The results were analyzed using UniFrac coupled with principal coordinate analysis (PCoA) to compare diversity, abundance, community structure, and specific bacterial groups in soil and rhizosphere samples. Permutational analysis of variance (PERMANOVA) analysis showed that soil temperature (P=0.001), rhizosphere temperature (P=0.001), rhizosphere salinity (P=0.032), and evapotranspiration (P=0.002) significantly affected beta diversity of soil and rhizosphere microbial communities. Furthermore, salinity had marginal effects (P=0.078) on soil beta diversity. However, temporal variability differentially affected rhizosphere microbial communities irrigated with saline wastewater. Therefore, microbial communities in soils impacted by saline irrigation water respond differently to irrigation water quality and season of application due to temporal effects associated with temperature., (Published by Elsevier B.V.)

To maintain high photosynthetic rates, plants must adapt to their light environment on a timescale of seconds to minutes. Therefore, the light-harvesting antenna system of photosystem II in thylakoid membranes, light-harvesting complex II (LHCII), has a feedback mechanism, which determines the proportion of absorbed energy dissipated as heat: non-photochemical chlorophyll fluorescence quenching (NPQ). This is crucial to prevent photo-oxidative damage to photosystem II (PSII) and is controlled by the transmembrane pH differences (ΔpH). High ΔpH activates NPQ by protonation of the protein PsbS and the enzymatic de-epoxidation of LHCII-bound violaxanthin to zeaxanthin. But the precise role of PsbS and its interactions with different LHCII complexes remain uncertain. We have investigated PsbS-LHCII interactions in native thylakoid membranes using magnetic-bead-linked antibody pull-downs. The interaction of PsbS with the antenna system is affected by both ΔpH and the level of zeaxanthin. In the presence of ΔpH alone, PsbS is found to be mainly associated with the trimeric LHCII protein polypeptides, Lhcb1, Lhcb2 and Lhcb3. However, a combination of ΔpH and zeaxanthin increases the proportion of PsbS bound to the minor LHCII antenna complex proteins Lhcb4, Lhcb5 and Lhcb6. This pattern of interaction is not influenced by the presence of PSII reactions centres. Similar to LHCII particles in the photosynthetic membrane, PsbS protein forms clusters in the NPQ state. NPQ recovery in the dark requires uncoupling of PsbS. We suggest that PsbS acts as a 'seeding' centre for the LHCII antenna rearrangement that is involved in NPQ.

The contents of soluble sugars (sucrose, fructose, glucose, maltose and raffinose), vitamin C and free amino acids (34 compounds, essential and non-essential) were quantified in open-field and greenhouse-grown spinaches in response to cold stress using liquid chromatography. In general, greenhouse cultivation produced nutritionally high value spinach in a shorter growing period, where the soluble sugars, vitamin C and total amino acids concentrations, including essential were in larger amounts compared to those grown in open-field scenarios. Further, low temperature exposure of spinach during a shorter growth period resulted in the production of spinach with high sucrose, ascorbate, proline, gamma-aminobutyric acid, valine and leucine content, and these constitute the most important energy/nutrient sources. In conclusion, cultivation of spinach in greenhouse at a low temperature (4-7°C) and exposure for a shorter period (7-21days) before harvest is recommended. This strategy will produce a high quality product that people can eat., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the photosynthetic apparatus of plants and algae, the major Light-Harvesting Complexes (LHCII) collect excitations and funnel these to the photosynthetic reaction center where charge separation takes place. In excess light conditions, remodeling of the photosynthetic membrane and protein conformational changes produces a photoprotective state in which excitations are rapidly quenched to avoid photodamage. The quenched states are associated with protein aggregation, however the LHCII complexes are also proposed to have an intrinsic capacity to shift between light harvesting and fluorescence-quenched conformational states. To disentangle the effects of protein-protein and protein-lipid interactions on the LHCII photoprotective switch, we compared the structural and fluorescent properties of LHCII lipid nanodiscs and proteoliposomes with very low protein-to-lipid ratios. We demonstrate that LHCII proteins adapta fully fluorescent state in nanodiscs and in proteoliposomes with highly diluted protein densities. Increasing the protein density induces a transition into a mildly-quenched state that reaches a plateau at a molar protein-to-lipid ratio of 0.001 and has a fluorescence yield reminiscent of the light-harvesting state in vivo. The low onset for quenching strongly suggests that LHCII-LHCII attractive interactions occur inside membranes. The transition at low protein densities does not involve strong changes in the excitonic circular-dichroism spectrum and is distinct from a transition occurring at very high protein densities that comprises strong fluorescence quenching and circular-dichroism spectral changes involving chlorophyll 611 and 612, correlating with proposed quencher sites of the photoprotective mechanisms., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The main physiological function of LHCII (light-harvesting pigment-protein complex of photosystem II), the largest photosynthetic antenna complex of plants, is absorption of light quanta and transfer of excitation energy toward the reaction centers, to drive photosynthesis. However, under strong illumination, the photosynthetic apparatus faces the danger of photodegradation and therefore excitations in LHCII have to be down-regulated, e.g., via thermal energy dissipation. One of the elements of the regulatory system, operating in the photosynthetic apparatus under light stress conditions, is a conversion of violaxanthin, the xanthophyll present under low light, to zeaxanthin, accumulated under strong light. In the present study, an effect of violaxanthin and zeaxanthin on the molecular organization and the photophysical properties of LHCII was studied in a monomolecular layer system with application of molecular imaging (atomic force microscopy, fluorescence lifetime imaging microscopy) and spectroscopy (UV-Vis absorption, FTIR, fluorescence spectroscopy) techniques. The results of the experiments show that violaxanthin promotes the formation of supramolecular LHCII structures preventing dissipative excitation quenching while zeaxanthin is involved in the formation of excitonic energy states able to quench chlorophyll excitations in both the higher (B states) and lower (Q states) energy levels. The results point to a strategic role of xanthophylls that are not embedded in a protein environment, in regulation of the photosynthetic light harvesting activity in plants.

How do plants cope with excess light energy? Crop health and stress tolerance are governed by molecular photoprotective mechanisms. Protective exciton quenching in plants is activated by membrane energization, via unclear conformational changes in proteins called antennas. Here we show that pH and salt gradients stimulate the response of such an antenna under low and high energization by all-atom Molecular Dynamics Simulations. Novel insight establishes that helix-5 (H5) conformation in CP29 from spinach is regulated by chemiosmotic factors. This is selectively correlated with the chl-614 macrocycle deformation and interactions with nearby pigments, that could suggest a role in plant photoprotection. Adding to the significance of our findings, H5 domain is conserved among five antennas (LHCB1-5). These results suggest that light harvesting complexes of Photosystem II, one of the most abundant proteins on earth, can sense chemiosmotic gradients via their H5 domains in an upgraded role from a solar detector to also a chemiosmotic sensor., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In this study, we analyzed seed germination, seedling growth, and physiological aspects after treatment with high voltage nanosecond pulsed plasma and micro DBD plasma in spinach (Spinacia oleracea L.), a green leafy vegetable known to have low germination rate. Both germination and dry weight of seedlings increased after high voltage pulse shots were applied to spinach seeds. However seeds treated with many shots (10 shots) showed a decrease in germination rate and seedling growth. Seeds treated with air DBD plasma exhibited slightly higher germination and subsequent seedling growth than those treated with N2 plasma. Seed surface was degenerated after treated with high voltage pulsed plasma and micro DBD plasma but no significant difference in the degree of degeneration was observed among micro DBD plasma treatment time. Level of GA3 hormone and mRNA expression of an amylolytic enzyme-related gene in seeds were elevated 1 day after treatment with high voltage pulsed plasma. The relative amount of chlorophyll and total polyphenols in spinach seedlings grown from seeds treated with air DBD plasma was increased in 30 s, 1 min, and 3 min treatments. Taken together, our results suggest a possibility that plasma can enhance seed germination by triggering biochemical processes in seeds., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Photosystem I (PSI) photoinhibition suppresses plant photosynthesis and growth. However, the mechanism underlying PSI photoinhibition has not been fully clarified. In this study, in order to investigate the mechanism of PSI photoinhibition in higher plants, we applied repetitive short-pulse (rSP) illumination, which causes PSI-specific photoinhibition in chloroplasts isolated from spinach leaves. We found that rSP treatment caused PSI photoinhibition, but not PSII photoinhibition in isolated chloroplasts in the presence of O2 However, chloroplastic superoxide dismutase and ascorbate peroxidase activities failed to protect PSI from its photoinhibition. Importantly, PSI photoinhibition was largely alleviated in the presence of methyl viologen, which stimulates the production of reactive oxygen species (ROS) at the stromal region by accepting electrons from PSI, even under the conditions where CuZn-superoxide dismutase and ascorbate peroxidase activities were inactivated by KCN. These results suggest that the ROS production site, but not the ROS production rate, is critical for PSI photoinhibition. Furthermore, we found that not only superoxide (O2 (-)) but also singlet oxygen ((1)O2) is involved in PSI photoinhibition induced by rSP treatment. From these results, we suggest that PSI photoinhibition is caused by both O2 (-) and (1)O2 produced within the thylakoid membranes when electron carriers in PSI become highly reduced. Here, we show, to our knowledge, new insight into the PSI photoinhibition in higher plants., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

The coupling factor CF1 is a catalytic part of chloroplast ATP synthase which is exposed to stroma whose viscosity is many-fold higher than that of reaction mixtures commonly used to measure kinetics of CF1-catalyzed ATP hydrolysis. This study is focused on the effect of medium viscosity modulated by sucrose or bovine serum albumin (BSA) on kinetics of Ca(2+)- and Mg(2+)-dependent ATP hydrolysis by CF1. These agents were shown to reduce the maximal rate of Ca(2+)-dependent ATPase without changing the apparent Michaelis constant (К m), thus supporting the hypothesis on viscosity dependence of CF1 activity. For the sulfite- and ethanol-stimulated Mg(2+)-dependent reaction, the presence of sucrose increased К m without changing the maximal rate that is many-fold as high as that of Ca(2+)-dependent hydrolysis. The hydrolysis reaction was shown to be stimulated by low concentrations of BSA and inhibited by its higher concentrations, with the increasing maximal reaction rate estimated by extrapolation. Sucrose- or BSA-induced inhibition of the Mg(2+)-dependent ATPase reaction is believed to result from diffusion-caused deceleration, while its BSA-induced stimulation is probably caused by optimization of the enzyme structure. Molecular mechanisms of the inhibitory effect of viscosity are discussed. Taking into account high protein concentrations in the chloroplast stroma, it was suggested that kinetic parameters of ATP hydrolysis, and probably those of ATP synthesis in vivo as well, must be quite different from measurements taken at a viscosity level close to that of water.

The photosynthetic apparatus of green plants is well known for its extremely high efficiency that allows them to operate under dim light conditions. On the other hand, intense sunlight may result in overexcitation of the light-harvesting antenna and the formation of reactive compounds capable of 'burning out' the whole photosynthetic unit. Non-photochemical quenching is a self-regulatory mechanism utilized by green plants on a molecular level that allows them to safely dissipate the detrimental excess excitation energy as heat. Although it is believed to take place in the plant's major light-harvesting complexes (LHC) II, there is still no consensus regarding its molecular nature. To get more insight into its physical origin, we performed high-resolution time-resolved fluorescence measurements of LHCII trimers and their aggregates across a wide temperature range. Based on simulations of the excitation energy transfer in the LHCII aggregate, we associate the red-emitting state, having fluorescence maximum at ∼700 nm, with the partial mixing of excitonic and chlorophyll-chlorophyll charge transfer states. On the other hand, the quenched state has a totally different nature and is related to the incoherent excitation transfer to the short-lived carotenoid excited states. Our results also show that the required level of photoprotection in vivo can be achieved by a very subtle change in the number of LHCIIs switched to the quenched state.

It has been shown by Khorobrykh et al. (Biochemistry (Moscow) 67:683-688, 2002); Yanykin et al. (Biochim Biophys Acta 1797:516-523, 2010); Khorobrykh et al. (Biochemistry 50:10658-10665, 2011) that Mn-depleted photosystem II (PSII) membrane fragments are characterized by an enhanced oxygen photoconsumption on the donor side of PSII which is accompanied with hydroperoxide formation and it was suggested that the events are related to the oxidative photoinhibition of PSII. Experimental confirmation of this suggestion is presented in this work. The degree of photoinhibition was determined by the loss of the capability of exogenous electron donors (Mn(2+) or sodium ascorbate) to the reactivation of electron transport [measured by the light-induced changes of chlorophyll fluorescence yield (∆F)] in Mn-depleted PSII membranes. The transition from anaerobic conditions to aerobic ones significantly activated photoinhibition of Mn-depleted PSII membranes both in the absence and in the presence of exogenous electron acceptor, ferricyanide. The photoinhibition of Mn-depleted PSII membranes was suppressed upon the addition of exogenous electron donors (Mn(2+), diphenylcarbazide, and ferrocyanide). The addition of superoxide dismutase did not affect the photoinhibition of Mn-depleted PSII membranes. It is concluded that the interaction of molecular oxygen (rather than superoxide anion radical formed on the acceptor side of PSII) with the oxidized components of the donor side of PSII reflects the involvement of O2 in the donor-side photoinhibition of Mn-depleted PSII membranes.

Plant growth-promoting rhizobacterium Bacillus subtilis UD1022 has been shown to trigger an induced systemic response in Arabidopsis thaliana. This interaction causes plant stomata to close, protecting the plant from infection by plant pathogens and thereby increasing crop yield. The purpose of this study was to determine whether UD1022 applied to the roots of plants is able to induce stomata closure in leafy greens as well as influence the persistence of human pathogens (Listeria and Salmonella) on plants. UD1022 induced stomata closure in the presence of human pathogens on both lettuce and spinach 3 h post-inoculation (p<0.0001). Results were confirmed by root inoculation with heat-killed UD1022, which did not induce stomata closure. Presence of UD1022 on lettuce roots significantly reduced the persistence of Listeria on plants after 3 days post-inoculation (p=0.02) but had less of an effect on the persistence of Salmonella. The results of this study indicate that plant growth-promoting rhizobacterium B. subtilis UD1022 may be able to prevent contamination by some human pathogens. This is the first study to investigate the use of plant growth-promoting rhizobacteria to control the persistence of human pathogens on plants.

The temperature dependence of plant respiratory rate (R) changes in response to growth temperature. Here, we used a modified Arrhenius model incorporating the temperature dependence of activation energy (Eo ), and compared the temperature dependence of R between cold-sensitive and cold-tolerant species. We analyzed the temperature dependences of leaf CO2 efflux rate of plants cultivated at low (LT) or high temperature (HT). In plants grown at HT (HT plants), Eo at low measurement temperature varied among species, but Eo at growth temperature in HT plants did not vary and was comparable to that in plants grown at LT (LT plants), suggesting that the limiting process was similar at the respective growth temperatures. In LT plants, the integrated value of loge R, a measure of respiratory capacity, in cold-sensitive species was lower than that in cold-tolerant species. When plants were transferred from HT to LT, the respiratory capacity changed promptly after the transfer compared with the other parameters. These results suggest that a similar process limits R at different growth temperatures, and that the lower capacity of the respiratory system in cold-sensitive species may explain their low growth rate at LT., (© 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.)

A series of fifteen new N-alkoxyphenylanilides of 3-hydroxynaphthalene-2-carboxylic acid was prepared and characterized. Primary in vitro screening of the synthesized compounds was performed against Staphylococcus aureus, three methicillin-resistant S. aureus strains, Mycobacterium tuberculosis H37Ra and M. avium subsp. paratuberculosis. Some of the tested compounds showed antibacterial and antimycobacterial activity against the tested strains comparable with or higher than that of the standards ampicillin or rifampicin. 3-Hydroxy-N-(2-propoxyphenyl)naphthalene-2-carboxamide and N-[2-(but-2-yloxy)-phenyl]-3-hydroxynaphthalene-2-carboxamide had MIC = 12 µM against all methicillin-resistant S. aureus strains; thus their activity is 4-fold higher than that of ampicillin. The second mentioned compound as well as 3-hydroxy-N-[3-(prop-2-yloxy)phenyl]-naphthalene-2-carboxamide had MICs = 23 µM and 24 µM against M. tuberculosis respectively. N-[2-(But-2-yloxy)phenyl]-3-hydroxynaphthalene-2-carboxamide demonstrated higher activity against M. avium subsp. paratuberculosis than rifampicin. Screening of the cytotoxicity of the most effective antimycobacterial compounds was performed using THP-1 cells, and no significant lethal effect was observed for the most potent compounds. The compounds were additionally tested for their activity related to inhibition of photosynthetic electron transport (PET) in spinach (Spinacia oleracea L.) chloroplasts. N-(3-Ethoxyphenyl)-3-hydroxynaphthalene-2-carboxamide (IC50 = 4.5 µM) was the most active PET inhibitor. The structure-activity relationships are discussed.

The increased salinity in greenhouses has become a problem of great concern. In this study, it was observed that the salt-induced oxidative damages (indicated by MDA, H2O2 and antioxidant enzymes, including POD, SOD and CAT) could be alleviated by application of NO gas. Consequently, although both photosynthesis and growth in plants were inhibited by NaCl stress, they were restored by NO gas application, and the fresh and dry biomasses of edible parts increased by 60% and 27% over NaCl stress treatment, respectively. Furthermore, gaseous NO application also significantly elevated the levels of several antioxidation-associated compounds such as proline, ascorbate, glutathione, total phenolics and flavonoids, as well as the total antioxidant capacity (indicated by DPPH scavenging activity) in NaCl-treated plants. Keeping in mind all of the above, we concluded that atmospheric application of trace amounts of nitric oxide gas could be an effective strategy for improving both biomass production and nutrition quality in spinach under salt stress., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: The modular body structure of plants enables detached plant organs, such as postharvest fruits and vegetables, to maintain active responsiveness to environmental stimuli, including daily cycles of light and darkness. Twenty-four hour light/darkness cycles entrain plant circadian clock rhythms, which provide advantage to plants. Here, we tested whether green leafy vegetables gain longevity advantage by being stored under light/dark cycles designed to maintain biological rhythms., Results: Light/dark cycles during postharvest storage improved several aspects of plant tissue performance comparable to that provided by refrigeration. Tissue integrity, green coloration, and chlorophyll content were generally enhanced by cycling of light and darkness compared to constant light or darkness during storage. In addition, the levels of the phytonutrient glucosinolates in kale and cabbage remained at higher levels over time when the leaf tissue was stored under light/dark cycles., Conclusions: Maintenance of the daily cycling of light and dark periods during postharvest storage may slow the decline of plant tissues, such as green leafy vegetables, improving not only appearance but also the health value of the crops through the maintenance of chlorophyll and phytochemical content after harvest.

Recent evidence has indicated the presence of novel plastoquinone-binding sites, QC and QD, in photosystem II (PSII). Here, we investigated the potential involvement of loosely bound plastosemiquinones in superoxide anion radical (O2-) formation in spinach PSII membranes using electron paramagnetic resonance (EPR) spin-trapping spectroscopy. Illumination of PSII membranes in the presence of the spin trap EMPO (5-(ethoxycarbonyl)-5-methyl-1-pyrroline N-oxide) resulted in the formation of O2-, which was monitored by the appearance of EMPO-OOH adduct EPR signal. Addition of exogenous short-chain plastoquinone to PSII membranes markedly enhanced the EMPO-OOH adduct EPR signal. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, the EMPO-OOH adduct EPR signal was suppressed by 50% when the urea-type herbicide DCMU (3-(3,4-dichlorophenyl)-1,1-dimethylurea) was bound at the QB site. However, the EMPO-OOH adduct EPR signal was enhanced by binding of the phenolic-type herbicide dinoseb (2,4-dinitro-6-sec-butylphenol) at the QD site. Both in the unsupplemented and plastoquinone-supplemented PSII membranes, DCMU and dinoseb inhibited photoreduction of the high-potential form of cytochrome b559 (cyt b559). Based on these results, we propose that O2- is formed via the reduction of molecular oxygen by plastosemiquinones formed through one-electron reduction of plastoquinone at the QB site and one-electron oxidation of plastoquinol by cyt b559 at the QC site. On the contrary, the involvement of a plastosemiquinone formed via the one-electron oxidation of plastoquinol by cyt b559 at the QD site seems to be ambiguous. In spite of the fact that the existence of QC and QD sites is not generally accepted yet, the present study provided more spectroscopic data on the potential functional role of these new plastoquinone-binding sites.