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[Objectives] In order to acquire accurate results of the expression of genes during different metabolism processes in plants, it is critical to perform the selection of suitable reference genes under different conditions. [Methods] We analyze the stabilities of 10 reference genes(Actin2, GAPDH, MSC27, 18S RNA, β-tublin, bZIP, UBQ, PPPrep, Ms03_50f03 and Ms03_69f07) within different tissues(roots, stems and leaves)of alfalfa (Medicago sativa L.) through geNorm and NormFinder softwares, which were collected at different times (samples were collected after UV-B irradiation for 0, 1, 2 and 5 d). [Results] The results illustrated that MSC27 and UBQ were suitable for alfalfa roots analysis upon UV-B irradiation, as revealed by their lowest stability values. Furthermore, when another reference gene was added, corresponding variation(V)value(V3/4)was not significantly altered when compared with that of V2/3, which only contained two reference genes. These results further suggested that two reference genes were enough in alfalfa roots analysis under UV-B stress. MSC27 and Actin2 was appropriated for the alfalfa stems analysis since the lowest M values were obtained. Meanwhile, the analysis of alfalfa leaves was advised to use Actin2 and GAPDH as internal genes under UV-B stress. The variation(V)value(V3/4)was higher than that of 2/3 when another reference gene was added, The 18S RNA and β-tublin had the heightest M values in the analysis of all three alfalfa tissues, which suggested that they were not suitable for reference genes. The results analyzed by Normfinder were almost the same as that of geNorm. The differences between these two softwares might be their different algorithms. [Conclusions] In alfalfa seedlings, MSC27 and UBQ were suitable in root analysis; MSC27 and Actin2 were appropriate in stem analysis; while we used Actin2 and GAPDH as internal genes to get more precise gene expression in leaf analysis. The results of the study have important practical value in the analysis of key genes under UV-B irradiation. [ABSTRACT FROM AUTHOR]

Key message: β-Cyclodextrin-hemin complex-induced tomato lateral root formation was associated with nitric oxide and heme oxygenase 1 by modulating cell cycle regulatory genes. Abstract: β-Cyclodextrin-hemin complex (β-CDH), a complex by combining β-cyclodextrin (β-CD) with hemin, a heme oxygenase 1 (HO1) inducer, was a trigger of cucumber adventitious root formation by enhancing HO1 gene expression. In this report, our results identified the previously unknown function of β-CDH in plants: the inducer of tomato lateral root (LR) formation. β-CDH-triggered LR formation is hemin-specific, since β-CD failed to induce LR development. Because nitric oxide (NO) is involved in LR formation, the correlation of β-CDH with NO and HO1 was investigated. Our analysis suggested that β-CDH induced an increase in endogenous NO production, followed by up-regulation of tomato HO1 gene and LR formation, all of which were mimicked by hemin and two NO-releasing compounds (SNP and GSNO). The induction of HO1 gene expression and LR formation triggered by β-CDH or hemin were significantly blocked by an inhibitor of HO1. Further results revealed that both β-CDH- and SNP-stimulated HO1 gene expression and thereafter LR formation were sensitive to the removal of NO with a potent NO scavenger, and the responses of SNP were significantly blocked by an inhibitor of HO1. Molecular evidence illustrated that representative cell cycle regulatory genes, including SlCDKA1, SlCYCA3;1, SlCYCA2;1, and SlCYCD3;1, were significantly up-regulated by β-CDH and SNP, but obviously blocked when seedlings were co-treated with the scavenger of NO or the inhibitor of HO1. In summary, our physiological and molecular evidence demonstrated that both NO and HO1 were involved in the β-CDH-induced LR formation with, at least partially, HO1 acting downstream of NO signaling. [ABSTRACT FROM AUTHOR]

Key message: Methane-rich water triggered adventitious rooting by regulating heme oxygenase1/carbon monoxide and calcium pathways in cucumber explants. Abstract: Heme oxygenase1/carbon monoxide (HO1/CO) and calcium (Ca) 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 Fe 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 Fe, 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 Ca-channel blocker and Ca chelator, the involvement of Ca 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 Ca pathways. [ABSTRACT FROM AUTHOR]

Background and Aims The activity of H+-ATPase is essential for energizing the plasma membrane. It provides the driving force for potassium retention and uptake through voltage-gated channels and for Na+ exclusion via Na+/H+ exchangers. Both of these traits are central to plant salinity tolerance; however, whether the increased activity of H+-ATPase is a constitutive trait in halophyte species and whether this activity is upregulated at either the transcriptional or post-translation level remain disputed.Methods The kinetics of salt-induced net H+, Na+ and K+ fluxes, membrane potential and AHA1/2/3 expression changes in the roots of two halophyte species, Atriplex lentiformis (saltbush) and Chenopodium quinoa (quinoa), were compared with data obtained from Arabidopsis thaliana roots.Key Results Intrinsic (steady-state) membrane potential values were more negative in A. lentiformis and C. quinoa compared with arabidopsis (−144 ± 3·3, −138 ± 5·4 and −128 ± 3·3 mV, respectively). Treatment with 100 mm NaCl depolarized the root plasma membrane, an effect that was much stronger in arabidopsis. The extent of plasma membrane depolarization positively correlated with NaCl-induced stimulation of vanadate-sensitive H+ efflux, Na+ efflux and K+ retention in roots (quinoa > saltbush > arabidopsis). NaCl-induced stimulation of H+ efflux was most pronounced in the root elongation zone. In contrast, H+-ATPase AHA transcript levels were much higher in arabidopsis compared with quinoa plants, and 100 mm NaCl treatment led to a further 3-fold increase in AHA1 and AHA2 transcripts in arabidopsis but not in quinoa.Conclusions Enhanced salinity tolerance in the halophyte species studied here is not related to the constitutively higher AHA transcript levels in the root epidermis, but to the plant’s ability to rapidly upregulate plasma membrane H+-ATPase upon salinity treatment. This is necessary for assisting plants to maintain highly negative membrane potential values and to exclude Na+, or enable better K+ retention in the cytosol under saline conditions. [ABSTRACT FROM AUTHOR]

Heme oxygenase1 (HO1) is involved in protecting plants from environmental stimuli. In this study, a sunflower ( Helianthus annuus L.) HO1 gene ( HaHO1) was cloned and sequenced. It was confirmed that HaHO1 encodes a precursor protein of 32.93 kDa with an N-terminal plastid transit peptide which was validated by subcellular localization. The amino acid sequence of HaHO1 shared high homology with other plant HO1s. The predicted three-dimensional structure showed a high degree of structural conservation as compared to the known HO1 crystal structures. Phylogenetic analysis revealed that HaHO1 clearly grouped with the plant HO1-like sequences. Moreover, the purified recombinant mature HaHO1 expressed in Escherichia coli exhibits HO activity. Thus, it was concluded that HaHO1 encodes a functional HO1 in sunflower. Additionally, HaHO1 gene was ubiquitously expressed in all tested tissues, and induced differentially during different growth stages after germination, and could be differentially induced by several stresses and hemin treatment. For example, a pretreatment with a low concentration of NaCl (25 mM) could lead to the induction of HaHO1 gene expression and thereafter a salinity acclamatory response. Above cytoprotective effect could be impaired by the potent HO1 inhibitor zinc protoporphyrin IX (ZnPPIX), which was further rescued by the addition of 50 % carbon monoxide aqueous solution (in particular) or bilirubin, two catalytic by-products of HO1, respectively. Similarly, a HO1 inducer, hemin, could mimic the salinity acclamatory response. Together, these findings strongly suggested that the up-regulation of HaHO1 might be required for the observed salinity acclimation in sunflower plants. [ABSTRACT FROM AUTHOR]

To investigate how light quality influences tomato ( Solanum lycopersicum L) seedlings, we examined changes in plant growth, chloroplast ultrastructure, photosynthetic parameters and some photosynthesis-related genes expression levels. For this, tomato plants were grown under different light qualities with the same photosynthetic photon flux density: red (R), blue (B), yellow (Y), green (G) and white (W) lights. Our results revealed that, compared with plants grown under W light, the growth of plants grown under monochromatic lights was inhibited with the growth reduction being more significant in the plants grown under Y and G lights. However, the monochromatic lights had their own effects on the growth and photosynthetic function of tomato seedlings. The plant height was reduced under blue light, but expression of rbcS, rbcL, psbA, psbB genes was up-regulated, and the ΦPSII and electron transport rate (ETR) values were enhanced. More starch grains were accumulated in chloroplasts. The root elongation, net photosynthetic rate (Pn), NPQ and rbcS and psbA genes expression were promoted under red light. Yellow light- and green light-illuminated plants grew badly with their lower Rubisco content and Pn value observed, and less starch grains accumulated in chloroplast. However, less influence was noted of light quality on chloroplast structure. Compared with yellow light, the values of ΦPSII, ETR, qP and NPQ of plants exposed to green light were significantly increased, suggesting that green light was beneficial to both the development of photosynthetic apparatus to some extent. [ABSTRACT FROM AUTHOR]

Haem oxygenase-1 (HO-1) and hydrogen peroxide (HO) are two key downstream signals of auxin, a well-known phytohormone regulating plant growth and development. However, the inter-relationship between HO-1 and HO 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 HO generation. Subsequently, LR formation was induced. NAA-induced HY1 expression is dependent on HO. This conclusion was supported by analyzing the removal of HO with ascorbic acid (AsA) and dimethylthiourea (DMTU), both of which could block NAA-induced HY1 expression and LR formation. HO-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 HO generation, since Znpp inhibition of HY1 blocked the NAA-induced HO production and LR formation. Genetic data demonstrated that hy1- 100 was significantly impaired in HO 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 HO production and LR formation, both of which were decreased by DMTU. Moreover, HO 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 HO 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 HO existing in auxin-induced LR formation in Arabidopsis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: This study investigated molecular mechanism of spermidine (Spd)-mediated enhancement of tolerance against salinity during alfalfa seed germination. Methods: A combination of histochemical, physiological and molecular approaches was used to study the effect of Spd on the alleviation of salt toxicity. Results: Exogenously applied Spd (100 μM) and a heme oxygenase-1 (HO-1) inducer hemin (10 μM) not only up-regulated alfalfa HO-1 gene expression and increased its protein level and activity, but also significantly alleviated the inhibitory effects of 100 mM NaCl on seed germination and seedling growth. K to Na ratio was also increased. Further results revealed that total, isozymatic activities or corresponding transcripts of antioxidant enzymes, including superoxide dismutase (SOD), ascorbate peroxidase (APX) and guaiacol peroxidase (POD), were induced differentially. These antioxidant behaviors were confirmed by histochemical staining for the detection of lipid peroxidation and the loss of plasma membrane integrity. Above cytoprotective role of Spd is specific for HO-1 because the inhibitor of HO-1 significantly suppressed above responses, and some of the inhibitory effects were reversed when 10 % carbon monoxide (CO)-saturated aqueous solution was added. Conclusions: Together, our results suggested that Spd-mediated alleviation of NaCl toxicity during alfalfa seed germination acts, at least partially, in a HO-1-dependent pathway. [ABSTRACT FROM AUTHOR]

Although molecular hydrogen can alleviate herbicide paraquat and Fusarium mycotoxins toxicity in plants and animals, whether or how molecular hydrogen influences pesticide residues in plants is not clear. Here, pot experiments in greenhouse revealed that degradation of carbendazim (a benzimidazole pesticide) in leaves could be positively stimulated by molecular hydrogen, either exogenously applied or with genetic manipulation. Pharmacological and genetic increased hydrogen gas could increase glutathione metabolism and thereafter carbendazim degradation, both of which were abolished by the removal of endogenous glutathione with its synthetic inhibitor, in both tomato and in transgenic Arabidopsis when overexpressing the hydrogenase 1 gene from Chlamydomonas reinhardtii. Importantly, the antifungal effect of carbendazim in tomato plants was not obviously altered regardless of molecular hydrogen addition. The contribution of glutathione-related detoxification mechanism achieved by molecular hydrogen was confirmed. Our results might not only illustrate a previously undescribed function of molecular hydrogen in plants, but also provide an environmental-friendly approach for the effective elimination or reduction of pesticides residues in crops when grown in pesticides-overused environmental conditions. [ABSTRACT FROM AUTHOR]

Despite substantial evidence on the separate roles of Arabidopsis nitric oxide-associated 1 (NOA1)-associated nitric oxide (NO) production and haem oxygenase 1 (HY1) expression in salt tolerance, their integrative signalling pathway remains largely unknown. To fill this knowledge gap, the interaction network among nitrate reductase (NIA/NR)- and NOA1-dependent NO production and HY1 expression was studied at the genetic and molecular levels. Upon salinity stress, the majority of NO production was attributed to NIA/NR/NOA1. Further evidence confirmed that HY1 mutant hy1-100, nia1/2/noa1, and nia1/2/noa1/hy1-100 mutants exhibited progressive salt hypersensitivity, all of which were significantly rescued by three NO-releasing compounds. The salinity-tolerant phenotype and the stronger NO production in gain-of-function mutant of HY1 were also blocked by the NO synthetic inhibitor and scavenger. Although NO- or HY1-deficient mutants showed a compensatory mode of upregulation of HY1 or slightly increased NO production, respectively, during 2 d of salt treatment, downregulation of ZAT10/12-mediated antioxidant gene expression (cAPX1/2 and FSD1) was observed after 7 d of treatment. The hypersensitive phenotypes and stress-related genes expression profiles were differentially rescued or blocked by the application of NO- (in particular) or carbon monoxide (CO)-releasing compounds, showing a synergistic mode. Similar reciprocal responses were observed in the nia1/2/noa1/hy1-100 quadruple mutant, with the NO-releasing compounds exhibit the maximal rescuing responses. Overall, the findings present the combination of compensatory and synergistic modes, linking NIA/NR/NOA1-dependent NO production and HY1 expression in the modulation of plant salt tolerance. [ABSTRACT FROM AUTHOR]

Aims: This study investigated the molecular mechanism of hydrogen-rich water (HRW)-mediated enhancement of tolerance against salinity stress during rice seed germination. Methods: A combination of physiological and molecular approaches was used to study the effect of HRW on the alleviation of salinity stress. Results: A 100-mM NaCl stress caused the increase of H 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. Conclusions: Together, our results suggested that exogenous HRW treatment on rice seeds may be a good option to alleviate salinity stress. [ABSTRACT FROM AUTHOR]

Lipoxygenases (LOXs) are enzymes involved in lipid peroxidation. Here we reported the identification, molecular and functional characterization of the gene encoding rice ( Oryza sativa L.) seed LOX3 (sLOX3). Via a map-based cloning strategy we identified Os03g0700400 as the candidate gene encoding sLOX3. Further functional complementary test and biochemical characterization of the recombinant Os03g0700400 protein verified the identification. The sLOX3 gene was highly expressed in roots, moderately in embryos and very weakly in leaves, leaf sheaths and stems. Transient expression experiment (in rice protoplasts) and subsequent laser confocal microscopic analysis demonstrated that the sLOX3 protein was localized into the cytosol. We next showed that overexpression of sLOX3 in a japonica sLOX3-normal rice cultivar, Wuyunjing 7 accelerated the decrease of seed germination ability when the seeds were routinely stored, which demonstrated that sLOX3 had a negative effect on seed longevity (storability). Meanwhile, an increased occurrence of embryo decay was observed in the same transgenic seeds, suggesting that sLOX3 might negatively affect seed longevity by facilitating colonization of particular seed pathogens. Our result forwarded the understanding of the effects of 9-LOX on rice seed longevity. [ABSTRACT FROM AUTHOR]

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 (HS), 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 HS production, and may play an important role in plant growth regulators and chemical stimuli responses. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aims: This study explored molecular mechanism of ascorbic acid (AsA)-mediated enhancement of plant tolerance against cadmium (Cd) stress. Methods: Complex pharmacological, histochemical and molecular approaches were applied to analyse the effect of AsA on the alleviation of Cd stress and corresponding signalling pathway. Results: Cd stress brought about severe oxidative damage and remarkable decrease in AsA content in alfalfa (Medicago sativa) seedling roots. Exogenous AsA not only increased AsA content in vivo, and strengthened the up-regulation of alfalfa heme oxygenase-1 (HO-1) transcript and HO activity triggered by Cd, but also significantly decreased Cd accumulation and oxidative damage, which was confirmed by the histochemical analysis. The responses of AsA were further impaired by the potent inhibitor of HO-1, zinc protoporphyrin IX (ZnPP), which were blocked further when 50 % saturation of carbon monoxide (CO) aqueous solution (in particular) or bilirubin (BR), two catalytic by-products of HO-1, was added, respectively. Molecular evidence illustrated that AsA-triggered the up-regulation of antioxidant enzyme genes, especially Mn-SOD and POD, were sensitive to ZnPP and reversed by CO. Conclusions: In short, above results suggested that cytoprotective roles triggered by AsA might be, at least partially, through HO-1-dependent fashion by the induction of antioxidant system and lowering Cd accumulation. [ABSTRACT FROM AUTHOR]

ABSTRACT Hydrogen gas (H2) was recently proposed as a novel antioxidant and signalling molecule in animals. However, the physiological roles of H2 in plants are less clear. Here, we showed that exposure of alfalfa seedlings to paraquat stress increased endogenous H2 production. When supplied with exogenous H2 or the heme oxygenase-1 (HO-1)-inducer hemin, alfalfa plants displayed enhanced tolerance to oxidative stress induced by paraquat. This was evidenced by alleviation of the inhibition of root growth, reduced lipid peroxidation and the decreased hydrogen peroxide and superoxide anion radical levels. The activities and transcripts of representative antioxidant enzymes were induced after exposure to either H2 or hemin. Further results showed that H2 pretreatment could dramatically increase levels of the MsHO-1 transcript, levels of the protein it encodes and HO-1 activity. The previously mentioned H2-mediated responses were specific for HO-1, given that the potent HO-1-inhibitor counteracted the effects of H2. The effects of H2 were reversed after the addition of an aqueous solution of 50% carbon monoxide (CO). We also discovered enhanced tolerance of multiple environmental stresses after plants were pretreated with H2. Together, these results suggested that H2 might function as an important gaseous molecule that alleviates oxidative stress via HO-1 signalling. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Sharp eyespot and Fusarium crown rot, mainly caused by soil-borne fungi Rhizoctonia cerealis and Fusarium pseudograminearum, are destructive diseases of major cereal crops including wheat (Triticum aestivum). However, the mechanisms underlying wheat-resistant responses to the two pathogens are largely elusive. In this study, we performed a genome-wide analysis of wall-associated kinase (WAK) family in wheat. As a result, a total of 140 TaWAK (not TaWAKL) candidate genes were identified from the wheat genome, each of which contains an N-terminal signal peptide, a galacturonan binding domain, an EGF-like domain, a calcium binding EGF domain (EGF-Ca), a transmembrane domain, and an intracellular Serine/Threonine protein kinase domain. By analyzing the RNA-sequencing data of wheat inoculated with R. cerealis and F. pseudograminearum, we found that transcript abundance of TaWAK-5D600 (TraesCS5D02G268600) on chromosome 5D was significantly upregulated, and that its upregulated transcript levels in response to both pathogens were higher compared with other TaWAK genes. Importantly, knock-down of TaWAK-5D600 transcript impaired wheat resistance against the fungal pathogens R. cerealis and F. pseudograminearum, and significantly repressed expression of defense-related genes in wheat, TaSERK1, TaMPK3, TaPR1, TaChitinase3, and TaChitinase4. Thus, this study proposes TaWAK-5D600 as a promising gene for improving wheat broad resistance to sharp eyespot and Fusarium crown rot (FCR) in wheat. [ABSTRACT FROM AUTHOR]