•An iTRAQ-based proteome-wide study was used to investigate the HRW response.•Shotgun MS/MS revealed proteins response to HRW under Cd stress in alfalfa roots.•248 Proteins showed a statistical significance among the 2377 proteins identified.•7 Main biological processes were detected associated with HRW-induced Cd tolerance.•Changes in lipid peroxide, NPTs contents, and ion homeostasis support our results.Recently, molecular hydrogen (H2) has emerged as a bio-regulator both in animals and plants. Normally, functions of endogenous generated H2 could be mimicked by exogenously applied hydrogen-rich water (HRW) or hydrogen-rich saline (particularly in animals). Although alfalfa seedlings showed more cadmium (Cd) resistance after the administration with HRW, corresponding molecular mechanism is still elusive. To address this gap, iTRAQ-based quantitative proteomics was used. The results showed that a total of 2377 proteins were identified with < 1% FDR, and 1254 protein abundance perturbations were confidently assessed. Total of 248 significant differential proteins were identified in Cd- and/or HRW-treated samples. Furthermore, 92 proteins from the 248 proteins were selected for further bioinformatics analysis. Interestingly, results indicated that they were classified into seven categories: defense and response to stress, sulfur compound metabolic process, amino acid and protein metabolic process, carbohydrate and energy metabolic process, secondary metabolic process, oxidation-reduction process, and metal ion homeostasis. In addition, the protein expression patterns were consistent with the results of decreased lipid peroxidation, increased non-protein thiols abundance, as well as iron and zinc content. These suggest that HRW alleviates Cd toxicity mainly by decreasing oxidative damage, enhancing sulfur compound metabolic process, and maintaining nutrient element homeostasis.Biological significanceContamination of soils by Cd has become a potential concern to crops. Medicago sativa is a widely used forage around the world. Recently, hydrogen gas (H2) was suggested as a candidate of signal molecule, and found to effectively attenuate Cd-induced damage in alfalfa seedlings. However, the underlying molecular mechanism still needs to be further elucidated. In the present work, an iTRAQ-based quantitative proteomics was firstly carried out, and the results revealed the main molecular targets and metabolic processes associated with Cd resistance conferred by H2. This study may expand our understanding of hydrogen gas-medicated heavy metal tolerance in plants.

Recent results discovered the protective roles of methane (CH4) against oxidative stress in animals. However, the possible physiological roles of CH4 in plants are still unknown. By using physiological, histochemical and molecular approaches, the beneficial role of CH4 in germinating alfalfa seeds upon copper (Cu) stress was evaluated. Endogenous production of CH4 was significantly increased in Cu-stressed alfalfa seeds, which was mimicked by 0.39 mM CH4. The pretreatment with CH4 significantly alleviated the inhibition of seed germination and seedling growth induced by Cu stress. Cu accumulation was obviously blocked as well. Meanwhile, α/β amylase activities and sugar contents were increased, all of which were consistent with the alleviation of seed germination inhibition triggered by CH4. The Cu-triggered oxidative stress was also mitigated, which was confirmed by the decrease of lipid peroxidation and reduction of Cu-induced loss of plasma membrane integrity in CH4-pretreated alfalfa seedlings. The results of antioxidant enzymes, including ascorbate peroxidase (APX), superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (POD) total or isozymatic activities, and corresponding transcripts (APX1/2, Cu/Zn SOD and Mn-SOD), indicated that CH4 reestablished cellular redox homeostasis. Further, Cu-induced proline accumulation was partly impaired by CH4, which was supported by the alternation of proline metabolism. Together, these results indicated that CH4 performs an advantageous effect on the alleviation of seed germination inhibition caused by Cu stress, and reestablishment of redox homeostasis mainly via increasing antioxidant defence.

Methane (CH4) is emerging as a candidate of signal molecule recently. However, whether or how CH4 enhances plant adaptation to aluminum (Al)-contaminated environment is still unknown. In this report, the physiological roles and possible molecular mechanisms of CH4 in the modulation of Al toxicity in alfalfa seedlings were characterized. Our results showed that, CH4 pretreatment could alleviate Al-induced seedling growth inhibition and redox imbalance. The defensive effects of CH4 against Al toxicity including the remission of Al-induced root elongation inhibition, nutrient disorder, and relative electrolyte leakage. Moreover, contents of organic acids, including citrate, malate, and oxalate, were increased by CH4. These results were paralleled by the findings of CH4 regulated organic acids metabolism and transport genes, citrate synthase, malate dehydrogenase, aluminum-activated malate transporter, and aluminum activated citrate transporter. Consistently, Al accumulation in seedling roots was decreased after CH4 treatment. In addition, Al-induced oxidative stress was also alleviated by CH4, through the regulation of the activities of anti-oxidative enzymes, such as ascorbate peroxidase, superoxide dismutase, and peroxidase, as well as their corresponding transcripts. Our data clearly suggested that CH4 alleviates Al toxicity by reducing Al accumulation in organic acid-dependent fashion, and reestablishing redox homeostasis.

•Cd induced melatonin production in alfalfa and Arabidopsis seedling roots.•Transgenic Arabidopsis lines overexpressing alfalfa SNAT (MsSNAT) exhibited high-level melatonin and enhanced Cd tolerance.•Cd accumulation in plants was decreased via melatonin-modulated heavy metal transporters.•Melatonin-reestablished redox homeostasis via miR398.Although melatonin-alleviated cadmium (Cd) toxicity both in animals and plants have been well studied, little is known about its regulatory mechanisms in plants. Here, we discovered that Cd stress stimulated the production of endogenous melatonin in alfalfa seedling root tissues. The pretreatment with exogenous melatonin not only increased melatonin content, but also alleviated Cd-induced seedling growth inhibition. The melatonin-rich transgenic Arabidopsis plants overexpressing alfalfa SNAT (a melatonin synthetic gene) exhibited more tolerance than wild-type plants under Cd conditions. Cd content was also reduced in root tissues. In comparison with Cd stress alone, ABC transporter and PCR2 transcripts in alfalfa seedlings, PDR8 and HMA4 in Arabidopsis, were up-regulated by melatonin. By contrast, Nramp6 transcripts were down-regulated. Changes in above transporters were correlated with the less accumulation of Cd. Additionally Cd-triggered redox imbalance was improved by melatonin. These could be supported by the changes of the Cu/Zn Superoxide Dismutase gene regulated by miR398a and miR398b. Histochemical staining, laser scanning confocal microscope, and H2O2 contents analyses showed the similar tendencies. Taking together, we clearly suggested that melatonin enhanced Cd tolerance via decreasing cadmium accumulation and reestablishing the microRNAs-mediated redox homeostasis.

There are limited data concerning the role of endogenous H2S in prolonging the postharvest of vegetables and fruits. Using a fluorescence microscope with a specific probe, we discovered that, during the senescence of postharvest daylily flower, endogenous H2S homeostasis was impaired. The activities of two important synthetic enzymes of H2S, l- and d-cysteine desulfhydrase, exhibited decreasing tendencies. However, NaHS (a H2S donor) not only blocked the decreased H2S production but also extended the postharvest life of daylilies. These beneficial roles were verified by the alleviation of lipid peroxidation and the increased activities of antioxidant enzymes. Meanwhile, the energy status was sustained, and the respiration rate was decreased. In contrast to NaHS, the addition of an inhibitor of H2S synthesis alone aggravated lipid peroxidation and lowered energy charge. Together, the present study implies that endogenous H2S alleviates senescence of postharvest daylilies via increasing antioxidant capacity and maintained energy status.Keywords: antioxidant capacity; daylily; endogenous hydrogen sulfide homeostasis; energy status; senescence;

The role of hydrogen gas (H2) in modulating cold tolerance was investigated.A combination of physiological and molecular approaches was used to study the effect of H2 on the alleviation of cold stress.Cold stress stimulated the production of H2 in rice seedlings. The pretreatment with exogenous H2 not only mimicked above physiological response, but also prevented cold-induced growth inhibition. With 0.39 mM H2 pretreatment, rice seedlings exhibited the maximum improving responses, including alleviating the decrease of chlorophyll contents and photosynthetic activity. The lipid peroxidation and the overproduction of reactive oxygen species (ROS) were decreased as well. These results were supported by the changes of total and isozymatic activities of antioxidant enzymes. In comparison with cold stress alone, miR398 transcripts were down-regulated by H2, and expression levels of its targets Cu/Zn-SOD1 (CSD1) and Cu/Zn-SOD2 (CSD2) were increased. By contrast, miR319 transcripts were differentially increased, showing a relatively negative correlation with its target genes PROLIFERATING CELL FACTOR5 (PCF5) and PROLIFERATING CELL FACTOR8 (PCF8).Our results suggested that endogenous H2 might contribute to the enhancement of cold tolerance, at least partially, by the reestablishment of redox homeostasis via miR398 and miR319.

The Arabidopsis heme oxygenase 1 (HY1) plays a significant role in the signal transduction of abiotic stimuli and hormonal response. To characterize the HY1 promoter, an approximately 1.8 kb of it (pHY1, -1666 to +132) and its deletion fragments (5D1, -1528 to +132; 5D2, -1109 to +132; 5D3, -688 to +132; 5D4, -169 to +132; 3D1, -1666 to +100; 3D2, -1666 to -1; and 3D3, -1666 to -170), were fused to the β-glucuronidase (GUS) reporter gene and transformed into Arabidopsis. The transgenic plants were subjected to several environmental stimuli (especially to mild salinity, iron deficiency, and mercury exposure). The results show that the region from +1 to +100 in the 5’-untranslated region were essential for HY1 basal promoter activity. The induced GUS activities under NaCl and H2O2 treatments were slowed down by the progressive 5’ deletion (from -1666 to -688) and correlated with the reduced numbers of myeloblastosis (MYB) binding sites (MBSs; -1542, -1333, -1078, and -177). The MBS-free promoter construct 5D4 (-169 to +132), however, fully lost the inducibility. Therefore, we propose that the MBS elements existing in the HY1 promoter might be crucial for salinity-induced HY1 up-regulation in an H2O2-dependent fashion. Moreover, the regions from -169 to -1 and -688 to -169 were presumed as the regulatory regions of HY1 promoter in response to iron deficiency and mercury exposure, respectively.

•H2 reestablished GA/ABA balance in germinating rice seeds under Al stress.•H2-promoted germination was sensitive to a GA biosynthesis inhibitor.•H2 could modulate miRNA expression.•Citrate efflux and miRNA-mediated antioxidant defense were modulated by H2.Although previous results showed that exogenous hydrogen (H2) alleviated aluminum (Al) toxicity, the detailed mechanism remains unclear. Here, we reported that the exposure of germinating rice seeds to Al triggered H2 production, followed by a decrease of GA/ABA ratio and seed germination inhibition. Compared to inert gas (argon), H2 pretreatment not only strengthened H2 production and alleviated Al-induced germination inhibition, but also partially reestablished the balance between GA and ABA. By contrast, a GA biosynthesis inhibitor paclobutrazol (PAC) could block the H2-alleviated germination inhibition. The expression of GA biosynthesis genes (GA20ox1 and GA20ox2) and ABA catabolism genes (ABA8ox1 and ABA8ox2), was also induced by H2. Above results indicated that GA/ABA might be partially involved in H2 responses. Subsequent results revealed that compared with Al alone, transcripts of miR398a and miR159a were decreased by H2, and expression levels of their target genes OsSOD2 and OsGAMYB were up-regulated. Whereas, miR528 and miR160a transcripts were increased differentially, and contrasting tendencies were observed in the changes of their target genes (OsAO and OsARF10). The transcripts of Al-tolerant gene OsSTAR1/OsSTAR2 and OsFRDL4 were up-regulated. Above results were consistent with the anti-oxidant defense, decreased Al accumulation, and enhanced citrate efflux. Together, our results provided insight into the mechanism underlying H2-triggered Al tolerance in plants.Download high-res image (150KB)Download full-size image

•Salt hypersensitivity of atsnat mutant is rescued by melatonin and H2O2.•Melatonin-mediated salt tolerance is sensitive to DPI, an inhibitor of NADPH oxidase.•AtrbohF is required for melatonin-induced salinity tolerance.•Ion homeostasis and redox homeostasis are reestablished by melatonin via AtrbohF.Although several literatures confirmed the beneficial roles of exogenous melatonin in the enhancement of salinity tolerance in plants, whether or how endogenous melatonin confers plant salinity tolerance is still elusive. In the report, we observed impaired melatonin level and salinity hypersensitivity in atsnat, the Arabidopsis melatonin synthesis mutant. Above hypersensitivity was rescued by melatonin or hydrogen peroxide. Meanwhile, melatonin-mediated salt tolerance in wild-type was abolished by an NADPH oxidase inhibitor, suggesting the possible role of NADPH oxidase-dependent reactive oxygen species (ROS). Genetic evidence further showed that the rapid stimulated RbohF transcripts and production of ROS elicited by melatonin in stressed wild-type plants were largely abolished by the mutation of AtrbohF. Meanwhile, salinity sensitivity of atrbohF mutant was not altered by melatonin, which was consistent with the higher Na+ content and the resulting greater Na+/K+ ratio, compared with those in wild-type plants. Further changes of SOS1, SOS2, and SOS3 transcripts suggested that the melatonin-triggered SOS-mediated Na+ efflux might be mediated by AtrbohF-dependent ROS. The addition of melatonin could intensify the increased antioxidant defence in stressed wild-type but not in atrbohF mutant, both of which were confirmed by the histochemical staining for ROS production and lipid peroxidation during the later period of stress. Collectively, our genetic and molecular evidence revealed that the AtrbohF-dependent ROS signaling is required for melatonin-induced salinity tolerance via the reestablishment of ion and redox homeostasis.Download high-res image (234KB)Download full-size image

β-Cyclodextrin–hemin (β-CDH) is a complex combining hemin with β-cyclodextrin (β-CD), which could improve hemin solubility. Our previous results showed that β-CDH, was able to enhance alfalfa tolerance against cadmium stress. However, whether or how β-CDH influences salinity tolerance is still elusive. In this report, we observed that similar to the beneficial responses of hemin rather than β-CD, the addition of β-CDH not only alleviated salinity-induced seedling growth inhibition (in particular), but also arrested chlorophyll degradation in tobacco seedlings. The efficiency of β-CDH against salinity stress compared to that of hemin, was confirmed, since the maximum beneficial responses against NaCl stress was obtained with 0.1 μM β-CDH and 10 μM hemin, respectively. Subsequent work showed that the redox imbalance caused by salinity stress could be improved by β-CDH. This was suggested by the reduced lipid peroxidation and hydrogen peroxide accumulation, as well as the induction of representative antioxidant genes, encoding superoxide dismutase, guaiacol peroxidase, and ascorbate peroxidase. Meanwhile, compared to control conditions, the ratio of K+ to Na+ was relatively low in NaCl-stressed tobacco seedlings. By contrast, the administration of β-CDH not only significantly blocked the increase of Na+, but also obviously increased K+, thus resulting in a high K+ to Na+ ratio in both shoot and root parts. Ion homeostasis is therefore reestablished. Together, our results suggested that β-CDH was able to improve salinity tolerance via the reestablishment of redox and ion homeostasis.

Hydrogen gas (H2) was recently suggested to perform multiple roles during plant response to abiotic stresses. However, physiological mechanisms and downstream targets remain elusive. Here, we showed that osmotic stress triggered by polyethylene glycol (PEG) could enhance hydrogenase-like activity and H2 production in alfalfa seedlings. After the administration of H2, the enhanced tolerance to PEG stress was confirmed, as evidence by the observation of the alleviation of root inhibition and the decrease in lipid peroxidation. H2 also rapidly elevated hydrogen peroxide (H2O2) levels, peaking at 5 h. When supplemented with exogenous H2O2, a heme oxygenase-1 (HO-1; a novel antioxidant enzyme) inducer hemin, and one of HO-1 catalytic products, carbon monoxide (CO), antioxidant enzymes and subsequent PEG tolerance were induced. The inhibition of NADPH oxidase and chemical scavenging of H2O2 could block H2-induced HO-1 expression and PEG tolerance. Simultaneously, a HO-1 inhibitor counteracted the effects of H2 except H2-induced H2O2 production. In summary, these results suggested that H2O2 might play a significant role in HO-1-dependent induction of plant osmotic stress tolerance triggered by H2.

Methane-rich water triggered adventitious rooting by regulating heme oxygenase1/carbon monoxide and calcium pathways in cucumber explants.Heme oxygenase1/carbon monoxide (HO1/CO) and calcium (Ca2+) were reported as the downstream signals in auxin-induced cucumber adventitious root (AR) formation. Here, we observed that application of methane-rich water (MRW; 80 % saturation) obviously induced AR formation in IAA-depleted cucumber explants. To address the universality, we checked adventitious rooting in soybean and mung bean explants, and found that MRW (50 and 10 % saturation, respectively) exhibited the similar inducing results. To further determine if the HO1/CO system participated in MRW-induced adventitious rooting, MRW, HO1 inducer hemin, its activity inhibitor zinc protoporphyrin IX (ZnPP), and its catalytic by-products CO, bilirubin, and Fe2+ were used to detect their effects on cucumber adventitious rooting in IAA-depleted explants. Subsequent results showed that MRW-induced adventitious rooting was blocked by ZnPP and further reversed by 20 % saturation CO aqueous solution. However, the other two by-products of HO1, bilirubin and Fe2+, failed to induce AR formation. Above responses were consistent with the MRW-induced increases of HO1 transcript and corresponding protein level. Further molecular evidence indicted that expression of marker genes, including auxin signaling-related genes and cell cycle regulatory genes, were modulated by MRW alone but blocked by the cotreatment with ZnPP, the latter of which could be significantly rescued by the addition of CO. By using the Ca2+-channel blocker and Ca2+ chelator, the involvement of Ca2+ pathway in MRW-induced adventitious rooting was also suggested. Together, our results indicate that MRW might serve as a stimulator of adventitious rooting, which was partially mediated by HO1/CO and Ca2+ pathways.

Heme oxygenase (HO; EC 1.14.99.3) is an important enzyme that yields biliverdin IXα (BV), carbon monoxide and iron. At least two kinds of HO subfamilies exist in plants. Our previous report revealed that rice (Oryza sativa L.) HO1 (SE5, also named as OsHO1), a major subfamily of HO, is an oxidative stress–response protein, especially upon paraquat exposure. However, whether there exists rice HO2, another subfamily of HO, is still unknown. In the present study, a rice HO2 gene (named as OsHO2) was cloned and characterized. Its genomic sequence consists of four exons and three introns, and encodes a 36.5 kDa protein precursor with a 4.9 kDa N-terminal transit leader peptide. Further results showed that OsHO2 has a conserved HO signature sequence and shares a high amino acid sequence similarity with other identified plant HO2s. The recombinant mature OsHO2 (mOsHO2) protein expressed in Escherichia coli did not exhibit HO activity, which was in contrast with that of mOsHO1. The results of subcellular localization of OsHO2 demonstrated that it was most likely localized in the chloroplasts. Real-time RT-PCR experiment revealed that although OsHO2 mRNA is a much less abundant than that of OsHO1, both of them were expressed in all tested tissues. Importantly, OsHO2 transcripts could be differentially induced by hemin (a substrate of HO), paraquat (in particular), and NaCl treatments. Together, the results suggested that OsHO2 might act downstream in the signal transduction pathways following abiotic stresses in rice.

Haem oxygenase-1 (HO-1) and hydrogen peroxide (H2O2) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and H2O2 in auxin-mediated lateral root (LR) formation is poorly understood. Herein, we revealed that exogenous auxin, 1-naphthylacetic acid (NAA), could simultaneously stimulate Arabidopsis HO-1 (HY1) gene expression and H2O2 generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on H2O2. This conclusion was supported by analyzing the removal of H2O2 with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. H2O2-induced LR formation was inhibited by an HO-1 inhibitor zinc protoporphyrin IX (Znpp) in wild-type and severely impaired in HY1 mutant hy1-100. Simultaneously, HY1 is required for NAA-mediated H2O2 generation, since Znpp inhibition of HY1 blocked the NAA-induced H2O2 production and LR formation. Genetic data demonstrated that hy1-100 was significantly impaired in H2O2 production and LR formation in response to NAA, compared with wild-type plants. The addition of carbon monoxide-releasing molecule-2 (CORM-2), the carbon monoxide (CO) donor, induced H2O2 production and LR formation, both of which were decreased by DMTU. Moreover, H2O2 and CORM-2 mimicked the NAA responses in the regulation of cell cycle genes expression, all of which were blocked by Znpp or DMTU, respectively, confirming that both H2O2 and CO were important in the early LR initiation. In summary, our pharmacological, genetic and molecular evidence demonstrated a close inter-relationship between HY1 and H2O2 existing in auxin-induced LR formation in Arabidopsis.

Here, α-Amy2/54 gene expression was used as a molecular probe to investigate the interrelationship among nitric oxide (NO), cyclic GMP (cGMP), and heme oxygenase-1 (HO-1) in GA-treated wheat aleurone layers. The inducible expressions of α-Amy2/54 and α-amylase activity were respectively amplified by two NO-releasing compounds, sodium nitroprusside (SNP) and spermine NONOate, in a GA-dependent fashion. Similar responses were observed when an inducer of HO-1, hemin—or one of its catalytic products, carbon monoxide (CO) in aqueous solution—was respectively added. The SNP-induced responses, mimicked by 8-bromoguanosine 3′,5′-cyclic monophosphate (8-Br-cGMP), a cGMP derivative, were NO-dependent. This conclusion was supported by the fact that endogenous NO overproduction was rapidly induced by SNP, and thereafter induction of α-Amy2/54 gene expression and increased α-amylase activity were sensitive to the NO scavenger. We further observed that the above induction triggered by SNP and 8-Br-cGMP was partially prevented by zinc protoporphyrin IX (ZnPPIX), an inhibitor of HO-1. These blocking effects were clearly reversed by CO, confirming that the above response was HO-1-specific. Further analyses showed that both SNP and 8-Br-cGMP rapidly up-regulated HO-1 gene expression and increased HO activity, and SNP responses were sensitive to cPTIO and the guanylate cyclase inhibitor 6-anilino-5,8-quinolinedione (LY83583). Molecular evidence confirmed that GA-induced GAMYB and ABA-triggered PKABA1 transcripts were up-regulated or down-regulated by SNP, 8-Br-cGMP or CO cotreated with GA. Contrasting changes were observed when cPTIO, LY83583, or ZnPPIX was added. Together, our results suggested that HO-1 is involved in NO- and cGMP-induced α-Amy2/54 gene expression in GA-treated aleurone layers.

A reliable result obtained by qRT-PCR highly depends on accurate transcript normalization using stably expressed reference genes. However, the transcript levels of traditional reference genes are not always stable. Also, the inaccurate normalization could easily lead to the false conclusions. In this report, by using geNorm and NormFinder algorithms, 12 candidate reference genes were evaluated in Arabidopsis under iron deficiency. Our results revealed that three novel reference genes (SAND, YLS8 and TIP41-like) were identified and validated as suitable reference genes for qRT-PCR normalization in both iron deprivation (the addition of Ferrozine to the medium) and starvation (withdrawal of iron from the medium) conditions. This conclusion was also confirmed by publicly available microarray data. In addition, when using SAND, YLS8 and TIP41-like as multiple reference genes, the expression patterns of FIT1 and IRT1, two iron deficiency marker genes, were approximately similar with that reported previously. However, a weaker inducible response was obtained from qRT-PCR by normalizating EF-1α alone. Together, we proposed that the combination of SAND, YLS8 and TIP41-like can be used for accurate normalization of gene expression in iron deficiency research. These results provide a valuable evidence for the importance of adequate reference genes in qRT-PCR normalization, insisting on the use of appropriate reference gene validation in all transcriptional analyses.

l-Cysteine desulfhydrase (DES; EC 4.4.1.1) is the most important enzyme that catalyzes the decomposition of l-cysteine to pyruvate, ammonia, and hydrogen sulfide (H2S), the latter of which has recently been recognized as the third gasotransmitter for multiple signaling events in plants. Previous results showed the existence of DES activity in Brassica napus; however, the gene encoding the true DES protein has not been characterized yet. Here, a rapeseed DES gene was isolated and sequenced. It shared high homology with Arabidopsis DES1, and encodes a polypeptide with 323 amino acids of 34.5 kDa. Subsequently, prokaryotic expression and biochemical analysis demonstrated that this protein predominantly catalyzes the breakdown of l-cysteine with the side reaction of l-cysteine synthesis [O-acetyl-l-serine(thiol)lyase activity], and was designated as BnDES1. Corresponding analysis of structural features was also in agreement with the above proposition. Molecular evidence showed that BnDES1 mRNA was widely expressed, but with the higher expression level in flowers. Further results showed that the BnDES1 transcripts were differentially up-regulated by several plant growth regulators and chemicals. Overall, the above findings provide evidence showing that BnDES1 is a potentially important enzyme responsible for the H2S production, and may play an important role in plant growth regulators and chemical stimuli responses.

This study investigated the molecular mechanism of hydrogen-rich water (HRW)-mediated enhancement of tolerance against salinity stress during rice seed germination.A combination of physiological and molecular approaches was used to study the effect of HRW on the alleviation of salinity stress.A 100-mM NaCl stress caused the increase of H2 release in germinating rice seeds. With respect to samples treated with 100 mM NaCl alone, exogenous HRW pretreatments differentially attenuated the inhibition of seed germination and seedling growth caused by salinity. Further results showed that both 50 % (in particular) and 100 % concentration of HRW could activate α/β-amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar. HRW also enhanced total, isozymatic activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase, catalase, and ascorbate peroxidase. These results were confirmed by the alleviation of oxidative damage, as indicated by a decrease of thiobarbituric acid reactive substances. Additionally, the ratio of potassium (K) to sodium (Na) in both the shoot and root parts was increased.Together, our results suggested that exogenous HRW treatment on rice seeds may be a good option to alleviate salinity stress.

Our previous results showed that β-cyclodextrin–hemin complex (CDH) exhibited a vital protective role against cadmium-induced oxidative damage and toxicity in alfalfa seedling roots by the regulation of heme oxygenase-1 (HO-1) gene expression. In this report, we further test whether CDH exhibited the hormonal-like response. The application of CDH and an inducer of HO-1, hemin, were able to induce the up-regulation of cucumber HO-1 gene (CsHO1) expression and thereafter the promotion of adventitious rooting in cucumber explants. The effect is specific for HO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPP) blocked the above responses triggered by CDH, and the inhibitory effects were reversed further when 30 % saturation of CO aqueous solution was added together. Further, molecular evidence showed that CDH triggered the increases of the HO-1-mediated target genes responsible for adventitious rooting, including one DnaJ-like gene (CsDNAJ-1) and two calcium-dependent protein kinase (CDPK) genes (CsCDPK1 and CsCDPK5), and were inhibited by ZnPP and reversed by CO. The calcium (Ca2+) chelator ethylene glycol-bis (2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) and the Ca2+ channel blocker lanthanum chloride (LaCl3) not only compromised the induction of adventitious rooting induced by CDH but also decreased the transcripts of above three target genes. However, the application of ascorbic acid (AsA), a well-known antioxidant in plants, failed to exhibit similar inducible effect on adventitious root formation. In short, above results illustrated that the response of CDH in the induction of cucumber adventitious rooting might be through HO-1-dependent mechanism and calcium signaling.Key message Physiological, pharmacological and molecular evidence showed that β-cyclodextrin–hemin complex (CDH) was able to induce cucumber adventitious rooting through heme oxygenase-1 (HO-1)-dependent mechanism and calcium signaling.

Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide (CO), and free iron (Fe2+)—and Arabidopsis and rice (Oryza sativa) HOs are involved in light signaling. Here, we identified that the rice PHOTOPERIOD SENSITIVITY 5 (SE5) gene, which encoded a putative HO with high similarity to HO-1 from Arabidopsis (HY1), exhibited HO activity, and localized in the chloroplasts. Rice RNAi mutants silenced for SE5 were generated and displayed early flowering under long-day conditions, consistent with phenotypes of the null mutation in SE5 gene reported previously (se5 and s73). The herbicide methyl viologen (MV), which produces reactive oxygen species (ROS), was applied to determine whether SE5 regulates oxidative stress response. Compared with wild-type, SE5 RNAi transgenic plants aggravated seedling growth inhibition, chlorophyll loss and ROS overproduction, and decreased the transcripts of some representative antioxidative genes. By contrast, administration of exogenous CO partially rescued corresponding MV hypersensitivity in the SE5 RNAi plants. Alleviation of seed germination inhibition, chlorophyll loss and ROS overproduction, as well as the induction of antioxidant defense were further observed when SE5 or HY1 was overexpressed in transgenic Arabidopsis plants, indicating that SE5 may be useful for molecular breeding designed to improve plant tolerance to oxidative stress.

Despite hydrogen sulfide (H2S) and nitric oxide (NO) are important endogenous signals or bioregulators involved in many vital aspects of plant growth and responses against abiotic stresses, little information was known about their interaction. In the present study, we evaluated the effects of H2S and NO on alfalfa (Medicago sativa L.) plants exposed to cadmium (Cd) stress. Pretreatment with an H2S donor sodium hydrosulfide (NaHS) and well-known NO donor sodium nitroprusside (SNP) decreased the Cd toxicity. This conclusion was supported by the decreases of lipid peroxidation as well as the amelioration of seedling growth inhibition and Cd accumulation, in comparison with the Cd-stressed alone plants. Total activities and corresponding transcripts of antioxidant enzymes, including superoxide dismutase, peroxidase and ascorbate peroxidase were modulated differentially, thus leading to the alleviation of oxidative damage. Effects of H2S above were reversed by 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO), the specific scavenger of NO. By using laser confocal scanning microscope combined with Greiss reagent method, further results showed that NO production increased significantly after the NaHS pretreatment regardless of whether Cd was applied or not, all of which were obviously inhibited by cPTIO. These decreases of NO production were consistent with the exaggerated syndromes associated with Cd toxicity. Together, above results suggested that NO was involved in the NaHS-induced alleviation of Cd toxicity in alfalfa seedlings, and also indicated that there exists a cross-talk between H2S and NO responsible for the increased abiotic stress tolerance.

Heme oxygenase-1 (HO1) is a heme-catabolizing enzyme induced by a variety of stress conditions. This article described the cloning and characterization of BrHO1 gene which codes for a putative HO1 from Chinese cabbage (Brassica rapa subsp. pekinensis). BrHO1 consists of three exons and encodes a protein precursor of 32.3 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of BrHO1 was 84% similar to Arabidopsis counterpart HY1. The three-dimensional structure of BrHO1 showed a high degree of structural conservation compared with the known HO1 crystal structures. Phylogenetic analysis revealed that BrHO1 clearly grouped with the HO1-like sequences. The recombinant BrHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXα (BV). Furthermore, the results of subcellular localization of BrHO1 demonstrated that BrHO1 gene product was most likely localized in the chloroplasts. BrHO1 was differently expressed in all tested tissues and could be induced upon osmotic and salinity stresses, cadmium (Cd) exposure, hydrogen peroxide (H2O2), and hemin treatments. Together, the results suggested that BrHO1 plays an important role in abiotic stress responses.

The molecular mechanisms and signal transduction pathways of hydrogen sulfide (H2S) in plant biology are still unclear. Here, by using pharmacological and biochemical approaches, we report that H2S promotes germination and alleviates salinity damage involving nitric oxide (NO) pathway.Upon 100 mM NaCl treatment, both H2S donor sodium hydrosulfide (NaHS) and NO donor sodium nitroprusside (SNP) at 100 μM could significantly attenuate the inhibition of alfalfa (Medicago sativa) seed germination and thereafter seedling growth inhibition. Meanwhile, the ratio of potassium (K) to sodium (Na) in the root parts was increased. Total, isozymatic activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), or ascorbate peroxidase (APX) were activated differentially, thus resulting in the alleviation of oxidative damage. The above protective roles of NaHS might be related to the induction of endogenous NO, because the addition of the specific scavenger of NO 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt (cPTIO) reversed above effects. Meanwhile, NaHS-triggered NO production was confirmed.Our observations indicate that H2S enhances plant responses against salinity stress by reducing oxidative damage, which might have a possible interaction with NO.

Results from our previous study suggested that haem oxygenase-1/carbon monoxide (HO-1/CO) acts as a downstream signal system in the auxin-induced pathway leading to cucumber (Cucumis sativus) adventitious root formation. The objective of this study was to test whether HO-1 is also involved in hydrogen sulfide (H2S)-induced adventitious root formation. Cucumber explants were treated with HO-1 inducer haemin and H2S donor sodium hydrosulfide (NaHS) in combination with the specific inhibitor of HO-1 zinc protoporphyrin IX (ZnPPIX), and their effects on cucumber adventitious root development in IAA-depleted explants were compared. The results showed that similar to inducible responses of haemin, NaHS brought about the induction of cucumber HO-1 transcripts (CsHO-1) and its protein levels, and thereafter adventitious root formation. A further experiment verified that H2S or HS- rather than other sulfur-containing components derived from NaHS was ascribed to the stimulation response. The inducible effect is specific for CsHO-1 because ZnPPIX significantly suppressed the above responses, and the inhibitory effects were reversed partially when 30% CO-saturated aqueous solution was added. Molecular evidence further suggested that the NaHS-triggered upregulation of target genes responsible for HO-1/CO-induced adventitious root formation, including CsDNAJ-1 and CsCDPK1/5, was inhibited significantly by ZnPPIX. These decreases were reversed obviously by the addition of CO aqueous solution. However, hypotaurine (HT), the H2S scavenger, could not influence the haemin- and CO-induced adventitious rooting in IAA-depleted cucumber explants. Together, the above results suggested that HO-1 was involved in H2S-induced cucumber adventitious root formation.

The aim of this study was to investigate whether presoaking with hemin, an inducer of heme oxygenase-1 (HO-1), could alleviate salinity damage during wheat seed germination in comparison with the pretreatment of a well-known nitric oxide (NO) donor sodium nitroprusside (SNP). The results showed that, compared with the samples upon 150 mM NaCl salt stress alone, both 10 μM hemin and 200 μM SNP pretreatments could (1) significantly attenuate the inhibition of seed germination and thereafter seedling growth; (2) induce HO expression; (3) enhance amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar; and (4) increase the potassium (K) to sodium (Na) ratio, particularly in the shoot parts. Hemin and SNP could also increase antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), guaiacol peroxidase (POD), and ascorbate peroxidase (APX), thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Moreover, semi-quantitative RT-PCR and isozymatic analysis illustrated that hemin and SNP pretreatment were able to up-regulate the expression of Mn-SOD (especially) and Cu/Zn-SOD gene, and activate SOD isozymatic activities. Since the addition of the NO scavenger methylene blue (MB) differentially reversed the above effects, the protective roles of hemin might be related to the induction of endogenous NO signal. Meanwhile, hemin-driven NO production was confirmed. Together, these results indicated that hemin exerted an advantageous effect on enhancing salinity tolerance during wheat seed germination, which might interact with NO.

Following previous findings that cadmium (Cd) induces heme oxygenase-1 (HO1) gene expression in alfalfa seedling roots, we now show that the decreased glutathione (GSH) and ascorbic acid (AsA) contents, induction of HO-1 gene expression and its protein level by Cd was mimicked by a GSH depletor diethylmaleate (DEM). Meanwhile, above Cd- or DEM-induced decreased GSH content followed by HO-1 up-regulation could be strengthened or reversed differentially by the application of a selective inhibitor of GSH biosynthesis l-buthionine-sulfoximine (BSO), or exogenous GSH and AsA, respectively. The antioxidative behavior of HO-1 induction was further confirmed by histochemical staining for the detection of loss of membrane integrity in a short period of treatment time. Additionally, the induction of HO-1 transcript was inhibited by the transcriptional inhibitor actinomycin D (ActD) or protein synthesis inhibitor cycloheximide (CX, especially). In contrast, the level of HO-2 transcript did not change upon various treatments. Together, above results suggested that Cd-induced up-regulation of HO-1 gene expression is associated with GSH depletion, which is at least existing transcriptional regulation level, thus leading to enhanced antioxidative capability transiently.

In animals, heme oxygenase (HO), a rate-limiting enzyme responsible for carbon monoxide (CO) production, was regarded as a protective system maintaining cellular homeostasis. It was also established that metal ions are powerful HO-inducing agents and cobalt chloride (CoCl2) was the first metal ion identified with an inducing property. Previous study suggests that CoCl2 stimulates adventitious root formation in tomato and cucumber cuttings. In this test, we discover that both CoCl2 and an inducer of HO-1, hemin, could lead to the promotion of lateral root development, as well as the induction of HO-1 protein expression, HO activity, or LeHO-1/2 transcripts, in lateral root initiation zone of tomato seedlings. The effect is specific for HO since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) blocked the above actions of CoCl2, and the inhibitory effect was reversed partially when 50% CO aqueous solution was added. However, the addition of ascorbic acid (AsA), a well-known antioxidant, exhibited no obvious effect on lateral root formation. Molecular evidence further showed that CoCl2-induced the up-regulation of target genes responsible for lateral root formation, including LeCDKA1, LeCYCA2;1, and LeCYCA3;1, was suppressed differentially by ZnPPIX. And these decreases were reversed further by the addition of CO. All together, these results suggest a novel role for HO in the CoCl2-induced tomato lateral root formation.

Heme oxygenase (HO, EC 1.14.99.3) catalyzes the oxidation of heme and performs vital roles in plant development and stress responses. Two HO isozymes exist in plants. Between these, HO-1 is an oxidative stress-response protein, and HO-2 usually exhibited constitutive expression. Although alfalfa HO-1 gene (MsHO1) has been investigated previously, HO2 is still poorly understood. In this study, we report the cloning and characterization of HO2 gene, MsHO2, from alfalfa (Medica sativa L.). The full-length cDNA of MsHO2 contains an ORF of 870 bp and encodes for 290 amino acid residues with a predicted molecular mass of 33.3 kDa. Similar to MsHO1, MsHO2 also appears to have an N-terminal transit peptide sequence for chloroplast import. Many conserved residues in plant HO were also conserved in MsHO2. However, unlike HO-1, the conserved histidine (His) required for heme-iron binding and HO activity was replaced by tyrosine (Tyr) in MsHO2. Further biochemical activity analysis of purified mature MsHO2 showed no HO activity, suggesting that MsHO2 may not be a true HO in nature. Semi-quantitative RT-PCR confirmed its maximum expression in the germinating seeds. Importantly, the expression levels of MsHO2 were up-regulated under sodium nitroprusside (SNP) and H2O2 (especially) treatment, respectively.

Recent studies suggest that carbon monoxide (CO), which is mainly produced by heme oxygenase (HO EC 1.14.99.3), may function as a physiological messenger or bioactive molecule by interacting with nitric oxide (NO) in animal cells. In this study, we report that application of the hematin and hemin, two heme molecules cleaved by HO to yield CO in animals, dose-dependently induced the significant increase in wheat root elongation as well as the actions of IAA and NO donor sodium nitroprusside (SNP). These responses were mimicked by the application of aqueous solution of CO with different saturation. Also, above heme molecule-induced effect is specific for CO since the potent inhibitor of HO-1, zinc protoporphyrin-IX (ZnPPIX) or CO scavenger hemoglobin (Hb) blocked the action of hematin and hemin, respectively. Further results proved that treatment with hematin or IAA could result in either the potent induction of HO-1 transcript or CO releasing in wheat root segments, both of which were reversed by the addition of ZnPPIX. ZnPPIX with lower concentration could prevent the elongation induced by IAA, while in the SNP-treatment the prevention of root growth occurred solely at higher concentrations. Also, wheat root segments elongation induced by IAA, SNP or hematin, was blocked by the specific NO scavenger, inhibitors of NO synthase (NOS) and guanylate cyclase (GC), respectively. Meanwhile, production of reactive oxygen species (ROS) could be demonstrated in the growing zone of wheat root segments treated by hematin or SNP using specific histochemical assay combined with the inhibitor investigation. Taken together, above results suggested that CO produced by HO might mediate the induction of growth elongation of wheat root segments by IAA, which might be also related to NO/cGMP- and even ROS-dependent pathways.

Carbon monoxide (CO) has recently proven to be an important bioactive or signaling molecule in mammalian cells. Its effects are mainly mediated by nitric oxide (NO) and cyclic GMP (cGMP). In Vicia faba leaves, CO production and heme oxygenase (HO) activity, an important CO synthetic enzyme, are first reported to increase in response to ABA treatment, which could result in stomatal closure. Interestingly, ABA-induced stomatal closure in V. faba guard cells is partially blocked when the synthetic CO inhibitor ZnPP, or the CO/NO scavenger Hb is added. Furthermore, we show that, exogenously applied CO donor, hematin, and CO aqueous solution not only result in the enhancement of CO release, but also time-dependently induce stomatal closure, and the latter is mimicked by the application of an NO donor SNP. The above-mentioned stomatal closure effects are differentially reversed by the addition of tungstate, a potent inhibitor of NO synthetic enzyme nitrate reductase (NR), the specific NO scavenger cPTIO, ZnPP, or Hb. During treatment for 4 h, SNP, 0.01% CO aqueous solution or hematin significantly triggers NO synthesis, whereas cPTIO, or tungstate approximately fully inhibits NO fluorescence. Additionally, application of the GC inhibitor ODQ blocks CO-induced stomatal closure. This inhibition could be reversed when 8-Br-cGMP is added. Thus, the above results suggest that CO produced by HO is involved in ABA-induced stomatal closure, and NO and cGMP may function as downstream intermediates in the CO signaling responsible for stomatal closure.

•The Bt gene is relatively more stable than the SPS gene under thermal treatment.•For Bt gene or SPS gene short DNA fragments are more stable than long DNA fragments.•The degradation of Bt gene and protein were positively correlated with the treatment violence.•The protein of Cry1Ab is more stable than protein of Cry1Ac and Cry1Ab/Ac.This study aimed to investigate the degradation of three transgenic Bacillus thuringiensis (Bt) genes (Cry1Ab, Cry1Ac, and Cry1Ab/Ac) and the corresponding encoded Bt proteins in KMD1, KF6, and TT51-1 rice powder, respectively, following autoclaving, cooking, baking, or microwaving. Exogenous Bt genes were more stable than the endogenous sucrose phosphate synthase (SPS) gene, and short DNA fragments were detected more frequently than long DNA fragments in both the Bt and SPS genes. Autoclaving, cooking (boiling in water, 30 min), and baking (200 °C, 30 min) induced the most severe Bt protein degradation effects, and Cry1Ab protein was more stable than Cry1Ac and Cry1Ab/Ac protein, which was further confirmed by baking samples at 180 °C for different periods of time. Microwaving induced mild degradation of the Bt and SPS genes, and Bt proteins, whereas baking (180 °C, 15 min), cooking and autoclaving led to further degradation, and baking (200 °C, 30 min) induced the most severe degradation. The findings of the study indicated that degradation of the Bt genes and proteins somewhat correlated with the treatment intensity. Polymerase chain reaction, enzyme-linked immunosorbent assay, and lateral flow tests were used to detect the corresponding transgenic components. Strategies for detecting transgenic ingredients in highly processed foods are discussed.

In our experiments, treatment of wheat seedling roots with varying concentrations of NaCl caused the inhibition of primary root growth in a dose-dependent manner, which was consistent with the progressive DNA laddering in the primary root tips. Upon 100, and 200 mM NaCl stress, the increase of carbon monoxide (CO) release and heme oxygenase (HO) activity was observed. By contraries, treatment with 400 mM NaCl is otherwise, that the result displays a decreasing tendency. Besides DNA laddering, 200 mM NaCl treatment exhibited the induction of other programmed cell death (PCD) associated with phenomenon in the primary root tips, such as TUNEL staining, nuclear condense and deformation. Further, HO's catalytic product CO was able to dose-dependently mitigate 200 mM NaCl-induced inhibition of root growth and delay PCD. These effects were prevented strongly by incubation with DETC (a Cu/Zn-SOD inhibitor), and differentially influenced by Tiron (a scavenger of superoxide anion) and apocynin (an inhibitor of NADPH oxidase). Meanwhile, 50% CO aqueous solution inhibited the overproduction of superoxide anion, NADPH oxidase activities and corresponding transcript, and enhanced superoxide dismutase (SOD) activity, Mn-SOD and Cu/Zn-SOD transcript. Taken together, the results suggested that CO might be involved in plant tolerance against salinity stress, and its alleviation of PCD and inhibition of root growth was related to the decrease of superoxide anion overproduction partially via up-regulation of SOD and down-regulation of NADPH oxidase expression.

Previous results revealed that haem oxygenase-1 (HO-1)/carbon monoxide (CO) system is involved in auxin-induced adventitious root formation. In this report, a cDNA for the gene ZmHO-1, encoding an HO-1 protein, was cloned from Zea mays seedlings. ZmHO-1 has a conserved HO signature sequence and shares highest homology with rice SE5 (OsHO-1) protein. We further discovered that N-1-naphthylacetic acid (NAA), haemin, and CO aqueous solution, led to the induction of ZmHO-1 expression as well as the thereafter promotion of lateral root development. These effects were specific for ZmHO-1 since the potent HO-1 inhibitor zinc protoporphyrin IX (ZnPPIX) differentially blocked the above actions. The addition of haemin and CO were able to reverse the auxin depletion-triggered inhibition of lateral root formation as well as the decreased ZmHO-1 transcripts. Molecular evidence showed that the haemin- or CO-mediated the modulation of target genes responsible for lateral root formation, including ZmCDK and ZmCKI2, could be blocked by ZnPPIX. Overexpression of ZmHO-1 in transgenic Arabidopsis plants resulted in promotion of lateral root development as well as the modulation of cell cycle regulatory gene expressions. Overall, our results suggested that a maize HO-1 gene is required for the lateral root formation.Highlights► A cDNA of haem oxygenase-1 (HO-1) was cloned from Zea mays seedling roots. ► The expression levels of ZmHO-1 gene and its corresponding protein were correlated with root development. ► The up-regulation of ZmHO-1 leads to lateral root formation by modulating cell cycle regulatory genes. ► Transgenic Arabidopsis with over-expression ZmHO-1 resulted in promotion of lateral root development.

Although carbon monoxide (CO) has always been regarded as a toxic gas, recent reports suggested that it is one of the products of heme oxygenase (HO; EC 1.14.99.3) catalysis in animals, and could confer beneficial cytoprotection against oxidative damage. Here, we investigated the effects of exogenous putative CO donor hematin and CO aqueous solution on rice seed germination under 100 mM NaCl salt stress. Both hematin and CO aqueous solution dose-dependently attenuated the inhibition of seed germination and seedling growth caused by salinity. Moreover, 1.0 μM hematin and 5% CO-saturated aqueous solution activated amylase activity, thus accelerating the formation of reducing sugar and total soluble sugar. Furthermore, hematin and CO aqueous solution induced catalase (CAT) and superoxide dismutase (SOD) activities, thus resulting in the alleviation of oxidative damage, as indicated by the decrease of thiobarbituric acid reactive substances (TBARS) content. Such above CO-induced effects were also confirmed by using semi-quantitative RT-PCR, including the up-regulation of expression patterns of α-amylase, CAT and Cu/Zn-SOD genes. Also, the similar function of hematin might contribute to endogenous HO-derived CO, since addition of the CO-specific synthetic inhibitor zinc protoporphyrin IX (ZnPPIX) and CO scavenger hemoglobin (Hb) reversed above effects. Meanwhile, further results also proved that treatment with hematin for 36 h could result in the potent induction of HO-1 transcript, HO activity and CO content in the germinating seeds. Collectively, these results indicated that CO performed an advantageous effect on attenuation of inhibition of seed germination and seedling growth induced by salt stress, and alleviated oxidative damage via activating anti-oxidant enzymes.

ABSTRACTUp-regulation of the gene that encodes intracellular heme oxygenase 1 (HO1) benefits plants under cadmium (Cd2+) stress; however, the molecular mechanisms remain unclear. Here, we elucidate the role of Arabidopsis HY1 (AtHO1) in Cd2+ tolerance by using genetic and molecular approaches. Analysis of two HY1 null mutants, three HY1 overexpression lines, HO double or triple mutants, as well as phyA and phyB mutants revealed the specific hypersensitivity of hy1 to Cd2+ stress. Supplementation with two enzymatic by-products of HY1, carbon monoxide (CO) and iron (Fe, especially), rescued the Cd2+-induced inhibition of primary root (PR) elongation in hy1-100. The mutation of HY1, which exhibited lower glutathione content than Col-0 in root tissues, was able to induce nitric oxide (NO) overproduction, Cd2+ accumulation, and severe Fe deficiency in root tissues. However, the contrasting responses appeared in 35S:HY1-4. Additionally, reduced levels of Ferric Reduction Oxidase 2 (FRO2) and Iron-Regulated Transporter 1 (IRT1) transcripts, and increased levels of Heavy Metal ATPase 2/4 (HMA2/4) transcripts bolster the notion that HY1 up-regulation ameliorates Fe deficiency, and might increase Cd2+ exclusion. Taken together, these results showed that HY1 plays a common link in Cd2+ tolerance by decreasing NO production and improving Fe homeostasis in Arabidopsis root tissues.SUMMARYHeme oxygenase 1 (HO1) is a potential contributor of Cd2+ tolerance; however, the molecular mechanisms are largely unclear. Using genetic and molecular approaches, we revealed that Arabidopsis HY1 (AtHO1) contributes to the alleviation of Cd2+ toxicity by decreasing NO production and improving Fe homeostasis in root tissues.

Our previous work showed that in cucumber (Cucumis sativus), auxin rapidly induces heme oxygenase (HO) activity and the product of HO action, carbon monoxide (CO), then triggers the signal transduction events leading to adventitious root formation. In this study, the cucumber HO-1 gene (named as CsHO1) was isolated and sequenced. It contains four exons and three introns and encodes a polypeptide of 291 amino acids. Further results show that CsHO1 shares a high homology with plant HO-1 proteins and codes a 33.3 kDa protein with a 65-amino transit peptide, predicting a mature protein of 26.1 kDa. The mature CsHO1 was expressed in Escherichia coli to produce a fusion protein, which exhibits HO activity. The CsHO1:GFP fusion protein was localized in the chloroplast. Related biochemical analyses of mature CsHO1, including Vmax, Km, Topt and pHopt, were also investigated. CsHO1 mRNA was found in germinating seeds, roots, stem, and especially in leaf tissues. Several well-known adventitious root inducers, including auxin, ABA, hemin, nitric oxide donor sodium nitroprusside (SNP), CaCl2, and sodium hydrosulfide (NaHS), differentially up-regulate CsHO1 transcripts and corresponding protein levels. These results suggest that CsHO1 may be involved in cucumber adventitious rooting.

This study tested whether an inducible isoform of heme oxygenase (HO, EC 1.14.99.3), HO-1, is involved in the cytokinin (CTK)-induced alleviation of senescence in detached wheat (Triticum aestivum L.) leaves during dark incubation. We discovered that exogenous supplement of 6-benzylaminopurine (6-BA) at 10 μM for 48 h not only delayed the dark-induced loss of chlorophyll and protein contents in detached wheat leaves, but also significantly increased HO activity in a time-dependent manner. This induction reached a maximum within 3 h of 6-BA supply, which was further confirmed by using semi-quantitative RT-PCR and protein gel blot analysis. Furthermore, the decreases in intracellular thiobarbituric acid reactive substances (TBARS) content, and the increases in the transcript level, total and isozymatic activities of some important antioxidant enzymes, such as catalase (CAT, EC 1.11.1.6), peroxidase (POD, EC 1.11.1.7), superoxide dismutase (SOD, EC 1.15.1.1), and ascorbate peroxidase (APX, EC 1.11.1.11), were observed. Reversed responses of chlorophyll, protein and TBARS contents, HO activity, and the expression of above antioxidant enzymes were observed when zinc protoporphyrin-IX (ZnPPIX), a potent HO-1 inhibitor, was added together with 6-BA. In contrast, HO-1 inducer hemin could partially mimic the effects of 6-BA. Together, the results suggest that HO-1 might be involved in the CTK-induced alleviation of senescence and lipid peroxidation in detached wheat leaves.

Hemin, a heme oxygenase-1 (HO-1) inducer, was shown to exert numerous beneficial physiological functions in animals. Our previous study suggests that HO-1/carbon monoxide (CO) acts as a novel downstream signal system in the auxin-induced adventitious rooting. The objective of this study was to test whether nitric oxide (NO) is involved in hemin-induced cucumber adventitious rooting. Applications of hemin or CO aqueous solution to auxin-depleted cucumber explant induced up-regulation of cucumber HO-1 transcripts (CsHO1), NO production, and thereafter adventitious root formation, and some above responses were blocked by the combination treatment with two nitric oxide synthase (NOS)-like enzyme inhibitors NG-nitro-l-arginine methylester hydrochloride and NG-nitro-l-arginine, a HO-1 specific inhibitor zinc protoporphyrin IX, and a specific NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide potassium salt. However, these blocking responses were not observed using tungstate, an inhibitor of nitrate reductase, another NO producing enzyme in plants. Furthermore, the guanylate cyclase inhibitors 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalin-1-one and 6-anilino-5,8-quinolinedione reduced root development induced by hemin, whereas the cell-permeable cyclic guanosine monophosphate (cGMP) derivative 8-Br-cGMP reversed this effect. Together, our results indicated that at least in our experimental conditions, NO might operate downstream of hemin promoting adventitious root formation probably in a cGMP-dependent manner.

To investigate the mechanism and signaling pathway of carbon monoxide (CO) and hematin in alleviating seed germination inhibition and lipid peroxidation, polyethylene glycol-6000 (PEG) was used to mimic osmotic stress in a series of experiments. The results showed that wheat seeds pretreated with a lower dose of PEG (12.5%) showed higher tolerance against osmotic stress as well as the up-regulation of heme oxygenase (HO, EC 1.14.99.3) and decreased lipid peroxidation during recuperation, compared to those with a higher dose of PEG (50%). Exposure of wheat seeds to 25% PEG, HO-1 inhibitor or specific scavenger of nitric oxide (NO) alone differentially led to seed germination inhibition. The PEG-induced inhibitory effects on seed germination were ameliorated by the HO-1 inducer hematin, CO or NO donor. Additionally, hematin was able to markedly boost the HO/CO system. However, the addition of the HO-1 inhibitor or the specific scavenger of NO not only reversed the protective effects conferred by hematin, but also blocked the up-regulation of HO/CO. In addition, hematin-driven NO production in wheat seeds under osmotic stress was confirmed. Based on these results, we conclude that the endogenous HO/CO signal system is required for the alleviation of osmotic stress-induced wheat seed germination inhibition and lipid peroxidation, which might have a possible interaction with NO.

Plant heme oxygenase (HO) catalyzes the oxygenation of heme to biliverdin, carbon monoxide, and free iron, and is regarded as a stress-responsive protein. Here, a cabbage HO1 gene (named as BoHO1) was isolated and characterized. BoHO1 shares a high degree homology with Arabidopsis AtHO1, and could locate in Arabidopsis chloroplast. BoHO1 mRNA was ubiquitously expressed in cabbage tissues, and was responsive to several stimuli and chemicals. Genetic evidence illustrated that over-expression of BoHO1 in transgenic Arabidopsis plants (35S:BoHO1-1 and 35S:BoHO1-2) significantly alleviated salinity stress-inhibited seedling growth, which were accompanied with the re-establishment of reactive oxygen species and ion homeostasis. Comparative proteomic analysis was subsequently performed. Results revealed that protein abundance related to light reactions was greatly suppressed by NaCl stress in wild-type, whereas was partially recovered in 35S:BoHO1-1. Salinity stress also strongly activated stress-related metabolic processes in wild-type, i.e. carbon and energy metabolism, ammonium detoxification, and protein turnover, and these induced tendencies were more intensive in 35S:BoHO1-1. Particularly, proteins related to glutathione metabolism and ion homeostasis were specifically enriched in NaCl-stressed 35S:BoHO1-1. On the basis of above results, we propose that BoHO1 could activate multiple stress-responsive pathways to help Arabidopsis regain cellular homeostasis, thus presenting enhanced adaptation to salinity stress.