Exogenous γ-aminobutyric acid (GABA) improves salt-inhibited nitrogen metabolism and the anaplerotic reaction of the tricarboxylic acid cycle by regulating GABA-shunt metabolism in maize seedlings.

Salinity stress hampers the growth of most crop plants and reduces yield considerably. In addition to its role in metabolism, γ-aminobutyric acid (GABA) plays a special role in the regulation of salinity stress tolerance in plants, though the underlying physiological mechanism remains poorly understood. In order to study the physiological mechanism of GABA pathway regulated carbon and nitrogen metabolism and tis relationship with salt resistance of maize seedlings, we supplemented seedlings with exogenous GABA under salt stress. In this study, we showed that supplementation with 0.5 mmol·L-1 (0.052 mg·g-1) GABA alleviated salt toxicity in maize seedling leaves, ameliorated salt-induced oxidative stress, and increased antioxidant enzyme activity. Applying exogenous GABA maintained chloroplast structure and relieved chlorophyll degradation, thus improving the photosynthetic performance of the leaves. Due to the improvement in photosynthesis, sugar accumulation also increased. Endogenous GABA content and GABA transaminase (GABA-T) and succinate semialdehyde dehydrogenase (SSADH) activity were increased, while glutamate decarboxylase (GAD) activity was decreased, via the exogenous application of GABA under salt stress. Meanwhile, nitrogen metabolism and the tricarboxylic acid (TCA) cycle were activated by the supply of GABA. In general, through the regulation of GABA-shunt metabolism, GABA activated enzymes related to nitrogen metabolism and replenished the key substrates of the TCA cycle, thereby improving the balance of carbon and nitrogen metabolism of maize and improving salt tolerance.

ZmERF21 directly regulates hormone signaling and stress-responsive gene expression to influence drought tolerance in maize seedlings.

Drought stress adversely impacts crop development and yield. Maize frequently encounters drought stress during its life cycle. Improvement of drought tolerance is a priority of maize breeding programs. Here, we identified a novel transcription factor encoding gene, APETALA2 (AP2)/Ethylene response factor (ERF), which is tightly associated with drought tolerance in maize seedlings. ZmERF21 is mainly expressed in the root and leaf and it can be highly induced by polyethylene glycol treatment. Genetic analysis showed that the zmerf21 mutant plants displayed a reduced drought tolerance phenotype, accompanied by phenotypical and physiological changes that are commonly observed in drought conditions. Overexpression of ZmERF21 in maize significantly increased the chlorophyll content and activities of antioxidant enzymes under drought conditions. RNA-Seq and DNA affinity purification sequencing analysis further revealed that ZmERF21 may directly regulate the expression of genes related to hormone (ethylene, abscisic acid) and Ca signaling as well as other stress-response genes through binding to the promoters of potential target genes. Our results thereby provided molecular evidence of ZmERF21 is involved in the drought stress response of maize.

Metabolomic and transcriptomic analyses reveal that sucrose synthase regulates maize pollen viability under heat and drought stress.

Maize pollen is highly sensitive to heat and drought, but few studies have investigated the combined effects of heat and drought on pollen viability. In this study, pollen's structural and physiological characteristics were determined after heat, drought, and combined stressors. Furthermore, integrated metabolomic and transcriptomic analyses of maize pollen were conducted to identify potential mechanisms of stress responses. Tassel growth and spikelet development were considerably suppressed, pollen viability was negatively impacted, and pollen starch granules were depleted during anthesis under stress. The inhibitory effects were more significant due to combined stresses than to heat or drought individually. The metabolic analysis identified 71 important metabolites in the combined stress compared to the other treatments, including sugars and their derivatives related to pollen viability. Transcriptomics also revealed that carbohydrate metabolism was significantly altered under stress. Moreover, a comprehensive metabolome-transcriptome analysis identified a central mechanism in the biosynthesis of UDP-glucose involved in reducing the activity of sucrose synthase SH-1 (shrunken 1) and sus1 (sucrose synthase 1) that suppressed sucrose transfer to UDP-glucose, leading to pollen viability exhaustion under stress. In conclusion, the lower pollen viability after heat and drought stress was associated with poor sucrose synthase activity due to the stress treatments.

Highly interwoven communities of a gene regulatory network unveil topologically important genes for maize seed development.

The complex interactions between transcription factors (TFs) and their target genes in a spatially and temporally specific manner are crucial to all cellular processes. Reconstruction of gene regulatory networks (GRNs) from gene expression profiles can help to decipher TF-gene regulations in a variety of contexts; however, the inevitable prediction errors of GRNs hinder optimal data mining of RNA-Seq transcriptome profiles. Here we perform an integrative study of Zea mays (maize) seed development in order to identify key genes in a complex developmental process. First, we reverse engineered a GRN from 78 maize seed transcriptome profiles. Then, we studied collective gene interaction patterns and uncovered highly interwoven network communities as the building blocks of the GRN. One community, composed of mostly unknown genes interacting with opaque2, brittle endosperm1 and shrunken2, contributes to seed phenotypes. Another community, composed mostly of genes expressed in the basal endosperm transfer layer, is responsible for nutrient transport. We further integrated our inferred GRN with gene expression patterns in different seed compartments and at various developmental stages and pathways. The integration facilitated a biological interpretation of the GRN. Our yeast one-hybrid assays verified six out of eight TF-promoter bindings in the reconstructed GRN. This study identified topologically important genes in interwoven network communities that may be crucial to maize seed development.

Proteomics analysis reveals that nitric oxide regulates photosynthesis of maize seedlings under water deficiency.

Nitric oxide (NO) is an important bioactive molecule that functions in regulating diverse abiotic stresses in plants, whereas its molecular mechanism remains obscure. In this study, treatment with 0.1 mM NO donor (sodium nitroprusside, SNP) significantly alleviated the inhibited growth induced by 15% polyethyleneglycol (PEG)-stimulated water deficiency (WD) for 3 days in maize seedlings, manifested by less decreased plant total fresh weight and dry weight. Comprehensive proteome analysis was further used to measure the expression profiles of leaf proteins of SNP-pretreated maize seedlings under WD conditions to explore the molecular mechanisms of NO-induced WD tolerance. Using 2-DE method, 135 protein spots showed significantly enhanced or reduced abundance, of which 102 spots were successfully identified MALDI-TOF/TOF MS. The identified protein species were associated with diverse functions, and most (52/83, 62.7%) of known protein species were related to photosynthetic processes. Compared to alone PEG treatment, the abundance of 25 identified protein species in SNP + PEG treatment were enhanced among the identified photosynthesis-related protein species. In addition, exogenous SNP application dramatically regulated chlorophyll α fluorescence kinetics e.g. the increase of maximum quantum yield of PSII (Fv/Fm), photosynthetic performance index (PI), and IP phase, whereas it remarkably reduced the polyphasic OJIP fluorescence transient, the accumulation of reactive oxygen species (H2O2 and O2•-) and malondialdehyde (MDA). These findings suggest that the NO-induced WD tolerance could be associated with improved photosynthetic capability in higher plants.

Nitric Oxide Enhancing Resistance to PEG-Induced Water Deficiency is Associated with the Primary Photosynthesis Reaction in Triticum aestivum L.

Photosynthesis is affected by water-deficiency (WD) stress, and nitric oxide (NO) is a free radical that participates in the photosynthesis process. Previous studies have suggested that NO regulates excitation-energy distribution of photosynthesis under WD stress. Here, quantitative phosphoproteomic profiling was conducted using iTRAQ. Differentially phosphorylated protein species (DEPs) were identified in leaves of NO- or polyethylene glycol (PEG)-treated wheat seedlings (D), and in control seedlings. From 1396 unique phosphoproteins, 2257 unique phosphorylated peptides and 2416 phosphorylation sites were identified. Of these, 96 DEPs displayed significant changes (≥1.50-fold, p < 0.01). These DEPs are involved in photosynthesis, signal transduction, etc. Furthermore, phosphorylation of several DEPs was upregulated by both D and NO treatments, but downregulated only in NO treatment. These differences affected the chlorophyll A⁻B binding protein, chloroplast post-illumination chlorophyll-fluorescence-increase protein, and SNT7, implying that NO indirectly regulated the absorption and transport of light energy in photosynthesis in response to WD stress. The significant difference of chlorophyll (Chl) content, Chl a fluorescence-transient, photosynthesis index, and trapping and transport of light energy further indicated that exogenous NO under D stress enhanced the primary photosynthesis reaction compared to D treatment. A putative pathway is proposed to elucidate NO regulation of the primary reaction of photosynthesis under WD.

Ultrastructural observation of mesophyll cells and temporal expression profiles of the genes involved in transitory starch metabolism in flag leaves of wheat after anthesis.

Transitory starch in cereal plant leaves is synthesized during the day and remobilized at night to provide a carbon source for growth and grain filling, but its mechanistic basis is still poorly understood. The objective of this study is to explore the regulatory mechanism for starch biosynthesis and degradation in plant source organs. Using transmission electron microscopy, we observed that during the day after anthesis, starch granules in mesophyll cells of wheat flag leaves accumulated in chloroplasts and the number of starch granules gradually decreased with wheat leaf growth. During the night, starch granules synthesized in chloroplasts during the day were completely or partially degraded. The transcript levels of 26 starch synthesis-related genes and 16 starch breakdown-related genes were further measured using quantitative real-time reverse transcription polymerase chain reaction. Expression profile analysis revealed that starch metabolism genes were clustered into two groups based on their temporal expression patterns. The genes in the first group were highly expressed and presumed to play crucial roles in starch metabolism. The genes in the other group were not highly expressed in flag leaves and may have minor functions in starch metabolism in leaf tissue. The functions of most of these genes in leaves were further discussed. The starch metabolism-related genes that are predominantly expressed in wheat flag leaves differ from those expressed in wheat grain, indicating that two different pathways for starch metabolism operate in these tissues. This provides specific information on the molecular mechanisms of transitory starch metabolism in higher plants.

Physiological, Ultrastructural and Proteomic Responses in the Leaf of Maize Seedlings to Polyethylene Glycol-Stimulated Severe Water Deficiency.

After maize seedlings grown in full-strength Hoagland solution for 20 days were exposed to 20% polyethylene glycol (PEG)-stimulated water deficiency for two days, plant height, shoot fresh and dry weights, and pigment contents significantly decreased, whereas malondialdehyde (MDA) content greatly increased. Using transmission electron microscopy, we observed that chloroplasts of mesophyll cells in PEG-treated maize seedlings were swollen, with a disintegrating envelope and disrupted grana thylakoid lamellae. Using two-dimensional gel electrophoresis (2-DE) method, we were able to identify 22 protein spots with significantly altered abundance in the leaves of treated seedlings in response to water deficiency, 16 of which were successfully identified. These protein species were functionally classified into signal transduction, stress defense, carbohydrate metabolism, protein metabolism, and unknown categories. The change in the abundance of the identified protein species may be closely related to the phenotypic and physiological changes due to PEG-stimulated water deficiency. Most of the identified protein species were putatively located in chloroplasts, indicating that chloroplasts may be prone to damage by PEG stimulated-water deficiency in maize seedlings. Our results help clarify the molecular mechanisms of the responses of higher plants to severe water deficiency.

The co-inoculation of Trichoderma viridis and Bacillus subtilis improved the aerobic composting efficiency and degradation of lignocellulose.

The aim of this study was to reveal the mechanism by which co-inoculation with both Trichoderma viridis and Bacillus subtilis improved the efficiency of composting and degradation of lignocellulose in agricultural waste. The results showed that co-inoculation with Trichoderma and Bacillus increased abundance of Bacteroidota to promote the maturation 7 days in advance. Galbibacter may be a potential marker of co-inoculation composting efficiency compost. The compost became dark brown, odorless, and had a carbon to nitrogen ratio of 16.40 and a pH of 8.2. Moreover, Actinobacteriota and Firmicutes still dominated the degradation of lignocellulose following inoculation with Trichoderma or Bacillus 35 days after composting. Bacterial function prediction analysis showed that carbohydrate metabolism was the primary metabolic pathway. In conclusion, co-inoculation with Trichoderma and Bacillus shortened the composting cycle and accelerated the degradation of lignocellulose. These findings provide new strategies for the efficient use of agricultural waste to produce organic fertilizers.

Reducing greenhouse gas intensity using a mixture of controlled-release urea and common urea combining suitable maize varieties in a summer maize system.

The one-time application of common urea blended with controlled-release urea (CRU) is considered effective for improving nitrogen use efficiency and grain yield and reducing the greenhouse gas emissions of summer maize in intensive agricultural systems. However, the trade-off between the economic and environmental performances of different blended fertilizer treatments for different maize varieties remains unclear. Therefore, a consecutive two-year field experiment was conducted in the North China Plain to study the effects of different ratios of CRU and common urea on the yield, nitrous oxide (N2O) emissions, yield-scaled total N2O emissions, greenhouse gas intensity (GHGI), and net ecosystem economic benefit (NEEB) in 2021 and 2022. Four N fertilizer treatments with equal rate at 180 kg N ha-1 were applied as N180U (all Urea), N180C1(1/3CRU), N180C2(2/3CRU), and N180C (all CRU), and two maize varieties (JNK728-yellow ripe variety and ZD958-green ripe variety) were used. The N180C1 and N180C2 treatments produced the highest grain yield in varieties JNK728 and ZD958 (9.4-11.5 t ha-1 and 9.0-11.0 t ha-1), respectively. Compared to the N180U treatment (conventional method), the N180C1 treatment reduced the GHGI (24.8 %-25.9 %) and increased the NEEB (33.1 %-33.4 %) in the JNK728 variety, whereas the N180C2 treatment reduced the GHGI (16.9 %-28.8 %) and increased the NEEB (27.2 %-48.1 %) in the ZD958 variety. The study concludes that a one-time application of blended nitrogen fertilizer in suitable varieties can minimize the GHGI and maximize the NEEB, which is an effective strategy for balancing yield and nitrogen efficiency in the summer maize system in the North China Plain.

Metagenomic insights into microbial variation and carbon cycling function in crop rotation systems.

The decomposition and utilization of plant-derived carbon by microorganisms and carbon fixation are crucial pathways for enhancing soil organic carbon (SOC) storage. However, a gap remains in our understanding of the impact of microorganisms on the decomposition of plant-derived carbon and their capacity for carbon fixation in crop rotation systems. Based on a 12-year experiment with wheat-maize (WM), wheat-cotton (WC), and wheat-soybean (WS) rotations, the microbial communities and carbon cycle function were investigated. The results indicated that WS rotation significantly increased SOC content compared to WM and WC. In addition, a significant increase was observed in microbially available carbon and microbial biomass carbon in the WS soil compared with those in the others. Further analysis of the microbial community factors that influenced SOC content revealed that WS rotation, in contrast to WM rotation, enhanced the diversity and richness of bacteria and fungi. Analysis of microbial carbon decomposition functions revealed an increase in starch, lignin, and hemicellulose decomposition genes in the WS soil compared to the others. The changes in carbon decomposition genes were primarily attributed to six bacterial genera, namely Nocardioides, Agromyces, Microvirga, Skermanella, Anaeromyxobacter, and Arthrobacter, as well as four fungal genera, namely Dendryphion, Staphylotrichum, Apiotrichum, and Abortiporus, which were significantly influenced by the crop rotation systems. In addition, microbial carbon fixation-related genes such as ACAT, IDH1, GAPDH, rpiA, and rbcS were significantly enriched in WS. Species annotation of differential carbon fixation genes identified 18 genera that play a role in soil carbon fixation variation within the crop rotation systems. This study highlights the impact of crop rotation systems on SOC content and alterations in specific microbial communities on carbon cycle function.

L-Arginine Alleviates the Reduction in Photosynthesis and Antioxidant Activity Induced by Drought Stress in Maize Seedlings.

Maize (Zea mays L.) is one of the most important food crops in the world. Drought is currently the most important abiotic factor affecting maize yield. L-arginine has emerged as a nontoxic plant growth regulator that enhances the tolerance of plants to drought. An experiment was conducted to examine the role of L-arginine in alleviating the inhibitory effects of drought on the photosynthetic capacity and activities of antioxidant enzymes when the plants were subjected to drought stress. The results showed that the biomass of maize seedlings decreased significantly under a 20% polyethylene glycol-simulated water deficit compared with the control treatment. However, the exogenous application of L-arginine alleviated the inhibition of maize growth induced by drought stress. Further analysis of the photosynthetic parameters showed that L-arginine partially restored the chloroplasts' structure under drought stress and increased the contents of chlorophyll, the performance index on an adsorption basis, and Fv/Fm by 151.3%, 105.5%, and 37.1%, respectively. Supplementation with L-arginine also reduced the oxidative damage caused by hydrogen peroxide, malondialdehyde, and superoxide ions by 27.2%, 10.0%, and 31.9%, respectively. Accordingly, the activities of ascorbate peroxidase, catalase, glutathione S-transferase, glutathione reductase, peroxidase, and superoxide dismutase increased by 11.6%, 108.5%, 104.4%, 181.1%, 18.3%, and 46.1%, respectively, under drought. Thus, these findings suggest that L-arginine can improve the drought resistance of maize seedlings by upregulating their rate of photosynthesis and their antioxidant capacity.

Water deficit aggravated the inhibition of photosynthetic performance of maize under mercury stress but is alleviated by brassinosteroids.

Mercury (Hg) significantly inhibits maize (Zea mays L.) production, which could be aggravated by water deficit (WD) due to climate change. However, there is no report on the maize in response to combined their stresses. This work was conducted for assessing the response and adaptive mechanism of maize to combined Hg and WD stress using two maize cultivars, Xianyu (XY) 335 and Yudan (YD) 132. The analysis was based on plant growth, physiological function, and transcriptomic data. Compared with the single Hg stress, Hg accumulation in whole plant and translocation factor (TF) under Hg+WD were increased by 64.51 % (1.44 mg kg-1) and 260.00 %, respectively, for XY 335; and 50.32 % (0.62 mg kg-1) and 220.02 %, respectively, for YD 132. Combined Hg and WD stress further increased the reactive oxygen species accumulation, aggravated the damage of the thylakoid membrane, and decreased chlorophyll content compared with single stress. For example, Chl a and Chl b contents of XY 335 were significantly decreased by 48.67 % and 28.08 %, respectively at 48 h after Hg+WD treatment compared with Hg stress. Furthermore, transcriptome analysis revealed that most of down-regulated genes were enriched in photosynthetic-antenna proteins, photosynthesis, chlorophyll and porphyrin metabolism pathways (PsbS1, PSBQ1 and FDX1 etc.) under combined stress, reducing light energy capture and electron transport. However, most genes related to the brassinosteroids (BRs) signaling pathway were up-regulated under Hg+WD stress. Correspondingly, exogenous BRs significantly enhanced the maize tolerance to stress by decreasing Hg accumulation and TF, and raising activities of antioxidant enzyme, the content of chlorophyll and photosynthetic performance. The PI, Fv/Fm and Fv/Fo of Hg+WD+BR treatment were increased by 29.88 %, 32.06 %, and 14.56 %, respectively, for XY 335 compared to Hg+WD. Overall, combined Hg and WD stress decreased photosynthetic efficiency by adversely affecting light absorption and electron transport, especially in stress-sensitive variety, but BRs could alleviate the inhibition of photosynthesis, providing a novel strategy for enhancing crop Hg and WD tolerance and food safety.

Mercury stress tolerance in wheat and maize is achieved by lignin accumulation controlled by nitric oxide.

Nitric oxide (NO) is an important phytohormone for plant adaptation to mercury (Hg) stress. The effect of Hg on lignin synthesis, NO production in leaf, sheath and root and their relationship were investigated in two members of the grass family - wheat and maize. Hg stress decreased growth and lignin contents, significantly affected phenylpropanoid and monolignol pathways (PAL, phenylalanine ammonia-lyase; 4-coumarate: CoA ligase, 4CL; cinnamyl alcohol dehydrogenase, CAD), with maize identified to be more sensitive to Hg stress than wheat. Among the tissue types, sheath encountered severe damage compared to leaves and roots. Hg translocation in maize was about twice that in wheat. Interestingly, total NO produced under Hg stress was significantly decreased compared to control, with maximum reduction of 43.4% and 42.9% in wheat and maize sheath, respectively. Regression analysis between lignin and NO contents or the activities of three enzymes including CAD, 4CL and PAL displayed the importance of NO contents, CAD, 4CL and PAL for lignin synthesis. Further, the gene expression profiles encoding CAD, 4CL and PAL provided support for the damaging effect of Hg on wheat sheath, and maize shoot. To validate NO potential to mitigate Hg toxicity in maize and wheat, NO donor and NO synthase inhibitor were supplemented along with Hg. The resulting phenotype, histochemical analysis and lignin contents showed that NO mitigated Hg toxicity by improving growth and lignin synthesis and accumulation. In summary, Hg sensitivity was higher in maize seedlings compared to wheat, which was associated with the lower lignin contents and reduced NO contents. External supplementation of NO is proposed as a sustainable approach to mitigate Hg toxicity in maize and wheat.

A transcriptomic analysis reveals the adaptability of the growth and physiology of immature tassel to long-term soil water deficit in Zea mays L.

Drought is a key threat to maize growth and yield. Understanding the mechanism of immature tassel (IT) response to long term drought is of paramount importance. Here, the maize inbred line PH6WC was tested under well-watered (CK) and two water deficit treatments (WD1 and WD2). The final IT length in the WD1 and WD2 treatments decreased by nearly 6.2% and 21.2% compared to the CK, respectively, and the average accumulation rate IT dry matter was 1.5-fold and 1.8-fold slower, respectively. Furthermore, RNA sequencing analysis was conducted on the IT sampled at 30 days after the WD treatments. In total, the cellular component in gene ontology (GO) analysis suggested that the differentially expressed genes were significantly enriched in three common terms (apoplast, plant-type cell wall, and anchored component of membrane) among the CK vs WD1, CK vs WD2, and WD1 vs WD2 comparisons. Next, a co-expression network analysis identified 44 modules that contained global expression genes. Finally, by combining the GO analysis with modules, nine genes involved in carbohydrate metabolism and the antioxidant system were screened out, and the six corresponding physiological parameters were all significantly increased under the WD treatments. These results showed that, although the IT length and dry matter decreased, the IT enhanced the adaptation to drought by regulating their own genetic and physiological changes.

Physiological and iTRAQ-based proteomic analyses reveal that melatonin alleviates oxidative damage in maize leaves exposed to drought stress.

To explain the underlying mechanism of melatonin-mediated drought stress responses in maize, maize pre-treated with or without melatonin was subjected to 20% PEG nutrient solution to induce drought stress. We found that exogenous melatonin significantly improved drought tolerance, demonstrated by improved photosynthesis, reduced ROS accumulation, enhanced activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and increased content of glutathione (GSH). Comparative iTRAQ proteomic analyses revealed a higher abundance of differentially expressed proteins (DEPs) in melatonin-treated maize under drought stress for carbon fixation in photosynthetic organisms, photosynthesis, biosynthesis of amino acids, and biosynthesis of secondary metabolites, compared to untreated plants. Changes in the above molecular mechanisms could explain the melatonin-induced physiological effects associated with drought tolerance. In summary, this study provides a more integrated picture about the effects of melatonin on the physiological and molecular mechanisms in maize seedlings responding to drought stress.

Cytokinin-induced photosynthetic adaptability of Zea mays L. to drought stress associated with nitric oxide signal: probed by ESR spectroscopy and fast OJIP fluorescence rise.

Nitric oxide (NO), as a diffusible molecule, performs important roles in diverse physiological processes. Interestingly, NO signaling is based on interactions with plant hormones. The aim of this study was, first, to test the effect of cytokinin (CTK) on the primary reaction of photosynthesis under drought stress, and then to examine whether NO is involved in CTK-induced photosynthetic resistance due to its role as a second messenger in stress response. Under drought stress, plants were treated with CTK, or CTK plus the NO scavenger (Hemoglobin [Hb]) for 6h. The effects of CTK and Hb on fast OJIP fluorescence rise were then examined. At the same time, NO and reactive oxygen species (ROS) signals in all the treatments were detected by electron spin resonance (ESR) spectroscopy. The results showed that CTK-regulated fluorescence transient rise under drought stress and increased the electron donation capacity of photosynthesis system (PS) II. The plant photosynthetic performance index (PI) on an absorption basis and corresponding three PI components (RC/ABS, P(TR,) and P(ET)) also increased. High NO signal intensity alleviated drought-induced ROS damage to plants; thus, the signal probably played a direct role in eliciting CTK regulation to energy absorption (RC/ABS) and excitation energy trapped (P(TR)) in response to drought. Although CTK stimulated more excitation energy conversion to electron transfer (P(ET)), because NO was probably bound to the plastoquinone pool (PQ) of the electron transport chain, CTK decreased electron transport to the acceptor side of PSII (see V(I), Sm and N). Furthermore, CTK stimulated more NO signal formation, probably mainly via a nitrate reductase (NR) source under the conditions of the study, and Hb prevented stimulation from CTK. However, these results will require confirmation from future studies.