Effect of pretreatment with hydrogen sulfide donor sodium hydrosulfide on heat tolerance in relation to antioxidant system in maize (Zea mays) seedlings

Hydrogen sulfide (H₂S) is a signal molecule that is involved in plant growth, development and the acquisition of stress tolerance including heat tolerance, but the mechanism of H₂S-induced heat tolerance is not completely clear. In present study, the effect of sodium hydrosulfide (NaHS), a H₂S donor, treatment on heat tolerance of maize seedlings in relation to antioxidant system was investigated. The results showed that NaHS treatment improved survival percentage of maize seedlings under heat stress in a concentration-dependent manner, indicating that H₂S treatment could improve heat tolerance of maize seedlings. To further study mechanism of NaHS-induced heat tolerance, catalase (CAT), guaiacol peroxidase (GPX), superoxide dismutase (SOD), glutathione reductase (GR) and ascorbate peroxidase (APX) activities, and glutathione (GSH) and ascorbic acid (AsA) contents in maize seedlings were determined. The results showed that NaHS treatment increased the activities of CAT, GPX, SOD and GR, and GSH and AsA contents as well as the ratio of reduced antioxidants to total antioxidants [AsA/(AsA+DHA) and GSH/(GSH +GSSG)] in maize seedlings under normal culture conditions compared with the control. Under heat stress, antioxidant enzymes activities, antioxidants contents and the ratio of the reduced antioxidants to total antioxidants in control and treated seedlings all decreased, but NaHS-treated seedlings maintained higher antioxidant enzymes activities and antioxidants levels as well as the ratio of reduced antioxidants to total antioxidants. All of above-mentioned results suggested that NaHS treatment could improve heat tolerance of maize seedlings, and the acquisition of this heat tolerance may be relation to enhanced antioxidant system activity.     

Hydrogen sulphide may be a novel downstream signal molecule in nitric oxide‐induced heat tolerance of maize (Zea mays L.) seedlings

Nitric oxide (NO) is a second messenger with multifunction that is involved in plant growth, development and the acquisition of stress tolerance. In recent years, hydrogen sulphide (H₂S) has been found to have similar functions, but crosstalk between NO and H₂S in the acquisition of heat tolerance is not clear. In this study, pretreatment with the NO donor sodium nitroprusside (SNP) improved the survival percentage of maize seedlings and alleviated an increase in electrolyte leakage and a decrease in tissue vitality as well as accumulation of malondialdehyde, indicating that pretreatment with SNP improved the heat tolerance of maize seedlings. In addition, pretreatment with SNP enhanced the activity of L‐cystine desulfhydrase, which, in turn, induced accumulation of endogenous H₂S, while application of H₂S donors, NaHS and GYY4137, increased endogenous H₂S content, followed by mitigating increase in electrolyte leakage and enhanced survival percentage of seedlings under heat stress. Interestingly, SNP‐induced heat tolerance was enhanced by application of NaHS and GYY4137, but was eliminated by inhibitors of H₂S synthesis DL‐propargylglycine, aminooxyacetic acid, potassium pyruvate and hydroxylamine, and the H₂S scavenger hypotaurine. All of the above‐mentioned results suggest that SNP pretreatment could improve heat tolerance, and H₂S may be a downstream signal molecule in NO‐induced heat tolerance of maize seedlings.     

Hydrogen sulfide donor sodium hydrosulfide-improved heat tolerance in maize and involvement of proline

Hydrogen sulfide (H₂S) has long been considered as a phytotoxin, but nowadays as a cell signal molecule involved in growth, development, and the acquisition of stress tolerance in higher plants. In the present study, hydrogen sulfide donor, sodium hydrosulfide (NaHS), pretreatment markedly improved germination percentage of seeds and survival percentage of seedlings of maize under heat stress, and alleviated an increase in electrolyte leakage of roots, a decrease in tissue vitality and an accumulation of malondialdehyde (MDA) in coleoptiles of maize seedlings. In addition, pretreatment of NaHS could improve the activity of Δ¹-pyrroline-5-carboxylate synthetase (P5CS) and lower proline dehydrogenase (ProDH) activity, which in turn induced accumulation of endogenous proline in maize seedlings. Also, application of proline could enhance endogenous proline content, followed by mitigated accumulation of MDA and increased survival percentage of maize seedlings under heat stress. These results suggest that sodium hydrosulfide pretreatment could improve heat tolerance of maize and the acquisition of this heat tolerance may be involved in proline.     

Signaling molecule methylglyoxal-induced thermotolerance is partly mediated by hydrogen sulfide in maize (<Emphasis Type="Italic">Zea mays</Emphasis> L.) seedlings

Methylglyoxal (MG) was traditionally viewed as toxic by-product of glycolysis and photosynthesis in plants, but now is emerging as a signaling molecule, which, similar to hydrogen sulfide (H₂S), participates in regulating seed germination, growth, development, and response to abiotic stress. However, whether exists an mutual effect between MG and H₂S in improving thermotolerance in plants is not found to be reported. In this paper, interplay between MG and H₂S in the formation of thermotolerance in maize seedlings was investigated. The results indicated that MG pretreatment elevated the survival percentage of maize seedlings under high-temperature stress, manifesting that MG could boost the thermotolerance of maize seedlings. Interestingly, MG-induced thermotolerance was reinforced by sodium hydrosulphide (NaHS, H₂S donor), while impaired by dl-propargylglycine (inhibitor of H₂S biosynthesis) and hypotaurine (scavenger of H₂S), respectively. In addition, H₂S could induce the thermotolerance of maize seedlings, which was impaired by aminoguanidine (AG) and N-acetyl-l-cysteine (NAC) (MG scavengers), respectively. Furthermore, MG stimulated the activity of a key enzyme in H₂S biosynthesis, l-cysteine desulfhydrase, which, in turn, triggered the elevation of endogenous H₂S in maize seedlings. In addition, H₂S increased the level of endogenous MG; this increase was crippled by AG and NAC. This paper, for the first time, reported that MG could improve the thermotolerance of maize seedlings, and its acquisition was, at least partly, mediated by H₂S.     

Hydrogen sulfide as a signal molecule in hematin-induced heat tolerance of tobacco cell suspension

Carbon monoxide (CO) is considered as a new emerging cell signal molecule which is involved in plant growth, development, and acquisition of stress tolerance. In recent years, hydrogen sulfide (H₂S) has been found to have similar functions, but crosstalk between CO and H₂S in the acquisition of heat tolerance in plants is not clear. In this study, pretreatment of tobacco (Nicotiana tabaccum L.) cells cultured in a suspension with a CO donor hematin significantly increased survival percentage of cells under a heat stress and regrowth ability after the heat stress, alleviated a decrease in cell vitality, and accumulation of malondialdehyde. In addition, treatment with hematin enhanced the activity of L-cysteine desulfhydrase, a key enzyme in H₂S biosynthesis, which in turn induced accumulation of endogenous H₂S in tobacco cells. Interestingly, hematin-induced heat tolerance was enhanced by addition of NaHS, a H₂S donor, but weakened by specific inhibitors of H₂S biosynthesis DL-propargylglycine or its scavenger hypotaurine. Furthermore, pretreatment with hemoglobin (a CO scavenger) and zinc protoporphyrin IX (a CO specific synthetic inhibitor) had no significant effect on NaHS-induced heat tolerance of tobacco cells. These results suggest that CO pretreatment could improve the heat tolerance of tobacco suspension cultured cells, and H₂S might exert its signal role downstream to CO-induced heat tolerance.     

Hydrogen sulfide alleviates aluminum toxicity in barley seedlings

Aims

Aluminum (Al) toxicity is one of the major factors that limit plant growth. Low concentration of hydrogen sulfide (H₂S) has been proven to function in physiological responses to various stresses. The objective of this study is to investigate the possible role of H₂S in Al toxicity in barley (Hordeum vulgare L) seedlings.

Methods

Barley seedlings pre-treated with sodium hydrosulfide (NaHS), a H₂S donor, and subsequently exposed to Al treatment were studied for their effects on root elongation, Al accumulation in seedlings, Al-induced citrate secretion and oxidative stress, and plasma membrane (PM) H⁺-ATPase expression.

Results

Our results showed that H₂S had significant rescue effects on Al-induced inhibition of root elongation which was correlated well with the decrease of Al accumulation in seedlings. Meanwhile, Al-induced citrate secretion was also significantly enhanced by NaHS pretreatment. Al-induced oxidative stress as indicated by lipid peroxidation and reactive oxygen species burst was alleviated by H₂S through the activation of the antioxidant system. Moreover, Al-induced reduction in PM H⁺-ATPase expression was reversed by exogenous NaHS.

Conclusions

Altogether, our results suggest H₂S plays an ameliorative role in protecting plants against Al toxicity by inducing the activities of antioxidant enzymes, increasing citrate secretion and citrate transporter gene expression, and enhancing the expression of PM H⁺-ATPase.     

Interaction of brassinosteroid functions and sucrose transporter SlSUT2 regulate the formation of arbuscular mycorrhiza

Transgenic tomato plants with reduced expression of the sucrose transporter SlSUT2 showed higher efficiency of mycorrhization suggesting a sucrose retrieval function of SlSUT2 from the peri-arbuscular space back into the cell cytoplasm plant cytoplasm thereby limiting mycorrhiza fungal development. Sucrose uptake in colonized root cells requires efficient plasma membrane-targeting of SlSUT2 which is often retained intracellularly in vacuolar vesicles. Protein-protein interaction studies suggested a link between SISUT2 function and components of brassinosteroid biosynthesis and signaling. Indeed, the tomato DWARF mutant d^x defective in BR synthesis¹ showed significantly reduced mycorrhization parameters.² The question has been raised whether the impact of brassinosteroids on mycorrhization is a general phenomenon. Here, we include a rice mutant defective in DIM1/DWARF1 involved in BR biosynthesis to investigate the effects on mycorrhization. A model is presented where brassinolides are able to impact mycorrhization by activating SUT2 internalization and inhibiting its role in sucrose retrieval.     

Endogenous salicylic acid accumulation is required for chilling tolerance in cucumber (Cucumis sativus L.) seedlings

Salicylic acid (SA) is an important plant hormone, and its exogenous application can induce tolerance to multiple environmental stresses in plants. In this study, we examine the potential involvement of endogenous SA in response to chilling in cucumber (Cucumis sativus L.) seedlings. A low temperature of 8 °C induces a moderate increase in endogenous SA levels. Chilling stimulates the enzymatic activities and the expression of genes for phenylalanine ammonia-lyase (PAL) and benzoic acid-2-hydroxylase rather than isochorismate synthase. This indicates that the PAL enzymatic pathway contributes to chilling-induced SA production. Cucumber seedlings pretreated with SA biosynthesis inhibitors accumulate less endogenous SA and suffer more from chilling damage. The expression of cold-responsive genes is also repressed by SA inhibitors. The reduction in stress tolerance and in gene expression can be restored by the exogenous application of SA, confirming the critical roles of SA in chilling responses in cucumber seedlings. Furthermore, the inhibition of SA biosynthesis under chilling stress results in a prolonged and enhanced hydrogen peroxide (H₂O₂) accumulation. The application of exogenous SA and the chemical scavenger of H₂O₂ reduces the excess H₂O₂ and alleviates chilling injury. In contrast, the protective effects of SA are negated by foliar spraying with high concentrations of H₂O₂ and an inhibitor of the antioxidant enzyme. These results suggest that endogenous SA is required in response to chilling stress in cucumber seedlings, by modulating the expression of cold-responsive genes and the precise induction of cellular H₂O₂ levels.     

Endosomal lipid flippases and their related diseases

 In eukaryotic cell membranes, phospholipids are asymmetrically distributed between the two leaflets of the lipid bilayer. For example, the extracellular leaflet of the plasma membrane (PM) is enriched with phosphatidylcholine and sphingomyelin, while the cytosolic leaflet of the PM is enriched with phosphatidylserine (PS) and phosphatidylethanolamine. The asymmetric distribution of PS in the PM is crucial for cell life, since PS in the extracellular leaflet of the PM is recognized as an “eat-me” signal by phagocytes. Inside the cells, a high PS concentration in the cytosolic leaflet of the PM is essential to facilitate various cellular events through the recruitment of signaling molecules such as protein kinase C and Akt.The asymmetric distribution of phospholipids is believed to be generated in part by phospholipid translocases, or “flippases.” The proteins responsible for flippase activity are type IV P-type ATPase (P₄-ATPases). P-type ATPases are multispan transmembrane pumps that use ATP hydrolysis as an energy source. P-type ATPases undergo autophosphorylation of a conserved aspartate residue during the catalytic cycle, hence the designation of “P”-type. P₄-ATPases are unique in that they are phospholipid transporters whereas other types of P-type ATPases are ion transporters.The human genome contains 14 P₄-ATPases, and mutations in some P₄-ATPases cause inherited genetic diseases. For example, mutations in ATP8B1 are associated with intrahepatic cholestasis and also cause hearing loss. Mutations in ATP8A2 are associated with a severe neurological disorder characterized by cerebellar ataxia, mental retardation, and dysequilibrium syndrome (CAMRQ).¹ Despite the accumulating evidence highlighting the physiological importance of P₄-ATPases, how dysfunction of P₄-ATPases causes diseases is poorly understood.In a recent study, we revealed the cellular function of the P₄-ATPase, ATP8A1.² ATP8A1 localizes at recycling endosomes (REs), an organelle that functions in recycling transport of internalized molecules back to the PM, thus defining the amount of proteins at the PM. PS is most concentrated in REs among intracellular organelles and we roughly estimated that 70 and 30% of PS are localized in the cytosolic and the luminal leaflets of RE membranes, respectively.² ATP8A1 generates the asymmetric transbilayer distribution of PS at REs. The knockdown of ATP8A1 halted recycling traffic from REs to the PM. At the mechanistic level, we found that EHD1, a dynamin-like membrane fission protein, lost its RE localization upon ATP8A1 knockdown and EHD1 knockdown also blocked recycling traffic. EHD1 bound PS in vitro and lost its membrane localization in cells that are defective in PS synthesis. Thus, we propose that PS flipping by ATP8A1 recruits EHD1 to RE membranes, thereby regulating the recycling traffic from REs to the PM (Fig. 1). Open in a separate windowFigure 1.Model of flippase-related diseases. Under normal conditions, flippases (e.g., ATP8A1 and ATP8A2) translocate PS to the cytosolic leaflet of RE membranes. PS recruits EHD1 to REs, and then EHD1 participates in the fission of RE membranes to generate transport vesicles that contain cell surface receptors. In flippase-dysfunctional situations, PS levels in the cytosolic leaflet of REs would be low. This impairs the PS/EHD1/membrane traffic axis, leading to a lower abundance of cell surface receptors that are critical for responses to extracellular ligands.ATP8A2 is a tissue-specific ATP8A1 paralogue. We found that a CAMRQ-causative mutation of ATP8A2 (I376M) lost its ATPase and flippase activity toward PS. ATP8A2 is not endogenously expressed in COS-1 cells. Interestingly, the phenotype that was caused by the loss of ATP8A1 in COS-1 cells, was restored by the exogenous expression of wild-type ATP8A2, but not I376M mutant ATP8A2. Moreover, cortical neurons prepared from ATP8A2 knockout mice showed lower abundance of transferrin receptors at the PM. Together, these results indicate that ATP8A2 functions in the recycling traffic in neurons, and that CAMRQ may result from the defect in recycling of important neurological receptor proteins from REs to the PM. One possible candidate protein is very low-density lipoprotein receptor (VLDLR). VLDLR is a receptor for reelin, an extracellular protein that guides neuronal migration in the cerebral cortex and cerebellum. VLDLR circulates between the PM and endosomes (possibly REs) by recycling traffic.³ Significantly, mutations in VLDLR gene are also linked to CAMRQ.^4,5 Therefore, impaired recycling traffic of VLDLR to the PM in neurons with dysfunctional ATP8A2 (I376M) may cause lower expression of VLDLR at the PM, leading to reduced reelin signaling, abnormal neuronal development, and neurological disorder.dATP8B, a P₄-ATPase in Drosophila melanogaster was recently reported to cause an impaired response to cVA pheromone (a sex-specific social cue) and mislocalization of the pheromone receptor in cVA-sensing neurons.⁶ The impaired response to the pheromone in dATP8B mutant was rescued by expressing bovine ATP8A2. Therefore, from insects to mammals, phospholipid flippases may define the localization of neuronal receptors to the PM.Lastly, our findings may explain the phenotype of ATP8A1 knockout mice.⁷ ATP8A1 knockout mice are vital but show deficiencies in hippocampus-dependent learning. Hippocampus-dependent learning involves modification of synaptic strength, and one cellular mechanism for tuning synaptic strength is long-term potentiation (LTP). During LTP, REs supply glutamate receptors to the post-synaptic membrane.⁸ Therefore, we speculate that impaired glutamate receptor traffic from REs to the post-synaptic membranes during LTP may underlie the deficiency in learning in ATP8A1 knockout mice. In agreement with this hypothesis, the dominant-negative form of EHD1 inhibits glutamate receptor traffic during LTP.⁸Many P₄-ATPases are expressed in the Golgi/endosomes and the PM. We expect that they contribute redundantly to the phospholipid asymmetry and membrane traffic through organelles. Simultaneous ablations of P₄-ATPases may dissect their roles and will give more insight into flippase-mediated cellular processes and -related diseases.     

H_2S对干旱胁迫下白三叶幼苗抗氧化防御能力的影响

以‘拉丁诺’白三叶(Trifolium repens cv.‘Ladino’)为试验材料,研究外源H2S处理对PEG6 000(聚乙二醇)模拟干旱胁迫下白三叶叶片相对含水量(RWC)、膜脂过氧化、活性氧成分、抗氧化酶、抗坏血酸-谷胱甘肽循环代谢和非酶抗氧化物质的影响,以揭示H_2S调控白三叶抗旱性的生理机制。结果显示:(1)0.2 mmol/L的外源NaHS(H_2S供体)能显著提高干旱胁迫下白三叶的叶片相对含水量,维持显著较低的电解质渗透率(EL)和丙二醛(MDA)含量。(2)与直接干旱胁迫相比,干旱胁迫下外源添加NaHS处理的白三叶叶片内超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性显著增强,抗坏血酸-谷胱甘肽循环代谢中关键酶抗坏血酸过氧化物酶(APX)、脱氢抗坏血酸还原酶(DHAR)、单脱水抗坏血酸还原酶(MDHAR)和谷胱甘肽还原酶(GR)活性及其抗氧化中间产物抗坏血酸(AsA)、谷胱甘肽(GSH)含量也显著提高。(3)叶片类黄酮、总酚和原花青素的含量在一定的胁迫时间范围内亦显著增加,并伴随着活性氧成分O_2~(-·)产生速率和H_2O_2水平降低。研究认为,外源H2S能通过促进干旱胁迫下白三叶体内的多重抗氧化防御能力来提高其幼苗的抗旱性。     

外源硫化氢和水杨酸对盐胁迫下加工番茄幼苗生长与生理特性的影响

以加工番茄KT-7为材料,在水培条件下,研究外源水杨酸(SA,0.15 mmol/L)、硫化氢(H2S)供体硫氢化钠(NaHS,50 mmol/L)对150 mmol/L NaCl胁迫下加工番茄幼苗的渗透调节、活性氧代谢和快速叶绿素荧光的影响,以探讨H2S和SA这2种信号分子协同作用、以及实际生产中缓解加工番茄幼苗盐胁...     

The tumor suppressor Lgl1 regulates front-rear polarity of migrating cells

Cell migration is a highly integrated, multistep process that plays an important role in physiological and pathological processes. The migrating cell is highly polarized, with complex regulatory pathways that integrate its component processes spatially and temporally.¹ The Drosophila tumor suppressor, Lethal (2) giant larvae (Lgl), regulates apical-basal polarity in epithelia and asymmetric cell division.² But little is known about the role of Lgl in establishing cell polarity in migrating cells. Recently, we showed that the mammalian Lgl1 interacts directly with non-muscle myosin IIA (NMIIA), inhibiting its ability to assemble into filaments in vitro.³ Lgl1 also regulates the cellular localization of NMIIA, the maturation of focal adhesions, and cell migration.³ We further showed that phosphorylation of Lgl1 by aPKCζ prevents its interaction with NMIIA and is important for Lgl1 and acto-NMII cytoskeleton cellular organization.⁴ Lgl is a critical downstream target of the Par6-aPKC cell polarity complex; we showed that Lgl1 forms two distinct complexes in vivo, Lgl1-NMIIA and Lgl1-Par6-aPKCζ in different cellular compartments.⁴ We further showed that aPKCζ and NMIIA compete to bind directly to Lgl1 through the same domain. These data provide new insights into the role of Lgl1, NMIIA, and Par6-aPKCζ in establishing front-rear polarity in migrating cells. In this commentary, I discuss the role of Lgl1 in the regulation of the acto-NMII cytoskeleton and its regulation by the Par6-aPKCζ polarity complex, and how Lgl1 activity may contribute to the establishment of front-rear polarity in migrating cells.     

miRNA-182 and the regulation of the glioblastoma phenotype - toward miRNA-based precision therapeutics

Glioblastoma (GBM) is an incurable cancer, with survival rates of just 14-16 months after diagnosis.¹ Functional genomics have identified numerous genetic events involved in GBM development. One of these, the deregulation of microRNAs (miRNAs), has been attracting increasing attention due to the multiple biologic processes that individual miRNAs influence. Our group has been studying the role of miR-182 in GBM progression, therapy resistance, and its potential as GBM therapeutic. Oncogenomic analyses revealed that miR-182 is the only miRNA, out of 470 miRNAs profiled by The Cancer Genome Atlas (TCGA) program, which is associated with favorable patient prognosis, neuro-developmental context, temozolomide (TMZ) susceptibility, and most significantly expressed in the least aggressive oligoneural subclass of GBM. miR-182 sensitized glioma cells to TMZ-induced apoptosis, promoted glioma initiating cell (GIC) differentiation, and reduced tumor cell proliferation via knockdown of Bcl2L12, c-Met and HIF2A.² To deliver miR-182 to intracranial gliomas, we have characterized Spherical Nucleic Acids covalently functionalized with miR-182 sequences (182-SNAs). Upon systemic administration, 182-SNAs crossed the blood-brain/blood-tumor barrier (BBB/BTB), reduced tumor burden, and increased animal subject survival.^2-4 Thus, miR-182-based SNAs represent a tool for systemic delivery of miRNAs and a novel approach for the precision treatment of malignant brain cancers.     

Comment on: HMGB1-dependent and -independent autophagy

HMGB1 (high mobility group box 1), a ubiquitously expressed DNA-binding nucleoprotein, has not only been attributed with important functions in the regulation of gene expression but is thought to function as an important damage-associated molecular pattern in the extracellular space. Recently, conditional Hmgb1 deletion strategies have been employed to overcome the perinatal mortality of global Hmgb1 deletion and to understand HMGB1 functions under disease conditions. From these studies, it has become evident that HMGB1 is not required for normal organ function. However, the different conditional ablation strategies have yielded contradictory results in some disease models. With nearly complete recombination in all transgenic mouse models, the main reason for opposite results is likely to lie within different targeting strategies. In summary, different targeting strategies need to be taken into account when interpreting HMGB1 functions, and further efforts need to be undertaken to compare these models side by side.We appreciate the thoughtful analysis on HMGB1-dependent and -independent autophagy by Sun and Tang.¹ However, we disagree with several statements in this review. Sun and Tang write “Mice with hepatocyte-specific deletion of Hmgb1 from Robert Schwabe''s lab are not complete conditional knockout mice; the protein level of HMGB1 in the liver is decreased by about 70%,” as well as “a major difference between Robert Schwabe''s engineered HMGB1 mice and other groups is the tissue-level expression of HMGB1 after knockout.”¹We would like to point out that livers are not solely composed of hepatocytes and that albumin-Cre mediated deletion of target genes in the liver cannot result in complete loss of hepatic mRNA or protein of target genes due to the presence of unrecombined nonparenchymal cells, unless the target gene is exclusively expressed in hepatocytes and/or cholangiocytes. The reduction of hepatic HMGB1 in our studies—reaching 90% and 72% at the mRNA and protein level, respectively—is precisely at the expected level for this conditional strategy, and similar to other studies that employed albumin-Cre for hepatocyte-specific knockout of other target genes.^2-5 Hepatocytes account only for approximately 52% of cells in the liver, with other cell types including Kupffer cells (∼18% of liver cells), hepatic stellate cells (˜8% of liver cells), endothelial cells (∼22% cells of liver cells) and cholangiocytes (<1 % of liver cells) contributing to the remainder.⁶ Accordingly, albumin-Cre-mediated reduction of mRNA and protein levels of target genes (i.e., Hmgb1 and HMGB1 in our study) in the liver cannot exceed the amount of mRNA and protein expressed by hepatocytes and cholangiocytes (which is typically about 70–90%,^2-5 due to higher mRNA and protein levels in hepatocytes than in other hepatic cell types). The high efficacy of our conditional approach is best demonstrated by almost complete loss of HMGB1 expression in the hepatocellular compartment of albumin-Cre mice—as evidenced by loss of HMGB1 expression in all HNF4α-positive cells and in isolated primary hepatocytes—whereas HMGB1 expression is retained in nonparenchymal cells, as demonstrated by costaining for Kupffer cell marker F4/80, endothelial cell marker endomucin, and hepatic stellate cell marker desmin.^7,8 The nearly perfect recombination rate in our mice was further confirmed by experiments that employed Mx1Cre for Hmgb1 deletion, which resulted in almost complete loss of hepatic Hmgb1 mRNA and HMGB1 protein.^7,8 Moreover, our transgenic mice show early postnatal mortality when bred with a germline Cre deleter,⁷ thus reproducing the phenotype of the global HMGB1 knockout.⁹In summary, our transgenic mouse model results in nearly perfect recombination efficiency with virtually complete loss of Hmgb1 mRNA and HMGB1 protein in all targeted cell types, and constitutes a valid tool for the assessment of HMGB1 functions in vivo. Findings from this model need to be taken into account for proper interpretation of the role of HMGB1 in the normal and diseased liver, and cannot be interpreted as a result of incomplete deletion efficiency. Hence, differences in targeting strategies (exons 2–4 by our approach, exons 2–3 in mice from Tang and colleagues) are likely to explain opposite findings, e.g. improvement of ischemia-reperfusion injury in our hands, but aggravation of liver damage in the study by Huang et al.^8,10 Further analysis needs to be performed to determine whether ablation of exons 2–3 versus exons 2–4 leads to complete loss of HMGB1 function.     

Multiwalled carbon nanotubes enhance human bone marrow mesenchymal stem cells’ spreading but delay their proliferation in the direction of differentiation acceleration

Investigating the ability of films of pristine (purified, without any functionalization) multiwalled carbon nanotubes (MWCNTs) to influence human bone marrow mesenchymal stem cells’ (hBMSCs) proliferation, morphology, and differentiation into osteoblasts, we concluded to the following: A. MWCNTs delay the proliferation of hBMSCs but increase their differentiation. The enhancement of the differentiation markers could be a result of decreased proliferation and maturation of the extracellular matrix B. Cell spread on MWCNTs toward a polygonal shape with many thin filopodia to attach to the surfaces. Spreading may be critical in supporting osteogenic differentiation in pre-osteoblastic progenitors, being related with cytoskeletal tension. C. hBMSCs prefer MWCNTs than tissue plastic to attach and grow, being non-toxic to these cells. MWCNTs can be regarded as osteoinductive biomaterial topographies for bone regenerative engineering.Cellular interaction with substrate and neighboring cells plays a critical role in osteoblast survival, proliferation, differentiation as well as bone remodeling. Regulated biophysical cues, such as nanotopography, have been shown to be integral for tissue regeneration in the stem cell niche. Multiwalled carbon nanotubes (MWCNTs) represent a nanomaterial that has won enormous popularity in nanotechnology, exhibiting extraordinary physicochemical properties and supporting the growth of different kinds of cells.^1-3Simultaneous enhancement of osteoblast cells’ proliferation and differentiation,^4,5 decrease of proliferation rates along with decreased differentiation⁶ or increased differentiation accompanied with decreased proliferation⁷ have been reported. Contradictory results concerning osteoblast cell adhesion, and morphology have also been reported. Osteoblast cell lines on CNTs have been found to elongate but not widen or displayed a spindle-shaped morphology.^8,9 Spreading and surface area covered were reduced.^8-10 On the contrary, Tutak et al.⁷ reported robust spreading on medium roughness CNTs networks.This variable behavior on CNTs is probably due to the various cell types used in these works. It is reported that primary human marrow stromal cells and cell lines use substantially different mechanisms to regulate adhesion and spreading on the substrate.¹¹In a recent work of ours, published in Annals of Biomedical Engineering,¹² it was found that MWCNTs can create an osteogenic environment for human bone marrow mesenchymal stem cells (hBMSCs), even without addition of exogenous factors, representing a suitable reinforcement for bone tissue engineering scaffolds.In the following, we will highlight and discuss some aspects of this work''s results, in the context of literature findings, and provide additional material in order to elucidate issues on the influence of MWCNTs on hBMSCs’ proliferation, morphology, and differentiation into osteoblasts.     

Photoperiodic control of sugar release during the floral transition: What is the role of sugars in the florigenic signal?

Florigen is a mobile signal released by the leaves that reaching the shoot apical meristem (SAM), changes its developmental program from vegetative to reproductive. The protein FLOWERING LOCUS T (FT) constitutes an important element of the florigen, but other components such as sugars, have been also proposed to be part of this signal.^1-5 We have studied the accumulation and composition of starch during the floral transition in Arabidopsis thaliana in order to understand the role of carbon mobilization in this process. In A. thaliana and Antirrhinum majus the gene coding for the Granule-Bound Starch Synthase (GBSS) is regulated by the circadian clock^6,7 while in the green alga Chlamydomonas reinhardtii the homolog gene CrGBSS is controlled by photoperiod and circadian signals.^8,9 In a recent paper¹⁰ we described the role of the central photoperiodic factor CONSTANS (CO) in the regulation of GBSS expression in Arabidopsis. This regulation is in the basis of the change in the balance between starch and free sugars observed during the floral transition. We propose that this regulation may contribute to the florigenic signal and to the increase in sugar transport required during the flowering process.     

Cadmium-Induced Hydrogen Sulfide Synthesis Is Involved in Cadmium Tolerance in Medicago sativa by Reestablishment of Reduced (Homo)glutathione and Reactive Oxygen Species Homeostases

Until now, physiological mechanisms and downstream targets responsible for the cadmium (Cd) tolerance mediated by endogenous hydrogen sulfide (H₂S) have been elusive. To address this gap, a combination of pharmacological, histochemical, biochemical and molecular approaches was applied. The perturbation of reduced (homo)glutathione homeostasis and increased H₂S production as well as the activation of two H₂S-synthetic enzymes activities, including _L-cysteine desulfhydrase (LCD) and _D-cysteine desulfhydrase (DCD), in alfalfa seedling roots were early responses to the exposure of Cd. The application of H₂S donor sodium hydrosulfide (NaHS), not only mimicked intracellular H₂S production triggered by Cd, but also alleviated Cd toxicity in a H₂S-dependent fashion. By contrast, the inhibition of H₂S production caused by the application of its synthetic inhibitor blocked NaHS-induced Cd tolerance, and destroyed reduced (homo)glutathione and reactive oxygen species (ROS) homeostases. Above mentioned inhibitory responses were further rescued by exogenously applied glutathione (GSH). Meanwhile, NaHS responses were sensitive to a (homo)glutathione synthetic inhibitor, but reversed by the cotreatment with GSH. The possible involvement of cyclic AMP (cAMP) signaling in NaHS responses was also suggested. In summary, LCD/DCD-mediated H₂S might be an important signaling molecule in the enhancement of Cd toxicity in alfalfa seedlings mainly by governing reduced (homo)glutathione and ROS homeostases.     

Central role of salicylic acid in resistance of safflower (Carthamus tinctorius L.) against salinity

The effects of salicylic acid (SA) on growth parameters and enzyme activities were investigated in salt-stressed safflower (Carthamus tinctorius L.). Twenty-five days after sowing, seedlings were treated with NaCl (0, 100, and 200?mM) and SA (1?mM), and were harvested at 21 days after treatments. Results showed that some growth parameters decreased under salinity, while malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) content, phenolic compounds, and some enzyme activities increased. SA application increased some growth parameters, MDA and H₂O₂ content, and enzyme activities except catalase (CAT), which was different from the other enzymes and SA significantly reduced CAT activity in plants. These results suggest that SA-induced tolerance to salinity may be related to regulation of antioxidative responses and H₂O₂ level. Our study suggested that the resistant safflower can direct reactive oxygen species from a threat to an opportunity by using SA. Therefore, exogenous application of SA played this role through regulation of the antioxidant system.     

H2S作为下游信号参与SA对低温弱光下黄瓜幼苗光合作用的调控

水杨酸(SA)和硫化氢(H₂S)在调控非生物胁迫下植物生长发育和生理代谢中均起着非常重要的作用,但二者作为信号分子在调控低温弱光下黄瓜光合作用中的互作关系还不清楚。本试验以黄瓜幼苗为试材,分别用SA和硫氢化钠(NaHS,H₂S供体)及其清除剂或抑制剂喷撒叶面,以适宜温光下去离子水处理为对照(CK),研究低温(8 ℃/5 ℃,昼/夜)弱光(100 μmol·m^-2·s^-1)下SA和H₂S对黄瓜幼苗光合作用的调控及互作关系。结果表明: SA可明显增强L-/D-半胱氨酸脱巯基酶(LCD、DCD)活性及其mRNA表达,促进内源H₂S产生;NaHS对苯丙氨酸解氨酶和异分支酸合成酶活性、mRNA表达量及内源SA含量影响不大。SA和NaHS可使低温弱光下黄瓜幼苗的光合速率、气孔导度和蒸腾速率明显提高,胞间CO₂浓度显著降低;同时增强核酮糖-1,5-二磷酸羧化酶、Rubisco活化酶、景天庚酮糖-1,7-二磷酸酯酶和果糖-1,6-二磷酸醛缩酶活性及其mRNA表达,促进光合碳同化;提高光下PSⅡ实际光化学效率和暗下PSⅡ最大光化学效率,从而减轻低温弱光胁迫对黄瓜幼苗的光合机构的损伤和生长量的影响。H₂S清除剂次牛磺酸(HT)可使SA对低温弱光下黄瓜幼苗的光合作用和生长促进效应明显减弱,而SA抑制剂多效唑和氨基茚磷酸对H₂S诱导的黄瓜幼苗光合机构对低温弱光的耐受性无显著影响,说明H₂S作为SA的下游信号,参与调控低温弱光下黄瓜幼苗的光合作用。