Hydrogen agronomy: research progress and prospects.

Agriculture is the foundation of social development. Under the pressure of population growth, natural disasters, environmental pollution, climate change, and food safety, the interdisciplinary "new agriculture" is becoming an important trend of modern agriculture. In fact, new agriculture is not only the foundation of great health and new energy sources, but is also the cornerstone of national food security, energy security, and biosafety. Hydrogen agronomy focuses mainly on the mechanism of hydrogen gas (H2) biology effects in agriculture, and provides a theoretical foundation for the practice of hydrogen agriculture, a component of the new agriculture. Previous research on the biological effects of H2 focused chiefly on medicine. The mechanism of selective antioxidant is the main theoretical basis of hydrogen medicine. Subsequent experiments have demonstrated that H2 can regulate the growth and development of plant crops, edible fungus, and livestock, and enhance the tolerance of these agriculturally important organisms against abiotic and biotic stresses. Even more importantly, H2 can regulate the growth and development of crops by changing the soil microbial community composition and structure. Use of H2 can also improve the nutritional value and postharvest quality of agricultural products. Researchers have also shown that the biological functions of molecular hydrogen are mediated by modulating reactive oxygen species (ROS), nitric oxide (NO), and carbon monoxide (CO) signaling cascades in plants and microbes. This review summarizes and clarifies the history of hydrogen agronomy and describes recent progress in the field. We also argue that emerging hydrogen agriculture will be an important direction in the new agriculture. Further, we discuss several scientific problems in hydrogen agronomy, and suggest that the future of hydrogen agronomy depends on contributions by multiple disciplines. Important future research directions of hydrogen agronomy include hydrogen agriculture in special environments, such as islands, reefs, aircraft, and outer space.

Effects of astaxanthin and emodin on the growth, stress resistance and disease resistance of yellow catfish (Pelteobagrus fulvidraco).

Yellow catfish (Pelteobagrus fulvidraco) has become a commercially important fish species in China and eastern Asia. High-density aquaculture has led to congestion and excessive stress and contributed to bacterial infection outbreaks that have caused high mortality. We investigated the effects of dietary supplementation with astaxanthin and emodin alone and in combination on the growth and stress resistance of yellow catfish. After 60 days of feeding, each group of fish (control, astaxanthin, emodin, and astaxanthin plus emodin (combination) groups) was exposed to acute crowding stress for 24 h, and a subsample of fish from the four groups was challenged with the bacterial septicemia pathogen Proteus mirabilis after the end of the crowding stress experiment. Compared with the control, the astaxanthin and emodin groups showed increases in serum total protein (TP), hepatic superoxide dismutase (SOD) activity and hepatic heat shock proteins 70 (HSP70) mRNA levels at 12 and 24 h after the initiation of crowding stress. The combination group exhibited increases in alanine aminotransferase (ALT) activity, aspartate aminotransferase (AST) activity, serum TP, hepatic SOD activity and hepatic HSP70 mRNA levels within 24 h after the initiation of crowding stress. However, decreases relative to the control were observed in the serum cortisol and glucose contents in the three treatment groups at 12 and 24 h after the initiation of crowding stress, in ALT and AST activity in the astaxanthin and emodin group at 24 h after the initiation of crowding stress, and in the serum lysozyme activity, serum alkaline phosphatase (ALP) activity, and hepatic catalase (CAT) and malondialdehyde (MDA) activity in the combination group at 24 h after the initiation of crowding stress. Additionally, the cumulative mortality after P. mirabilis infection was lower in all three treatment groups (57.00%-70.33%) than in the control (77.67%). Dietary supplementation with astaxanthin and emodin decreased the specific growth rate (SGR) and weight gain (WG) of healthy yellow catfish, although significant differences in mortality were not observed. These results indicate that dietary supplementation with 80 mg/kg astaxanthin and 150 mg/kg emodin can improve the anti-oxidative capabilities, hepatic HSP70 levels, and resistance to acute crowding stress of yellow catfish. Finally, an appropriate strategy for enhance yellow catfish stress resistance and disease resistance is proposed.

Genetic variation in Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis, three endemic and endangered species in China using SSR and TRAP markers.

Leeches are not only important medicinal animals worldwide but also are endangered. We aimed to (i) explore the level of genetic diversity within/among populations of three leeches, (ii) assess genetic differentiation among these three leeches, and (iii) discuss an appropriate strategy for conserving leech germplasm. A total of 315 individuals of Whitmania pigra, Hirudo nipponica and Poecilobdella manillensis from 21 populations were collected in China and Vietnam. The genetic structure and genetic diversity among and within the 21 populations were evaluated using target region amplified polymorphism (TRAP) and simple sequence repeat (SSR) markers. Sixteen pairs of TRAP primers generated a total of 398 fragments, of which 396 (99.50%) were polymorphic; fourteen pairs of SSR primers generated a total of 60 fragments, of which 59 (98.33%) were polymorphic. Shannon's index (I) and Nei's gene diversity index (H) for the three leeches were high at the species level (I=0.4980 and H=0.3323 for TRAPs, I=0.4487 and H=0.2969 for SSRs in W. pigra; I=0.4147/0.3769, H=0.2788/0.2566 for H. nipponica; and I=0.4616/0.4717, H=0.3099/0.3203 for P. manillensis). However, low genetic diversity was determined at the population level; the average genetic diversity measures within populations were H=0.1767/0.1376, I=0.2589/0.2043 for W. pigra, H=0.2149/0.2021, I=0.3184/0.3000 for H. nipponica and H=0.2850/0.2724, I=0.4152/0.3967 for P. manillensis. We conclude that there was limited gene exchange within/among populations and species, as the gene flow number (Nm) was 0.5493/0.5807. However, for all three species, the genetic diversity was different at the population level. Gene differentiation (Gst) and Nm were 0.4682 /0.5364 and 0.5678/0.4321 for W. pigra, 0.2294/0.2127 and 1.6797/1.8512 for H. nipponica and 0.1214/0.1496 and 3.6202/2.8412 for P. manillensis. STRUCTURE analysis, Unweighted Pair-Group Method with Arithmetic means (UPGMA) cluster analysis and Principal Coordinates Analysis (PCOA) all yielded similar results. The isolation-by-distance pattern was not significant for any of the three species by the Mantel test. These data emphasize the need for management, conservation, and rehabilitation of this animal species. Finally, an appropriate strategy for conserving leech is proposed.

Novosphingobium chloroacetimidivorans sp. nov., a chloroacetamide herbicide-degrading bacterium isolated from activated sludge.

Strain BUT-14(T), a Gram-reaction-negative, non-spore-forming, ellipse-shaped bacterium, was isolated from activated sludge of a chloroacetamide-herbicides-manufacturing wastewater treatment facility. The strain was able to degrade more than 90% of butachlor, acetochlor and alachlor (100 mg l(-1)) within 5 days of incubation. The taxonomic position was investigated using a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain BUT-14(T) was a member of the genus Novosphingobium and showed the highest sequence similarities to Novosphingobium soli DSM 22821(T) (97.9%), N. naphthalenivorans KACC 15258(T) (97.4%), N. pentaromativorans JCM 12182(T) (97.4%) and N. barchaimii DSM 25411(T) (97.1%) and lower (<97%) sequence similarities to all other species of the genus Novosphingobium. Chemotaxonomic analysis revealed that strain BUT-14(T) possessed Q-10 as the predominant ubiquinone, spermidine as the major polyamine and C(18 : 1)ω7c (46.9%), C(17 : 1)ω6c (17.9%), summed feature 3, C(14 : 0) 2-OH (4.4%), C(15 : 0) 2-OH (3.1%) and C(16 : 0) (5.51%) as the major fatty acids. The polar lipids included lipid, glycolipid, phosphatidylglycerol, phospholipid, phosphatidylethanolamine, phosphatidylcholine, sphingoglycolipid and phospatidyldimethylethanolamine. Strain BUT-14(T) showed low DNA-DNA relatedness with N. soli DSM 22821(T) (41.5±2.9%), N. naphthalenivorans JCM 12182(T) (49.2±4.2%), N. pentaromativorans KACC 12295(T) (53.2±1.9%) and N. barchaimii DSM 25411 (51.2±4.5%). The DNA G+C content was 66±0.3 mol%. The combination of phylogenetic analysis, phenotypic characteristics, chemotaxonomic data and DNA-DNA hybridization supports the suggestion that strain BUT-14(T) represents a novel species of the genus Novosphingobium, for which the name Novosphingobium chloroacetimidivorans sp. nov. is proposed. The type strain is BUT-14(T) ( = CCTCC AB 2013086(T) = KACC 17147(T) = JCM 19923(T)).

In vivo imaging of nitric oxide and reactive oxygen species using laser scanning confocal microscopy.

Both nitric oxide (NO) and reactive oxygen species (ROS) are versatile molecules that mediate a variety of cellular responses in plants. In this chapter, methods for imaging NO and ROS using laser scanning confocal microscopy (LSCM) are presented. Arabidopsis roots, dyed with DAF-FM or H(2)DCF, are observed using the Leica TCS-SP2 LSCM. NO or ROS production are imaged and their kinetic changes monitored with the laser excitation and emission wavelengths at 488 nm and between 500 and 530 nm, respectively. In addition, Leica software is employed to visualize and calculate the fluorescence intensity data.

ZmHO-1, a maize haem oxygenase-1 gene, plays a role in determining lateral root development.

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.

Isolation and characterization of a heme oxygenase-1 gene from Chinese cabbage.

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 (H(2)O(2)), and hemin treatments. Together, the results suggested that BrHO1 plays an important role in abiotic stress responses.

Characterization of a wheat heme oxygenase-1 gene and its responses to different abiotic stresses.

In animals and recently in plants, heme oxygenase-1 (HO1) has been found to confer protection against a variety of oxidant-induced cell and tissue injuries. In this study, a wheat (Triticum aestivum) HO1 gene TaHO1 was cloned and sequenced. It encodes a polypeptide of 31.7 kD with a putative N-terminal plastid transit peptide. The amino acid sequence of TaHO1 was found to be 78% similar to that of maize HO1. Phylogenetic analysis revealed that TaHO1 clusters together with the HO1-like sequences in plants. The purified recombinant TaHO1 protein expressed in Escherichia coli was active in the conversion of heme to biliverdin IXa (BV), and showed that the V(max) was 8.8 U·mg(-1) protein with an apparent K(m) value for hemin of 3.04 µM. The optimum Tm and pH were 35 °C and 7.4, respectively. The result of subcellular localization of TaHO1 showed that the putative transit peptide was sufficient for green fluorescent protein (GFP) to localize in chloroplast and implied that TaHO1 gene product is at least localized in the chloroplast. Moreover, we found that TaHO1 mRNA could be differentially induced by the well-known nitric oxide (NO) donor sodium nitroprusside (SNP), gibberellin acid (GA), abscisic acid (ABA), hydrogen peroxide (H(2)O(2)) and NaCl treatments. Therefore, the results suggested that TaHO1 might play an important role in abiotic stress responses.

Cloning and characterization of a heme oxygenase-2 gene from alfalfa (Medicago sativa L.).

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 H(2)O(2) (especially) treatment, respectively.

Molecular cloning and expression of a cucumber (Cucumis sativus L.) heme oxygenase-1 gene, CsHO1, which is involved in adventitious root formation.

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), CaCl(2), 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.

Molecular cloning, characterization, and expression of an alfalfa (Medicago sativa L.) heme oxygenase-1 gene, MsHO1, which is pro-oxidants-regulated.

It has been documented that plant heme oxygenase-1 (HO-1; EC 1.14.99.3) is both development- and stress-regulated, thus it plays a vital role in light signalling and stress responses. In this study, an alfalfa (Medica sativa L.) HO-1 gene MsHO1 was isolated and sequenced. It contains four exons and three introns within genomic DNA sequence and encodes a polypeptide with 283 amino acids. MsHO1 had a conserved HO signature sequence and showed high similarity to other HOs in plants, especially HO-1 isoform. The MsHO1:GFP fusion protein was localized in the chloroplast. Further biochemical activity analysis of mature MsHO1, which was expressed in Escherichia coli, showed that the Vmax was 48.78 nmol biliverdin-IXα (BV) h⁻¹ nmol⁻¹ protein with an apparent Km value for hemin of 2.33 µM, and the optimum Tm and pH were 37 °C and 7.2, respectively. Results of semi-quantitative RT-PCR and western blot showed that the expressions of MsHO1 were higher in alfalfa stems and leaves than those in germinating seeds and roots. Importantly, MsHO1 gene expression and protein level were induced significantly by some pro-oxidant compounds, including hemin and nitric oxide (NO) donor sodium nitroprusside (SNP). In conclusion, MsHO1 may play an important role in oxidative responses.

Evidence of Arabidopsis salt acclimation induced by up-regulation of HY1 and the regulatory role of RbohD-derived reactive oxygen species synthesis.

In Arabidopsis thaliana, a family of four genes (HY1, HO2, HO3 and HO4) encode haem oxygenase (HO), and play a major role in phytochrome chromophore biosynthesis. To characterize the contribution of the various haem oxygenase isoforms involved in salt acclimation, the effects of NaCl on seed germination and primary root growth in Arabidopsis wild-type and four HO mutants (hy1-100, ho2, ho3 and ho4) were compared. Among the four HO mutants, hy1-100 displayed maximal sensitivity to salinity and showed no acclimation response, whereas plants over-expressing HY1 (35S:HY1) exhibited tolerance characteristics. Mild salt stress stimulated biphasic increases in RbohD transcripts and production of reactive oxygen species (ROS) (peaks I and II) in wild-type. ROS peak I-mediated HY1 induction and subsequent salt acclimation were observed, but only ROS peak I was seen in the hy1-100 mutant. A subsequent test confirmed the causal relationship of salt acclimation with haemin-induced HY1 expression and RbohD-derived ROS peak II formation. In atrbohD mutants, haemin pre-treatment resulted in induction of HY1 expression, but no similar response was seen in hy1-100, and no ROS peak II or subsequent salt acclimatory responses were observed. Together, the above findings suggest that HY1 plays an important role in salt acclimation signalling, and requires participation of RbohD-derived ROS peak II.

Heme oxygenase is involved in cobalt chloride-induced lateral root development in tomato.

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 (CoCl(2)) was the first metal ion identified with an inducing property. Previous study suggests that CoCl(2) stimulates adventitious root formation in tomato and cucumber cuttings. In this test, we discover that both CoCl(2) 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 CoCl(2), 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 CoCl(2)-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 CoCl(2)-induced tomato lateral root formation.

Endogenous hydrogen peroxide plays a positive role in the upregulation of heme oxygenase and acclimation to oxidative stress in wheat seedling leaves.

Pretreatment of lower H(2)O(2) doses (0.05, 0.5 and 5 mM) for 24 h was able to dose-dependently attenuate lipid peroxidation in wheat seedling leaves mediated by further oxidative damage elicited by higher dose of H(2)O(2) (150 mM) for 6 h, with 0.5 mM H(2)O(2) being the most effective concentrations. Further results illustrated that 0.5 mM H(2)O(2) pretreatment triggered the biphasic production of H(2)O(2) during a 24 h period. We also noticed that only peak I (0.25 h) rather than peak II (4 h) was approximately consistent with the enhancement of heme oxygenase (HO) activity, HO-1 gene expression. Meanwhile, enhanced superoxide dismutase (SOD) activity, Mn-SOD and Cu,Zn-SOD transcripts might be a potential source of peak I of endogenous H(2)O(2). Further results confirmed that 0.5 mM H(2)O(2) treatment for 0.5 h was able to upregulate HO gene expression, which was detected by enzyme activity determination, semi-quantitative reverse transcription-polymerase chain reaction and western blotting. Meanwhile, the application of N,N'-dimethylthiourea, a trap for endogenous H(2)O(2), not only blocked the upregulation of HO, but also reversed the corresponding oxidation attenuation. Together, the above results suggest that endogenous H(2)O(2) production (peak I) plays a positive role in the induction of HO by enhancing its mRNA level and protein expression, thus leading to the acclimation to oxidative stress.

The heme oxygenase/carbon monoxide system is involved in the auxin-induced cucumber adventitious rooting process.

Indole acetic acid (IAA) is an important regulator of adventitious rooting via the activation of complex signaling cascades. In animals, carbon monoxide (CO), mainly generated by heme oxygenases (HOs), is a significant modulator of inflammatory reactions, affecting cell proliferation and the production of growth factors. In this report, we show that treatment with the auxin transport inhibitor naphthylphthalamic acid prevented auxin-mediated induction of adventitious rooting and also decreased the activity of HO and its by-product CO content. The application of IAA, HO-1 activator/CO donor hematin, or CO aqueous solution was able to alleviate the IAA depletion-induced inhibition of adventitious root formation. Meanwhile, IAA or hematin treatment rapidly activated HO activity or HO-1 protein expression, and CO content was also enhanced. The application of the HO-1-specific inhibitor zinc protoporphyrin IX (ZnPPIX) could inhibit the above IAA and hematin responses. CO aqueous solution treatment was able to ameliorate the ZnPPIX-induced inhibition of adventitious rooting. Molecular evidence further showed that ZnPPIX mimicked the effects of naphthylphthalamic acid on the inhibition of adventitious rooting, the down-regulation of one DnaJ-like gene (CSDNAJ-1), and two calcium-dependent protein kinase genes (CSCDPK1 and CSCDPK5). Application of CO aqueous solution not only dose-dependently blocked IAA depletion-induced inhibition of adventitious rooting but also enhanced endogenous CO content and up-regulated CSDNAJ-1 and CSCDPK1/5 transcripts. Together, we provided pharmacological, physiological, and molecular evidence that auxin rapidly activates HO activity and that the product of HO action, CO, then triggers the signal transduction events that lead to the auxin responses of adventitious root formation in cucumber (Cucumis sativus).

[Effects of exogenous nitric oxide donor on germination and activities of hydrolytic enzymes in wheat seed under osmotic stress].

Results in this paper showed that the effect of nitric oxide (NO) donor, sodium nitroprusside (SNP), which could promote the germination of wheat seeds under osmotic stress, was through enhancing imbibition using water content determination and imbibition analysis of wheat seed. SNP could also enhance amylase isozyme I activities, accelerate the liquefaction of endosperms, while have no effect on esterase. Moreover, SNP could strongly increase the sugars, such as the fructose, glucose and sucrose contents in wheat seeds during early germination (12 h) under normal conditions. When the seeds were treated with SNP with or without PTIO, an NO scavenger, and different exogenous sugars, i.e. fructose, glucose and sucrose, it was found that these sugars might activate amylase isozyme I activity mediated by the signal molecule NO to start the early stage of germination.

[The effect of exogenous glucose, fructose and NO donor sodium nitroprusside (SNP) on rice seed germination under salt stress].

The effects and the relationship between soaking rice seed of exogenous glucose, fructose and nitric oxide (NO) donor sodium nitroprusside (SNP) on the seed germination under salt stress were investigated. The results showed that employment of exogenous fructose, glucose and NO donor SNP could significantly promote the germination index and the early germination rate of rice seed under salt stress. Also, SNP pretreatment enhanced endogenous glucose and fructose contents. Effects of exogenous glucose and fructose combined with different concentrations of SNP on the rice seed germination and seedling growth under salt stress were surveyed. The results also showed that there existing positive effect of application of glucose plus SNP on the rice seed germination in comparison with existing a few negative effects of fructose plus SNP pretreatment. Additionally, the alleviation of inhibition of early rice seedling growth under salt stress by SNP could be modulated by glucose and fructose pretreatment, also the effect of glucose was stronger.

A rapid response of beta-amylase to nitric oxide but not gibberellin in wheat seeds during the early stage of germination.

The effects of nitric oxide (NO) and gibberellic acid (GA(3)) on the responses of amylases in wheat (Triticum aestivum L.) seeds (caryopses) were investigated during the first 12 h of germination. GA(3) had no effects on the activities of alpha-amylase (EC 3.2.1.1) or beta-amylase (EC 3.2.1.2), either in intact seeds or embryoless halves within 12 h. In contrast, addition of sodium nitroprusside (SNP), an NO donor, was able to induce a rapid increase in beta-amylase activity without affecting alpha-amylase. Furthermore, the rapid response of beta-amylase to SNP in wheat seeds could be attributed to NO and was approximately dose-dependent. Some other aspects of SNP induction of amylase isozymes were also characterized. Further investigations showed that SNP might play an interesting role in the dissociation of free beta-amylase from small homopolymers or heteropolymers. Furthermore, SNP also directly induced the release of bound beta-amylase from glutenin and its crude enzyme preparation. However, the slight increase in protease also induced by SNP might not be responsible for this action. Interestingly, based on the fact that the rapid response of beta-amylase to NO also existed in seeds of other species, such as barley, soybean, rice and watermelon, it might be a universal event in early seed germination.

[Exogenous nitric oxide alleviates osmotic stress-induced membrane lipid peroxidation in wheat seedling leaves].

When wheat (Triticum aestivum L. Yangmai 158) seedling (with three fully expanded leaves) roots were treated with 15% PEG-6000 in combination with different concentrations (0.1 and 0.5 mmol/L) of exogenous nitric oxide donor sodium nitroprusside (SNP) and NO(-)(3)/NO(-)(2) (control), the enhancement of lipoxygenase (LOX) activity in wheat seedling leaves under osmotic stress was delayed at the lower concentration of SNP treatment (0.1 mmol/L), while the generation rate of O(-.)(2), the enhancement of membrane permeability and the accumulation of MDA and H(2)O(2)were also alleviated. SNP 0.1 mmol/L significantly enhanced the activity of superoxide dismutase (SOD), and accelerated the accumulation of proline. In comparison with these changes, the effects of higher concentration of SNP (0.5 mmol/L) and corresponding control (NO(-)(3)/NO(-)(2)) were not so obvious. The above results indicate that lower concentrations of in vitro nitric oxide (NO) can protect wheat seedling leaves from membrane lipid peroxidation caused by osmotic stress.

[Effects of nitric oxide on root growth and its oxidative damage in wheat seedling under salt stress].

Effects of nitric oxide (NO), a substance newly found to have protective functions in plants, on root growth of wheat (Triticum aestivum L. Yangmai 158) seedlings under salt stress were studied. Sodium nitroprusside (SNP), an NO donor, markedly alleviated the inhibitory effect of salt on root elongation at salt concentrations around 150 mmol/L, but was ineffective when NaCl concentration was at 300 mmol/L or higher. It was most effective at 0.05-0.1 mmol/L, and had harmful effect at 0.30-5 mmol/L. Furthermore, when wheat seedling roots were treated with 150 mmol/L NaCl in combination with 0.05 mmol/L SNP and NO scavenger (hemoglobin, Hb), NO(-)(2)/NO(-)(3) and K(3)Fe(CN)(6) as controls, both of which were the two other major products besides NO when SNP dissolved in water, respectively, activities of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) were enhanced to some extent. While, the generation of O(-.)(2) and the accumulation of MDA and H(2)O(2) were alleviated. DNA laddering was observed when wheat seedling roots exposed to 150 mmol/L NaCl for 6 d, also suggesting that salt stress might induce oxidative damage in root-tips. In combination with hemoglobin, NO(-)(2)/NO(-)(3) and K(3)Fe(CN)(6) as controls, the results showed that NO could block DNA ladders. Above results suggest that it is NO rather than any substances which is protecting root-tip cells of wheat seedlings from oxidative damage caused by salt stress.