施氮量对小麦叶片硝酸还原酶活性、一氧化氮含量和气体交换的影响

研究了不同施氮量对冬小麦分蘖到抽穗期叶片硝酸还原酶(NR)活性、一氧化氮(NO)含量、气体交换参数和籽粒产量的影响.结果表明:叶片光合速率（P_n）、蒸腾速率（T_r）、瞬时水分利用效率（IWUE）和产量均随施氮量的增加呈先升高后降低的趋势,在180 kg·hm^-2氮处理时达到最高.随施氮量的增加,叶片NR活性提高; 在分蘖期和拔节期,叶片NR活性与NO含量呈显著线性相关（R²≥0.68,n=15）,NO含量和气孔导度（G_s）呈显著正二次相关（R²≥0.43,n=15）;低氮处理下,NR活性较低使叶片NO含量维持在较低水平,促进气孔开放,高氮处理下,NR活性较高使叶片NO含量增加,诱导气孔关闭;在抽穗期叶片NR活性和NO含量无显著相关关系,虽然NO含量和G_s也呈显著正二次相关（R²≥0.36,n=15）,但不能通过施氮提高NR活性来影响叶片NO含量,进而调节叶片气孔行为.合理施氮使小麦叶片NO含量维持在较低水平,可提高叶片G_s、T_r和IWUE,增强作物抗旱能力,促进光合作用,提高小麦产量.     

Spectroscopic and kinetic studies of Nor1, a cytochrome P450 nitric oxide reductase from the fungal pathogen Histoplasma capsulatum

The fungal respiratory pathogen Histoplasma capsulatum evades the innate immune response and colonizes macrophages during infection. Although macrophage production of the antimicrobial effector nitric oxide (NO) restricts H. capsulatum growth, the pathogen is able to establish a persistent infection. H. capsulatum contains a P450 nitric oxide reductase homologue (NOR1) that may be important for detoxifying NO during infection. To characterize the activity of this putative P450 enzyme, a 404 amino acid fragment of Nor1p was expressed in Escherichia coli and purified to homogeneity. Spectral characterization of Nor1p indicated that it was similar to other fungal P450 nitric oxide reductases. Nor1p catalyzed the reduction of NO to N₂O using NADH as the direct reductant. The K_M for NO was determined to be 20 μM and the k_cat to be 5000 min⁻¹. Together, these results provide evidence for a protective role of a P450 nitric oxide reductase against macrophage-derived NO.     

Phytoglobins in the nuclei,cytoplasm and chloroplasts modulate nitric oxide signaling and interact with abscisic acid

Symbiotic hemoglobins provide O₂ to N₂‐fixing bacteria within legume nodules, but the functions of non‐symbiotic hemoglobins or phytoglobins (Glbs) are much less defined. Immunolabeling combined with confocal microscopy of the Glbs tagged at the C‐terminus with green fluorescent protein was used to determine their subcellular localizations in Arabidopsis and Lotus japonicus. Recombinant proteins were used to examine nitric oxide (NO) scavenging in vitro and transgenic plants to show S‐nitrosylation and other in vivo interactions with NO and abscisic acid (ABA) responses. We found that Glbs occur in the nuclei, chloroplasts and amyloplasts of both model plants, and also in the cytoplasm of Arabidopsis cells. The proteins show similar NO dioxygenase activities in vitro, are nitrosylated in Cys residues in vivo, and scavenge NO in the stomatal cells. The Cys/Ser mutation does not affect NO dioxygenase activity, and S‐nitrosylation does not significantly consume NO. We demonstrate an interaction between Glbs and ABA on several grounds: Glb1 and Glb2 scavenge NO produced in stomatal guard cells following ABA supply; plants overexpressing Glb1 show higher constitutive expression of the ABA responsive genes Responsive to ABA (RAB18), Responsive to Dehydration (RD29A) and Highly ABA‐Induced 2 (HAI2), and are more tolerant to dehydration; and ABA strongly upregulates class 1 Glbs. We conclude that Glbs modulate NO and interact with ABA in crucial physiological processes such as the plant's response to dessication.     

Indirect determination of nitric oxide production by reduction of nitrate with a freeze-thawing-resistant nitrate reductase from Escherichia coli MC1061

Preparation of a nitrate reductase lysate of Escherichia coli MC1061 to measure nitrate and nitrite in biologic fluids is described. To obtain the crude bacterial lysate containing nitrate reductase activity, E. coli MC1061 was subjected to 16-20 freeze-thawing cycles, from -70 to 60 degrees C, until nitrite reductase activity was < or = 25%. Nitrate reductase activity was detected mainly in the crude preparation. To validate the nitrate reduction procedure, standard nitrate solutions (1.6-100 microM) were incubated with the nitrate reductase preparation for 3 h at 37 degrees C, and nitrite was estimated by the Griess reaction in a microassay. Nitrate solutions were reduced to nitrite in a range of 60-70%. Importantly, no cofactors were necessary to perform nitrate reduction. The biological samples were first reduced with the nitrate reductase preparation. After centrifugation, samples were deproteinized with either methanol/ether or zinc sulfate and nitrite was quantified. The utility of the nitrate reductase preparation was assessed by nitrate+nitrite determination in serum of animals infected with the protozoan Entamoeba histolytica or the bacteria E. coli and in the supernatant of cultured lipopolysaccharide-stimulated RAW 264.7 mouse macrophages. Our results indicate that the nitrate reductase-containing lysate provides a convenient tool for the reduction of nitrate to determine nitrate+nitrite in biological fluids by spectrophotometric methods.     

Expression of a constitutively active nitrate reductase variant in tobacco reduces tobacco‐specific nitrosamine accumulation in cured leaves and cigarette smoke

Burley tobaccos (Nicotiana tabacum) display a nitrogen‐use‐deficiency phenotype that is associated with the accumulation of high levels of nitrate within the leaf, a trait correlated with production of a class of compounds referred to as tobacco‐specific nitrosamines (TSNAs). Two TSNA species, 4‐(methylnitrosamino)‐1‐(3‐pyridyl)‐1‐butanone (NNK) and N‐nitrosonornicotine (NNN), have been shown to be strong carcinogens in numerous animal studies. We investigated the potential of molecular genetic strategies to lower nitrate levels in burley tobaccos by overexpressing genes encoding key enzymes of the nitrogen‐assimilation pathway. Of the various constructs tested, only the expression of a constitutively active nitrate reductase (NR) dramatically decreased free nitrate levels in the leaves. Field‐grown tobacco plants expressing this NR variant exhibited greatly reduced levels of TSNAs in both cured leaves and mainstream smoke of cigarettes made from these materials. Decreasing leaf nitrate levels via expression of a constitutively active NR enzyme represents an exceptionally promising means for reducing the production of NNN and NNK, two of the most well‐documented animal carcinogens found in tobacco products.     

Overexpression of class 1 plant hemoglobin genes enhances symbiotic nitrogen fixation activity between Mesorhizobium loti and Lotus japonicus

Plant hemoglobins (Hbs) have been divided into three groups: class 1, class 2, and truncated Hbs. The various physiological functions of class 1 Hb include its role as a modulator of nitric oxide (NO) levels in plants. To gain more insight into the functions of class 1 Hbs, we investigated the physical properties of LjHb1 and AfHb1, class 1 Hbs of a model legume Lotus japonicus and an actinorhizal plant Alnus firma , respectively. Spectrophotometric analysis showed that the recombinant form of the LjHb1 and AfHb1 proteins reacted with NO. The localization of LjHb1 expression was correlated with the site of NO production. Overexpression of LjHb1 and AfHb1 by transformed hairy roots caused changes in symbiosis with rhizobia. The number of nodules formed on hairy roots overexpressing LjHb1 or AfHb1 increased compared with that on untransformed hairy roots. Furthermore, nitrogenase activity as acetylene-reduction activity (ARA) of LjHb1- or AfHb1 -overexpressing nodules was higher than that of the vector control nodules. Microscopic observation with a NO-specific fluorescent dye suggested that the NO level in LjHb1 - and AfHb1 -overexpressing nodules was lower than that of control nodules. Exogenous application of a NO scavenger enhanced ARA in L. japonicus nodule s , whereas a NO donor inhibited ARA. These results suggest that the basal level of NO in nodules inhibits nitrogen fixation, and overexpression of class 1 Hbs enhances symbiotic nitrogen fixation activity by removing NO as an inhibitor of nitrogenase.     

Comparison of effects of nitric oxide synthase (NOS) inhibitors on plasma nitrite/nitrate levels and tissue NOS activity in septic organs

An excessive production of nitric oxide (NO) by NO synthase (NOS) is considered to contribute to circulatory disturbance, tissue damage, and refractory hypotention, which are often observed in septic disorders. It is anticipated that a selective inducible NOS (iNOS) inhibitor with excellent pharmacokinetics may be potentially effective as a novel and potent therapeutic intervention in sepsis. We examined whether or not a selective iNOS inhibitor shows iNOS selectivity at the tissue level, when administered systemically. The effects of four NOS inhibitors on plasma nitrite/nitrate (NOx) and tissue NOS levels were compared in major organs (lungs, liver, heart, kidneys, and brain) 6 hr after the injection of E. coli lipopolysaccharide (LPS) into male Wistar-King rats. The rats treated with the three iNOS inhibitors (N-(3-(aminomethyl)benzyl)acetamidine (1400W), (1 S, 5 S, 6 R, 7 R )-2-aza-7-chloro-3-imino-5-methylbicyclo [4.1.0] heptane hydrochloride (ONO-1714), and aminoguanidine) administered 1 hr after LPS injection, showed dose-dependent decreases in plasma NOx levels and NOS activity in the lungs. The non-selective NOS inhibitor (N(G)-methyl-L-arginine (L-NMMA)) had an effect only at the maximum dose. The differences in in vitro iNOS selectivity among these drugs did not correlate with iNOS selectivity at the tissue level. The relationship between plasma NOx levels and NOS activity in the lungs showed a linear relationship with or without the NOS inhibitors. In conclusion, the iNOS selectivity of these drugs does not seem to differ at the tissue level. Plasma NOx levels may be a useful indicator of lung NOS activity.     

Babo1, formerly Vop1 and Cop1/2, is no eyespot photoreceptor but a basal body protein illuminating cell division in Volvox carteri

In photosynthetic organisms many processes are light dependent and sensing of light requires light‐sensitive proteins. The supposed eyespot photoreceptor protein Babo1 (formerly Vop1) has previously been classified as an opsin due to the capacity for binding retinal. Here, we analyze Babo1 and provide evidence that it is no opsin. Due to the localization at the basal bodies, the former Vop1 and Cop1/2 proteins were renamed V.c. Babo1 and C.r. Babo1. We reveal a large family of more than 60 Babo1‐related proteins from a wide range of species. The detailed subcellular localization of fluorescence‐tagged Babo1 shows that it accumulates at the basal apparatus. More precisely, it is located predominantly at the basal bodies and to a lesser extent at the four strands of rootlet microtubules. We trace Babo1 during basal body separation and cell division. Dynamic structural rearrangements of Babo1 particularly occur right before the first cell division. In four‐celled embryos Babo1 was exclusively found at the oldest basal bodies of the embryo and on the corresponding d‐roots. The unequal distribution of Babo1 in four‐celled embryos could be an integral part of a geometrical system in early embryogenesis, which establishes the anterior–posterior polarity and influences the spatial arrangement of all embryonic structures and characteristics. Due to its retinal‐binding capacity, Babo1 could also be responsible for the unequal distribution of retinoids, knowing that such concentration gradients of retinoids can be essential for the correct patterning during embryogenesis of more complex organisms. Thus, our findings push the Babo1 research in another direction.     

Changes in nitric oxide levels and their relationship with callose deposition during the interaction between soybean and Soybean mosaic virus

• The present study aimed to investigate changes in nitric oxide (NO) level and its relationship with callose deposition during the interaction between soybean and Soybean mosaic virus (SMV).
• Soybean cv. ‘Jidou 7’ and SMV strains N3 and SC‐8 were used to constitute incompatible and compatible combinations. Intracellular NO was labelled with the NO‐specific fluorescence probe DAF‐FM DA. Confocal laser scanning microscopy (CLSM) was then used to observe changes in NO production during SMV infection‐induced defence responses in soybean.
• The results showed NO fluorescence increased rapidly at 2–72 h post‐inoculation, peaked at 72 h and then decreased in the incompatible combination. However, in the compatible combination, extremely weak NO fluorescence appeared in the early stage (2–24 h) post‐inoculation, but was not observed thereafter. Injections of the NO scavenger c‐PTIO prior to inoculation postponed the onset of NO production to 48 or 72 h post‐inoculation. The same occurred when injections of NR or NOS inhibitors were applied prior to inoculation. The observation of callose fluorescence in the incompatible combination revealed that either the elimination or reduction of NO in the early stage led to a delay in callose formation, enabling the virus to cause systemic infection.
• Together with our previous findings, this study indicates that viral infection could induce NO production and callose deposition during the incompatible interaction between soybean and SMV. The production of NO involves NR and NOS enzymatic pathways, and NO mediates the process of callose deposition at plasmodesmata.

     

外源一氧化氮供体硝普钠浸种对盐胁迫下小麦幼苗碳氮代谢及抗氧化系统的影响

预先用0.1mmol/L的SNP(硝普钠,NO供体)浸种,研究NO预处理对120mmol/L NaCl胁迫下两小麦品种(扬麦12和淮麦17)幼苗叶片抗氧化系统、碳氮代谢及蛋白酶活性的影响。结果表明,NO预处理能有效地抑制NaCl胁迫下小麦幼苗叶片超氧阴离子释放(O.2-)和过氧化氢(H2O2)积累,提高超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性,降低丙二醛(MDA)含量,提高叶绿素、类胡萝卜素和可溶性总糖含量。另外,NO预处理显著提高叶片可溶性蛋白质含量,以及内肽酶和羧肽酶活性。分析表明,NO有利于维持盐胁迫下小麦碳氮代谢正常运转,从而促进植株生长,提高小麦幼苗株高、鲜重和干重。试验条件下,NO对淮麦17的促进作用大于扬麦12。     

Structure and activity of the denitrifying community in a maize-cropped field fertilized with composted pig manure or ammonium nitrate

One alternative to mineral fertilization is to use organic fertilizers. Our aim was to compare the impacts of 7-year applications of composted pig manure and ammonium nitrate on the structure and activity of the denitrifying community. Mineralization and organization of N, denitrification rates and N2O/N2 ratio were also investigated. Fourteen months after the last application, the potential denitrifying activity (+319%), N mineralization (+110%) and organization (+112%) were higher under pig compost than under ammonium nitrate fertilization. On the other hand, the N2O/(N2O+N2) ratio was lower (P<0.05, n=5) under organic fertilization. These effects of organic fertilization were in accordance with its higher total carbon content and microbial biomass. Fingerprints and clone library analyses showed that the structure of the denitrifying community was affected by the fertilization regime. Our results reveal that organic or mineral fertilizer applications could affect both structure and activity of the denitrifying community, with a possible influence on in situ N2O fluxes. These effects of the fertilization regime persisted for at least 14 months after the last application.