Phytochrome-Dependent Regulation of ZFP6 and ZFPH Impacts Photomorphogenesis in Arabidopsis thaliana.

Phytochromes (phy) are key regulators of photomorphogenesis in plants. Among the different phys characterized in higher plants (i.e., phyA to phyE), phyA and phyB primarily regulate phenotypic responses in plants under far-red (FR) and red (R) conditions, respectively. Recent findings suggest that some zinc finger proteins (ZFPs) are involved in plant light-modulated morphogenesis. However, the interaction(s) between phyA, phyB and ZFP homologs potentially involved in photomorphogenesis, as well as their phenotypic and molecular effects in Arabidopsis seedlings exposed to R and FR light remain to be elucidated fully. Prior analyses with phytochrome chromophore deficient lines indicated that ZFP6 expression is misregulated compared to levels in Col-0 wild type (WT). Here, we used plants with phytochrome chromophore or apoprotein (specifically phyA and phyB) deficiencies, lines with mutations in ZFP6 and ZFP6 HOMOLOG (ZFPH) genes, and plants overexpressing ZFP6 to examine regulatory interactions between phytochromes, ZFP6, and ZFPH. Our results indicate that phytochromes are required for downregulation of ZFP6 and ZFPH and suggest a role for light-regulated control of ZFP levels in phytochrome-dependent photomorphogenesis. Conversely, PHYB is downregulated in zfp6 mutants under R light. Analyses of a zfp6zfph double mutant confirmed disruption in photomorphogenic phenotypes, including the regulation of hypocotyl elongation in seedlings grown under FR light. In addition, PIF3 and PIF4 levels are transcriptionally regulated by ZFP6 and ZFPH in a gibberellic acid-dependent manner. ZFP6 overexpression resulted in opposite phenotypic responses to those observed in the zfp6 and zfph mutants grown in FR and R light, as well as a reduction in the rosette size of mature ZFP6 OX plants relative to WT under white light. Based on these observations, we provide insight into how phy and ZFPs interact to regulate specific aspects of light-dependent processes in Arabidopsis.

Soil and foliar exposure of soybean (Glycine max) to Cu: Nanoparticle coating-dependent plant responses.

In this study, we investigated the effects of citric acid (CA) coated copper oxide nanoparticles (CuO NPs) and their application method (foliar or soil exposure) on the growth and physiology of soybean (Glycine max). After nanomaterials exposure via foliar or soil application, Cu concentration was elevated in the roots, leaves, stem, pod, and seeds; distribution varied by plant organ and surface coating. Foliar application of CuO NPs at 300 mg/L and CuO-CA NPs at 75 mg/L increased soybean yield by 169.5% and 170.1%, respectively. In contrast, foliar and soil exposure to ionic Cu with all treatments (75 and 300 mg/L) had no impact on yield. Additionally, CuO-CA NPs at 300 mg/L significantly decreased Cu concentration in seeds by 46.7%, compared to control, and by 44.7%, compared to equivalent concentration of CuO NPs. Based on the total Cu concentration, CuO NPs appeared to be more accessible for plant uptake, compared to CuO-CA NPs, inducing a decrease in protein content by 56.3% and inhibiting plant height by 27.9% at 300 mg/kg under soil exposure. The translocation of Cu from leaf to root and from the root to leaf through the xylem was imaged by two-photon microscopy. The findings indicate that citric acid coating reduced CuO NPs toxicity in soybean, demonstrating that surface modification may change the toxic properties of NPs. This research provides direct evidence for the positive effects of CuO-CA NPs on soybean, including accumulation and in planta transfer of the particles, and provides important information when assessing the risk and the benefits of NP use in food safety and security.

Copper oxide (CuO) nanoparticles affect yield, nutritional quality, and auxin associated gene expression in weedy and cultivated rice (Oryza sativa L.) grains.

Weedy rice grows competitively with cultivated rice and significantly diminishes rice grain production worldwide. The different effects of Cu-based nanomaterials on the production of weedy and cultivated rice, especially the grain qualities are not known. Grains were collected from weedy and cultivated rice grown for four months in field soil amended with nanoscale CuO (nCuO), bulk CuO (bCuO), and copper sulfate (CuSO4) at 0, 75, 150, 300, and 600 mg Cu/kg soil. Cu translocation, essential element accumulation, yield, sugar, starch, protein content, and the expression of auxin associated genes in grains were determined. The grains of weedy and cultivated rice were differentially impacted by CuO-based compounds. At ≥300 mg/kg, nCuO and bCuO treated rice had no grain production. Treatment at 75 mg/kg significantly decreased grain yield as compared to control with the order: bCuO (by 88.7%) > CuSO4 (by 47.2%) ~ nCuO (by 38.3% only in cultivated rice); at the same dose, the Cu grain content was: nCuO ~ CuSO4 > bCuO > control. In weedy grains, K, Mg, Zn, and Ca contents were decreased by 75 and 150 mg/kg nCuO by up to 47.4%, 34.3%, 37.6%, and 60.0%, but no such decreases were noted in cultivated rice, and Fe content was increased by up to 88.6%, and 53.2%. In rice spikes, nCuO increased Mg, Ca, Fe, and Zn levels by up to 118.1%, 202.6%, 133.8%, and 103.9%, respectively. Nanoscale CuO at 75 and 150 mg/kg upregulated the transcription of an auxin associated gene by 5.22- and 1.38-fold, respectively, in grains of weedy and cultivated rice. The biodistribution of Cu-based compounds in harvested grain was determined by two-photon microscopy. These findings demonstrate a cultivar-specific and concentration-dependent response of rice to nCuO. A potential use of nCuO at 75 and 150 mg/kg in cultivar-dependent delivery system was suggested based on enhanced grain nutritional quality, although the yield was compromised. This knowledge, at the physiological and molecular level, provides valuable information for the future use of Cu-based nanomaterials in sustainable agriculture.

The gluconeogenic glucose-6-phosphatase gene is expressed during oxygen-limited conditions in the white shrimp Penaeus (Litopenaeus) vannamei: Molecular cloning, membrane protein modeling and transcript modulation in gills and hepatopancreas.

The white shrimp Penaeus (Litopenaeus) vannamei is the most economically important crustacean species cultivated in the Western Hemisphere. This crustacean shifts its metabolism to survive under extreme environmental conditions such as hypoxia, although for a limited time. Glucose-6-phosphatase (G6Pase) is a key enzyme contributing to maintain blood glucose homeostasis through gluconeogenesis and glycogenolysis. To our knowledge, there are no current detailed studies about cDNA or gene sequences of G6Pase from any crustacean reported. Herein we report the shrimp P. (L.) vannamei cDNA and gene sequences. The gene contains seven exons interrupted by six introns. The deduced amino acid sequence has 35% identity to other homolog proteins, with the catalytic amino acids conserved and phylogenetically close to the corresponding invertebrate homologs. Protein molecular modeling predicted eight transmembrane helices with the catalytic site oriented towards the lumen of the endoplasmic reticulum. G6Pase expression under normoxic conditions was evaluated in hepatopancreas, gills, and muscle and the highest transcript abundance was detected in hepatopancreas. In response to different times of hypoxia, G6Pase mRNA expression did not change in hepatopancreas and became undetectable in muscle; however, in gills, its expression increased after 3 h and 24 h of oxygen limitation, indicating its essential role to maintain glycemic control in these conditions.

Soil-aged nano titanium dioxide effects on full-grown carrot: Dose and surface-coating dependent improvements on growth and nutrient quality.

Rutile titanium dioxide nanoparticles (nTiO2) were weathered in field soil at 0, 100, 200, and 400 mg Ti/kg soil for four months. Two types of nTiO2 with different surface coatings (hydrophilic and hydrophobic), uncoated nTiO2 (pristine), and the untreated control were included. Thereafter, carrot seeds (Daucus carota L.) were sown in those soils and grown in a growth chamber for 115 days until full maturity. A comparison was made between this and our previous unaged study, where carrots were treated in the same way in soil with freshly amended nTiO2. The responses of plants depended on the nTiO2 surface coating and concentration. The aged hydrophobic and hydrophilic-coated nTiO2 induced more positive effects on plant development at 400 and 100 mg Ti/kg soil, respectively, compared with control and pristine treatments. Taproot and leaf fresh biomass and plant height were improved by up to 64%, 40%, and 40% compared with control, respectively. Meanwhile, nutrient elements such as Fe in leaves, Mg in taproots, and Ca, Zn, and K in roots were enhanced by up to 66%, 64%, 41, 143% and 46%, respectively. However, the contents of sugar, starch, and some other metal elements in taproots were negatively affected, which may compromise their nutritional quality. Taken together, the overall growth of carrots was benefited by the aged nTiO2 depending on coating and concentration. The aging process served as a potential sustainable strategy to alleviate the phytotoxicity of unweathered nanoparticles.

Soil-Weathered CuO Nanoparticles Compromise Foliar Health and Pigment Production in Spinach (Spinacia oleracea).

In this study, spinach plants exposed to fresh/unweathered (UW) or weathered (W) copper compounds in soil were analyzed for growth and nutritional composition. Plants were exposed for 45 days to freshly prepared or soil-aged (35 days) nanoparticulate CuO (nCuO), bulk-scale CuO (bCuO), or CuSO4 at 0 (control), 400, 400, and 40 mg/kg of soil, respectively. Foliar health, gas exchange, pigment content (chlorophyll and carotenoid), catalase and ascorbate peroxidase enzymes, gene expression, and Cu bioaccumulation were evaluated along with SEM imagery for select samples. Foliar biomass was higher in UW control (84%) and in UW ionic treatment (87%), compared to the corresponding W treatments (p ≤ 0.1). Root catalase activity was increased by 110% in UW bCuO treatment as compared to the W counterpart; the value for the W ionic treatment was increased by 2167% compared to the UW counterpart (p ≤ 0.05). At 20 days post-transplantation, W nCuO-exposed plants had ∼56% lower carotenoid content compared to both W control and the UW counterpart (p ≤ 0.05). The findings indicate that over the full life cycle of spinach plant the weathering process significantly deteriorates leaf pigment production under CuO exposure in particular and foliar health in general.

Metabolomic analysis reveals dose-dependent alteration of maize (Zea mays L.) metabolites and mineral nutrient profiles upon exposure to zerovalent iron nanoparticles.

Nanoscale zero-valent iron (nZVI) has been widely applied in the environmental field to degrade organic pollutants. The potential risk posed from nZVI on crop species is not well understood and is critical for sustainable application in the future. In this study, maize (Zea mays L.) plants were cultivated in field soils mixed with nZVI at 0, 50, and 500 mg/kg soil for four weeks. Upon exposure to 500 mg/kg nZVI, ICP-MS results showed that Fe accumulated by roots and translocated to leaves was increased by 36% relative to untreated controls. At 50 mg/kg, root elongation was enhanced by 150-200%; at 500 mg/kg, pigments, lipid peroxidation, and polyphenolic levels in leaves were increased by 12, 87 and 23%, respectively, whereas the accumulation of Al, Ca, and P were decreased by 62.2%, 19.7%, and 13.3%, respectively. A gas chromatography-mass spectrometry (GC-MS) based metabolomics analysis of maize roots revealed that antioxidants and stress signaling-associated metabolites were downregulated at 50 mg/kg, but were upregulated at 500 mg/kg. At 50 mg/kg, the content of glutamate was increased by 11-fold, whereas glutamine was decreased by 99% with respect to controls. Interestingly, eight metabolic pathways were disturbed at 50 mg/kg, but none at 500 mg/kg. This metabolic reprogramming at the lower dose represented potential risks to the health of exposed plants, which could be particularly important although no phenotypic impacts were noted. Overall, metabolites analysis provides a deeper understanding at the molecular level of plant response to nZVI and is a powerful tool for full characterization of risk posed to crop species as part of food safety assessment.

Effects of different surface-coated nTiO2 on full-grown carrot plants: Impacts on root splitting, essential elements, and Ti uptake.

The production and environmental release of surface-modified titanium dioxide nanoparticles (nTiO2) have increased. Hence, crops may be directly exposed to the nTiO2 in soil. In this study, we grew carrots in soils amended with pristine, hydrophilic and hydrophobic surface-coated nTiO2 at 100, 200, and 400 mg kg-1 until full-plant maturity. The content of Ti in plant secondary roots treated with different nTiO2 at 400 mg kg-1 was in the order of hydrophobic > hydrophilic > pristine treatments, with values of 140.1, 100.5, and 64.3 mg kg-1, respectively. The fresh biomass of the taproot was significantly decreased by all nTiO2 forms at 400 mg kg-1 by up to 56 %, compared to control. Pristine nTiO2 at 100 mg kg-1 enhanced the fresh weight of leaves by 51 % with respect to control. Remarkably, an abnormal increase of taproot splitting was found in plants treated with all nTiO2 forms. In carrots treated with the surface-coated nTiO2, the accumulation of Ca, Mg, Fe, and Zn increased in leaves; but Mg, Mn, and Zn decreased in taproots. These results suggest that future regulation of nTiO2 release into soils should consider its surface coating properties since the phytotoxicity effects depend on nTiO2 outer structure.

Antioxidant and defense genetic expressions in corn at early-developmental stage are differentially modulated by copper form exposure (nano, bulk, ionic): Nutrient and physiological effects.

In the present study, Zea mays seedlings grown under nano Cu(OH)2 (nCu), bulk Cu(OH)2 (bCu), and ionic CuSO4 (iCu) compound exposure were harvested after six days. The nutritional profile was determined to be significantly disrupted in the roots by 1000 ppm bCu treatment, resulting in a 58.7% reduction in potassium compared to the control. In the shoots, a significant decrease of manganese was observed for 10 and 1000 ppm iCu treatments with 55.7% and 64.2% reductions, respectively. The overall protein content and catalase (CAT) enzymatic activity, however, remained unaffected in either roots or shoots, while an absence of polyphenol oxidase (PPO) activity was observed for all samples. The genetic expression of defense-related genes, metallothionein (MT), CAT, ascorbate peroxidase (APX), and PPO was assessed. The genetic expression of MT was upregulated 50-fold in roots treated with 1000 ppm bCu. There were no significant differences in CAT transcripts among the various treatments, while APX was upregulated 28 and 19-fold in shoots treated with 10 ppm bCu and 10 ppm nCu, respectively. Meanwhile, APX mRNA levels were downregulated five-fold in shoots treated with 1000 ppm iCu. Thus, indicating that the role of APX in plant defense was reinforced in seedlings exposed to low concentration of particulate Cu compounds. Remarkably, no PPO expression was found in any of the treatments and controls, which suggests this enzyme is expressed only under specific external factors or seedlings have an "immature" cascade signaling activation of the PPO system. Taken together, these results show that bCu and nCu treatments at a low concentration do not compromise vital cell machinery but rather elicit the enhancement of defense responses as observed through the increase in APX expression. Furthermore, under optimal concentrations, these Cu treatments show promise in enhancing corn defense responses, which can ultimately lead to increases in future global crop yields.

Copper nanowires as nanofertilizers for alfalfa plants: Understanding nano-bio systems interactions from microbial genomics, plant molecular responses and spectroscopic studies.

The recent application of nano copper (Cu) compounds in the agrosystem has shown potential to improve the physiological performance and agronomical parameters of crops. We grew alfalfa (Medicago sativa) in potting mix amended with bulk, nano, and ionic Cu compounds at 80 and 280 mg Cu/kg; then, we evaluated plant performance at physiological and molecular levels. Plants treated with bulk/nano Cu presented better agronomical responses. The P and S content was reduced in bulk and ionic Cu-exposed plants, compared to controls (p ≤ .05). All Cu forms increased the content of Fe and Zn in roots and Fe in leaves, compared to controls (p ≤ .05). Leaf-superoxide dismutase expression was augmented ~27-fold and rubisco mRNA was unaffected in bulk/nano Cu-treated plants, compared to controls (p ≤ .05). Bulk/nano Cu incremented the relative abundance of microorganisms involved in the elemental uptake. These results indicate that nano Cu improved the physiology of alfalfa and can be considered as potential nanofertilizers.

Bok choy (Brassica rapa) grown in copper oxide nanoparticles-amended soils exhibits toxicity in a phenotype-dependent manner: Translocation, biodistribution and nutritional disturbance.

The comparative toxicity of nano/bulk cupric oxide (CuO) and ionic copper (Cu) in Rosie and Green bok choy (Brassica rapa) varieties, with higher and lower anthocyanin contents, respectively, was investigated. Both phenotypes were cultivated for 70 days in natural soil amended with nano CuO (nCuO), bulk CuO (bCuO), and Cu chloride (CuCl2) at 75, 150, 300, and 600 mg Cu/kg soil. Essential elements in tissues, agronomical parameters, chlorophyll content, and Cu distribution in leaf were determined. In both varieties, nCuO treatments significantly increased Cu uptake in roots, compared with bCuO and CuCl2 (p ≤ 0.05). At all treatment concentrations, Rosie variety had more Cu than Green. More physiological impairments such as chlorophyll and leaf biomass reduction were observed in treated-Rosie varieties, compared to Green plants. The adverse effects were higher in nCuO-treated plants than their bCuO- or ionic Cu-exposed counterparts. Different distribution patterns of the translocated Cu in leaf midrib and parenchyma depended on particle size and plant phenotype, as demonstrated by two-photon microscopy. The different effects of CuO-based compounds in Rosie and Green varieties may be related to the anthocyanin content. These findings help to understand the factors involved in nanoparticles uptake and translocation to plant edible parts.

Improvement of nutrient elements and allicin content in green onion (Allium fistulosum) plants exposed to CuO nanoparticles.

With the exponential growth of nanomaterial production in the last years, nano copper (Cu)-based compounds are gaining more consideration in agriculture since they can work as pesticides or fertilizers. Chinese scallions (Allium fistulosum), which are characterized by their high content of the antioxidant allicin, were the chosen plants for this study. Spectroscopic and microscopic techniques were used to evaluate the nutrient element, allicin content, and enzyme antioxidant properties of scallion plants. Plants were harvested after growing for 80 days at greenhouse conditions in soil amended with CuO particles [nano (nCuO) and bulk (bCuO)] and CuSO4 at 75-600 mg/kg]. Two-photon microscopy images demonstrated the particulate Cu uptake in nCuO and bCuO treated roots. In plants exposed to 150 mg/kg of the Cu-based compounds, root Cu content was higher in plants treated with nCuO compared with bCuO, CuSO4, and control (p ≤ 0.05). At 150 mg/kg, nCuO increased root Ca (86%), root Fe (71%), bulb Ca (74%), and bulb Mg (108%) content, compared with control (p ≤ 0.05). At the same concentration, bCuO reduced root Ca (67%) and root Mg (33%), compared with control (p ≤ 0.05). At all concentrations, nCuO and CuSO4 increased leaf allicin (56-187% and 42-90%, respectively), compared with control (p ≤ 0.05). The antioxidant enzymes were differentially affected by the Cu-based treatments. Overall, the data showed that nCuO enhances nutrient and allicin contents in scallion, which suggests they might be used as a nanofertilizer for onion production.

Study of organochlorine pesticides and heavy metals in soils of the Juarez valley: an important agricultural region between Mexico and the USA.

The Juarez Valley is an important agricultural region in northern Mexico, conveniently organized into three modules (I to III). For decades, their soils have been exposed to organochlorine pesticides (OCPs) and also have been irrigated with wastewaters, which may contain heavy metals. Nowadays, there is very limited information regarding the presence of OCPs and heavy metals in these soils. Thus, the aim of this study was to diagnose these soils for OCPs and heavy metal content by using gas chromatography coupled with electron micro-capture detector and atomic absorption spectrometry, respectively. The results indicated that 4,4'-dichlorodiphenyldichloroethylene and 4,4'-dichlorodiphenyltrichloroethane were primarily disseminated across the three modules since they were found in 100% and 97% of the analyzed soils, respectively. According to international regulations, none of the determined OCP concentrations are out of the limits. Additionally, the Cu, Zn, Fe, Pb, and Mn were found in all sampled soils from the three modules. The highest concentration of Fe was found in module II (1902.7 ± 332.2 mg kg-1), followed by Mn in module III (392.43 ± 74.43 mg kg-1), Zn in module I (38.36 ± 26.57 mg kg-1), Pb in module II (23.48 ± 6.48 mg kg-1), and Cu in module I (11.04 ± 3.83 mg kg-1) (p ≤ 0.05). These values did not exceed the limits proposed by international standards. The Cd was detected in most of the analyzed soils and all their values, with an average of 2 mg kg-1, surpassed the Mexican standards (0.35 mg kg-1). This study has mapped the main OCPs and heavy metals in the Juarez Valley and can serve as a starting point to further monitor the behave of xenobiotics. Since these recalcitrant compounds might be bio-accumulated in biological systems, further analytical methods, as well as remediation techniques, should be developed.

Can abiotic stresses in plants be alleviated by manganese nanoparticles or compounds?

Abiotic stress has become one of the most challenging problems for agriculture as the world population keeps increasing dramatically. Crop stress management using manganese (Mn) compounds has been recently employed to reduce the negative effects caused by drought, harsh temperature, and salinity. In response to abiotic stress, an adequate supply of Mn has shown to remediate plant manganese deficiency, induce Mn superoxide dismutase at the transcriptional level to face reactive oxygen species production, and stimulate manganese-dependent proteins to maintain cell integrity. Lately, nanoparticles (NPs) have been explored in agriculture applications. Recent studies have implied that Mn NPs may help plants to overcome abiotic stresses at higher efficiency and lower toxicity, compared to their bulk or ionic counterparts. Although studies have shown that Mn compounds promote crop growth and alleviate abiotic stress, many questions related to Mn-plant networking, their mode of signaling, and the Mn-dependent regulation processes need to be answered.

A comparative metagenomic and spectroscopic analysis of soils from an international point of entry between the US and Mexico.

The Paso del Norte region is characterized by its dynamic industries and active agriculture. Throughout the years, urban and agricultural soils from this region have been exposed to xenobiotics, heavy metals, and excess of hydrocarbons. In this study, samples of urban [domestic workshops (DW)] and agricultural-intended (AI) soils from different sites of Ciudad Juárez, Mexico were evaluated for their fertility, element content, and microbial diversity. Chemical analyses showed that nitrites, nitrates, P, K, Mg, and Mn were predominantly higher in AI soils, compared to DW soils (p ≤ 0.05). The composition of soil microbial communities showed that Proteobacteria phylum was the most abundant in both soils (67%, p ≤ 0.05). In AI soils, Paracoccus denitrificans was reduced (p ≤ 0.05), concurring with an increment in nitrates, while the content of nitrogen was negatively correlated with the rhizobium group (r2 = -0.65, p ≤ 0.05). In DW soils, the Firmicutes phylum represented up to ~25%, and the relative abundance of Proteobacteria strongly correlated with a higher Cu content (r2 = 0.99, p ≤ 0.0001). The monotypic genus Sulfuricurvum was found only in oil-contaminated soil samples. Finally, all samples showed the presence of the recently created phylum Candidatus saccharibacteria. These results describe the productivity parameters of AI soils and its correlation to the microbial diversity, which are very important to understand and potentiate the productivity of soils. The data also suggest that soils impacted with hydrocarbons and metal(oid)s allow the reproduction of microorganisms with the potential to alleviate contaminated sites.

Toxicity of copper hydroxide nanoparticles, bulk copper hydroxide, and ionic copper to alfalfa plants: A spectroscopic and gene expression study.

Bulk Cu compounds such as Cu(OH)2 are extensively used as pesticides in agriculture. Recent investigations suggest that Cu-based nanomaterials can replace bulk materials reducing the environmental impacts of Cu. In this study, stress responses of alfalfa (Medicago sativa L.) seedlings to Cu(OH)2 nanoparticle or compounds were evaluated. Seeds were immersed in suspension/solutions of a Cu(OH)2 nanoform, bulk Cu(OH)2, CuSO4, and Cu(NO3)2 at 25 and 75 mg/L. Six days later, the germination, seedling growth, and the physiological and biochemical responses of sprouts were evaluated. All Cu treatments significantly reduced root elongation (average = 63%). The ionic compounds at 25 and 75 mg/L caused a reduction in all elements analyzed (Ca, K, Mg, P, Zn, and Mn), excepting for S, Fe and Mo. The bulk-Cu(OH)2 treatment reduced K (48%) and P (52%) at 75 mg/L, but increased Zn at 25 (18%) and 75 (21%) mg/L. The nano-Cu(OH)2 reduced K (46%) and P (48%) at 75 mg/L, and also P (37%) at 25 mg/L, compared with control. Confocal microscopy images showed that all Cu compounds, at 75 mg/L, significantly reduced nitric oxide, concurring with the reduction in root growth. Nano Cu(OH)2 at 25 mg/L upregulated the expression of the Cu/Zn superoxide dismutase gene (1.92-fold), while ionic treatments at 75 mg/L upregulated (∼10-fold) metallothionein (MT) transcripts. Results demonstrated that nano and bulk Cu(OH)2 compounds caused less physiological impairments in comparison to the ionic ones in alfalfa seedlings.

Phosphoenolpyruvate carboxykinase cytosolic and mitochondrial isoforms are expressed and active during hypoxia in the white shrimp Litopenaeus vannamei.

Hypoxic zones in marine environments are spreading around the world affecting the survival of many organisms. Marine animals have several strategies to respond to hypoxia, including the regulation of gluconeogenesis. Phosphoenolpyruvate carboxykinase (PEPCK) is a key regulatory enzyme of gluconeogenesis. The objective of this work was to study two isoforms of PEPCK, one mitochondrial (PEPKC-M) and one cytosolic (PEPCK-C), from the white shrimp Litopenaeus vannamei and the response to hypoxia. Both PEPCK isoforms are 72 kDa proteins and have 92% identity at the amino acid level. The mitochondrial isoform has a N-terminal signal peptide for mitochondrial import. Gene expression and enzymatic activity in subcellular fractions were detected in gills, hepatopancreas and muscle in normoxic and hypoxic conditions. Expression of PEPCK-C was higher than PEPCK-M in all the tissues and induced in response to hypoxia at 48 h in hepatopancreas, while the enzymatic activity of PEPCK-M was higher than PEPCK-C in gills and hepatopancreas, but not in muscle and also increased in response to hypoxia in hepatopancreas but decreased in gills and muscle. During limiting oxygen conditions, shrimp tissues obtain energy by inducing anaerobic glycolysis, and although gluconeogenesis implies energy investment, due to the need to maintain glucose homeostasis, these gluconeogenic enzymes are active with contrasting behaviors in the cytosol and mitochondrial cell compartments and appear to be up-regulated in hepatopancreas indicating this tissue pivotal role in gluconeogenesis during the response to hypoxia.

Role of HIF-1 on phosphofructokinase and fructose 1, 6-bisphosphatase expression during hypoxia in the white shrimp Litopenaeus vannamei.

HIF-1 is a transcription factor that controls a widespread range of genes in metazoan organisms in response to hypoxia and is composed of α and ß subunits. In shrimp, phosphofructokinase (PFK) and fructose bisphosphatase (FBP) are up-regulated in hypoxia. We hypothesized that HIF-1 is involved in the regulation of PFK and FBP genes in shrimp hepatopancreas under hypoxia. Long double stranded RNA (dsRNA) intramuscular injection was utilized to silence simultaneously both HIF-1 subunits, and then, we measured the relative expression of PFK and FBP, as well as their corresponding enzymatic activities in hypoxic shrimp hepatopancreas. The results indicated that HIF-1 participates in the up-regulation of PFK transcripts under short-term hypoxia since the induction caused by hypoxia (~1.6 and ~4.2-fold after 3 and 48h, respectively) is significantly reduced in the dsRNA animals treated. Moreover, PFK activity was significantly ~2.8-fold augmented after 3h in hypoxia alongside to an ~1.9-fold increment in lactate. However, when animals were dsRNA treated, both were significantly reduced. On the other hand, FBP transcripts were ~5.3-fold up-regulated in long-term hypoxic conditions (48h). HIF-1 is involved in this process since FBP transcripts were not induced by hypoxia when HIF-1 was silenced. Conversely, the FBP activity was not affected by hypoxia, which suggests its possible regulation at post-translational level. Taken together, these results position HIF-1 as a prime transcription factor in coordinating glucose metabolism through the PFK and FBP genes among others, in shrimp under low oxygen environments.

Expression of fructose 1,6-bisphosphatase and phosphofructokinase is induced in hepatopancreas of the white shrimp Litopenaeus vannamei by hypoxia.

Marine organisms are exposed to hypoxia in natural ecosystems and during farming. In these circumstances marine shrimp survive and synthesize ATP by anaerobic metabolism. Phosphofructokinase (PFK) and fructose 1,6-bisphosphatase (FBP) are key enzymes in carbohydrate metabolism. Here we report the cDNA of FBP from the shrimp Litopenaeus vannamei hepatopancreas and expression of PFK and FBP under normoxia and hypoxia. Hypoxia induces PFK and FBP expression in hepatopancreas but not in gills and muscle. Induction in hepatopancreas of the glycolytic and gluconeogenic key enzymes, PFK and FBP, suggests that PFK could be a key factor for increasing anaerobic rate, while FBP is probably involved in the activation of gluconeogenesis or the pentose-phosphates pathway during hypoxia in the highly active metabolism of hepatopancreas.