Association of heat shock induced resistance to disease with increased accumulation of insoluble extensin and ethylene synthesis

Cucumber seedlings (Cucumis sativus L. cv Marketer), heat-shocked for 40 s at 50 °C, acquire disease resistance to the fungal pathogen Cladosporium cucumerinum E11. and Arth. The resistance develops 15 to 21 h after heat shock treatment and is associated with stimulated ethylene production and changes in the plant cell wall. There was a two-fold increase in the production of ethylene and in its precursor 1-aminocyclopropane-1-carboxylic acid by 6 h after the heat shock. Heat shock also enhanced the accumulation of insoluble extensin, a hydroxyproline-rich glycoprotein found in cell walls of seedlings. A heat shock treatment of seedlings 24 h before inoculation with C. cucumerinum resulted in a doubling of extensin content at 72 h after the inoculation. Cell walls from heat shocked seedlings were up to 55% more resistant to degradation by enzymes from C. cucumerinum than were cell walls from unshocked seedlings, but increased lignin depositions did not appear responsible. The accumulation of extensin after heat shock and its crosslinking by peroxidase is discussed as a possible mechanism of induced resistance to C. cucumerinum.     

Kinetics of the reaction of the first metabolite of fenitropan with some low molecular weight thiols

2-Nitro-I-phenylpropen-3-yl acetate (NPPA), the first metabolite of the fungicide fenitropan, reacted readily with glutathione in aqueous ethanol solutions at room temperature, producing at least two products. The first reaction step, which is believed to be a bimolecular nucleophilic (Michael) addition, had a strongly pH-dependent reaction rate. Second-order rate constants were calculated for the reactions of NPPA at pH 4 with glutathione and with some other low molecular weight thiols. Nucleophilic reactivity of the reacting thiolate anions is linearly related to the pK_a values of the thiols. However, as the concentration of thiolate anion is also determined by the thiol pK_a, there is no simple relationship between second-order rate constants and thiol pK_a. Thus the blocking rate of the various essential sulphydryl groups in fungi by NPPA is determined by the sulphydryl pK_a values and the ambient pH value.     

ROS and NO production in compatible and incompatible tomato-<Emphasis Type="Italic">Meloidogyne incognita</Emphasis> interactions

Nitric oxide (NO) has been shown to be an essential regulatory molecule in plant response to pathogen infection in synergy with reactive oxygen species (ROS). At the present, nothing is known about the role of NO in disease resistance to nematode infection. We used a resistant tomato cultivar with different sensitivity to avirulent and virulent populations of the root-knot nematode Meloidogyne incognita to investigate the key components involved in oxidative and nitrosative metabolism. We analyzed the superoxide radical production, hydrogen peroxide content, and nitric oxide synthase (NOS)-like and nitrate reductase activities, as potential sources of NO. A rapid NO accumulation and ROS production were found at 12 h after infection in compatible and incompatible tomato-nematode interactions, whereas the amount of NO and ROS gave different results 24 and 48 h after infection amongst compatible and incompatible interactions. NOS-like arginine-dependent enzyme rather than nitrate reductase was the main source of NO production, and NOS-like activity increased substantially in the incompatible interaction. We can envisage a functional overlap of both NO and ROS in tomato defence response to nematode invasion, NO and H₂O₂ cooperating in triggering hypersensitive cell death. Therefore, NO and ROS are key molecules which may help to orchestrate events following nematode challenge, and which may influence the host cellular metabolism.     

Approaches to relieving aeration stress in waterlogged plants

A fertile soil contains sufficient water-filled pores to replenish losses by evapotranspiration and to sustain growth. However, too many such pores can be detrimental because the exchange of gases between the soil, plant roots and the aerial environment is impaired by the water they contain (waterlogging). The consequences include an accumulation of metabolically generated gases (such as ethylene), oxygen deficiency and the production of potentially toxic substances. Knowledge of these effects, and their consequences for growth, hormone production and inorganic nutrient uptake, enhance the prospects for ameliorating flooding injury by prophylactic or restorative means. Theexamples described are (a) the use of more porousmedia for plant containers in the nutrient film technique (NFT) of glasshouse crop production; (b) fungistatic, acid neutralising and oxygen-releasing seed coatings; (c) boron and nitrogen fertilisation; (d)foliar applications of cytokinins and gibberellins; (e) the inhibition of ethylene action or stimulation of ethylene production in aquatic species.     

<Emphasis Type="Italic">N</Emphasis>,<Emphasis Type="Italic">N</Emphasis>-dimethylsphingosine,an inhibitor of sphingosine kinase,induces phytoalexin production and hypersensitive cell death of Solanaceae plants without generation of reactive oxygen species

Plant recognition of elicitors derived from pathogens induces various resistant reactions, including production of reactive oxygen species, hypersensitive cell death and accumulation of phytoalexins. Previously, we isolated a ceramide elicitor from Phytophthora infestans, which activates O₂ ⁻ production of potato suspension-cultured cells. In this study, we employed nine ceramide-related chemicals to test their elicitor activity. Although, none of the tested chemicals induced O₂ ⁻ production, N,N-dimethylsphingosine (DMS) induced accumulation of phytoalexin in potato tubers. In potato, tobacco and Nicotiana benthamiana, DMS also induced rapid cell death. DMS-treated potato cells stained with 4′,6-diamidino-2-phenylindole (DAPI) showed chromatin condensation, and isolated DNA from DMS-treated cells had ladder pattern, confirming that DMS-induced plant cell death is a hypersensitive reaction-like programmed cell death. Involvement of ceramide signaling in induction of plant defense reactions is discussed. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effect of atrazine and methabenzthiazuron on oxygen evolution and cell growth of Chlorella pyrenoidosa

Cultures of Chlorella pyrenoidosa Pringsheim were grown photoautotrophically in the presence of two concentrations (0·25 and 0·50 μm ) of atrazine and methabenzthiazuron. The cell density and chlorophyll content were greatly reduced in presence of both herbicides. These herbicides affected net photsynthesis equally without affecting oxygen uptake. However, cell division was more inhibited by atrazine than by methabenzthiazuron, but chlorophyll content per cell was more inhibited by methabenzthiazuron than by atrazine.     

Plant cell cultures as a model for the biochemistry of crop selectivity

Plant cell cultures have been used to study the metabolic degradation of 4-amino-5-methyl-2-(tert-butylaminocarbonyl)-1,2,4-triazolin-3-one. The biochemical basis of selectivity was shown to reside in effective metabolic conjugation. The herbicide was eliminated from cell cultures of beet (which is tolerant to it) by conjugation (N-glycolisation), but this occured to only a limited extent with cell cultures of non-target plants such as soybean.     

Two phases of intracellular reactive oxygen species production during victorin-induced cell death in oats

Reactive oxygen species (ROS) are thought to be involved in various forms of programmed cell death (PCD) in animal and plant cells. PCD, along with the production of ROS, occurs during plant–pathogen interactions. Here we show that victorin, a host-specific toxin produced by Cochliobolus victoriae, which causes victoria blight of oats, induces two phases of intracellular ROS production in victorin-sensitive oat mesophyll cells. The initial production of ROS is restricted at mitochondria and not accompanied with cellular oxidative damage. Later production of ROS is dispersed into cells concomitant with lipid peroxidation, chloroplast dysfunction, and cell death. Superoxide dismutase can clearly suppress the initial ROS production and delay the progression of cell death. These data indicate that the initial ROS production may be involved in the cell death induction process, and the later ROS production may play important roles in events leading to cellular disruption.     

Stimulation of respiratory pathways in tomato roots infested by Meloidogyne incognita

Respiration of tomato roots susceptible and resistant to Meloidogyne incognita was measured during infestation. No significant changes in respiratory rate occurred in susceptible tomato roots, during infestation by M. incognita. In resistant tomato roots, a pronounced increase of both cyanide-sensitive and cyanide-resistant oxidases, was observed during nematode attack. The time-course of the respiration during 12 days, after nematode inoculation, showed that resistant tomato roots responded with a rapid increase in cyanide-sensitive and cyanide-resistant respiration as invading nematodes progressed; no changes were observed in the susceptible tomato roots.Change in the rate of oxygen uptake paralleled an increase in nematode density in resistant tomato roots; oxygen uptake rose linearly to an infestation level of 50 juveniles for each seedling, above which value it declined. The physiological significance of the alternative respiratory pathway is discussed.     

Alternative Respiration: a Biochemical Mechanism of Resistance to Azoxystrobin (ICIA 5504) in Septoria tritici

The mechanism of resistance to ICIA 5504 (azoxystrobin) in a Septoria tritici mutant raised in the laboratory has been investigated. This mutant was approximately 10 times less sensitive than the wild-type strain in in-vitro tests towards spore germination or fungal growth. Glucose oxidation in whole cells was inhibited in the wild type (80% inhibition at 0·1 μg ml^-1), whereas in the resistant mutant, oxygen uptake was stimulated (50% stimulation at 1·0 μg ml^-1). Respiration of the wild-type strain was inhibited by antimycin A and cyanide but not that of the mutant. These results indicate the existence of an efficient alternative respiratory pathway in the mutant, which was inhibited by the addition of 2 mM salicylhydroxamate (SHAM). Using mitochondria, antimycin A and ICIA 5504 did not completely inhibit NADH oxidation in either strain. Addition of SHAM inhibited part of the antimycin- and ICIA 5504-insensitive oxygen uptake only in mutant mitochondria. For complete inhibition of oxygen reduction, SHAM and cyanide need to be present. Thus, three systems of electron transfer from exogenous NADH to oxygen are present in S. tritici mitochondria: the cytochrome pathway which is sensitive to ICIA 5504 and antimycin A inhibition in both strains, the system of NADH-cytochrome c reductase which bypasses the methoxyacrylate inhibition at the cytochrome bc₁ complex, and the alternative oxidase which is inhibited by SHAM, and which is partially functioning only in mitochondria isolated from the ICIA 5504-resistant mutant. When the S. tritici isolates were tested for their in-vivo sensitivity to ICIA 5504 on wheat, the resistant strain was controlled better than the wild type. This indicates that the decreased ATP formation by the alternative pathway of respiration was inadequate for efficient parasitic growth on the host. © 1997 SCI.     

Interaction with phospholipids as a possible mode of action of 3-phenylindole on Aspergillus niger

Low concentrations of 3-PI(3-phenylindole) inhibit several uptake and biosynthetic processes in liquid cultures of Aspergillus niger. The incorporation of ³²P_i into phospholipids, [¹⁴C]uridine into nucleic acids, and [¹⁴C]phenylalanine into proteins was inhibited and the uptake of all three precursors was reduced. Studies on an in vitro interaction between 3-phenylindole and phospholipids showed, that at about equimolar concentrations 3-phenylindole prevents phospholipids from swelling in water. At the same ratio a decrease of the absorption intensity of the NH-band of 3-phenylindole was observed in ir spectra. Moreover, the maxima of the uv spectrum of 3-phenylindole shifted after addition of a sonicated phospholipid suspension. It is suggested that binding to phospholipids is the first effect of 3-phenylindole. Apparently this affects, consequently, several membrane-bound reactions, i.e., transport and biosynthetic processes.     

ABA signaling inhibits oxalate-induced production of reactive oxygen species and protects against <Emphasis Type="Italic">Sclerotinia sclerotiorum</Emphasis> in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Oxalic acid is an essential virulence factor of Sclerotinia sclerotiorum that elicits wilting symptoms in infected host plants. Foliar wilting in response to oxalic acid is known to be dependent on an increase in stomatal conductance. To determine whether stomatal regulation controls susceptibility to S. sclerotiorum, abscisic acid-insensitive and open stomata mutants of Arabidopsis thaliana were analyzed. Whereas abscisic acid-insensitive mutants were hypersusceptible to S. sclerotiorum, open stomata mutants were as susceptible as wild type. It was concluded that stomatal regulation does not control susceptibility to S. sclerotiorum because open stomata mutants are known to only impair guard cells whereas abscisic acid-insensitive mutants also affect other cell types. Guard cell-independent processes also control sensitivity to oxalic acid because oxalic acid was more toxic to abscisic acid-insensitive mutants than to open stomata mutants. To explore a possible mechanism of toxicity, production of reactive oxygen species was measured in plant cells after exposure to oxalic acid. Oxalic acid was found to elicit reactive oxygen species production independently of abscisic acid. Nevertheless, cancellation of reactive oxygen species elicitation after co-stimulation of wild-type guard cells with oxalic acid and abscisic acid provided evidence for antagonistic interaction between both molecules.     

In vitro production of pycnidia by Septoria tritici

The in vitro production of pycnidia bySeptoria tritici was examined on six media reported to induce the formation of fruiting bodies. Among 26 freshly isolated cultures from various parts of the world, consistent differences in growth type were found which were only partially influenced by nutritional and environmental conditions. Cultures with yeast-like growth produced hardly any pycnidia or pseudopycnidia, while cultures with intermediate or mycelial growth types produced them frequently. Incubation in continuous darkness induced intermediate to mycelial growth types rather than yeast-like growth types in some cultures, and concomitantly the production of more pycnidia. Potato-dextrose agar induced intermediate to mycelial growth types and production of (pseudo)pycnidia more often than V8 agar and wheat leaf extract agar, which had previously been reported to be especially beneficial to (pseudo) pycnidium formation byS. tritici. Isolates with a consistently yeast-like growth type, producing (virtually) no fructifications under any of the experimental conditions, were slightly stimulated to form pseudopycnidia on water agar supplemented with sterile pieces of maize, wheat or carnation leaves.     

Recovery of solubilized δ‐endotoxin from Bacillus thuringiensis subsp kurstaki fermentation broth

Insecticidal δ‐endotoxin proteins, degraded from parasporal crystals by protease, were recovered by a simple procedure using heat treatment, solubilization, and ultrafiltration of a fermentation broth of Bacillus thuringiensis subsp kurstaki HD‐1. A 68 kDa insecticidal protein was obtained and characterized by SDS‐PAGE. The procedure described gave a nearly quantitative recovery of toxicity. Furthermore, bioassay results on larvae of the diamondback moth (Plutella xylostella) showed that the 68 kDa δ‐endotoxin fraction (P1) was the principal insecticidal component to this target insect. A similar molecular mass polypeptide P2 (65 kDa) which was solubilized together with P1 from the parasporal crystals, gave relatively low mortality. © 2000 Society of Chemical Industry     

A plastidic glucose-6-phosphate dehydrogenase is responsible for hypersensitive response cell death and reactive oxygen species production

Glucose-6-phosphate dehydrogenase (G6PDH) has been implicated in the supply of reduced nicotine amide cofactors for resistance to biotic and abiotic stresses. Here, we show participation of the plastidic P2 isoform of G6PDH in plant immunity. A cytosolic isoform (NbG6PDH-Cyto) and two plastidic isoforms (NbG6PDH-P1 and NbG6PDH-P2) cloned from Nicotiana benthamiana were localized in cytosol and chloroplasts, respectively. Hypersensitive response (HR) cell death and NADPH oxidase (RBOH; respiratory burst oxidase homolog)-dependent reactive oxygen species (ROS) production after recognition of INF1 elicitin, secreted by oomycete Phytophthora infestans, decreased in NbG6PDH-P2-silenced plants, but not in NbG6PDH-Cyto- and NbG6PDH-P1-silenced plants. Silencing of the cytosolic NAD kinase NbNADK1, which phosphorylates NADH to form NADPH, compromised HR cell death and ROS production, and concomitant silencing with NbG6PDH-P2 reduced HR cell death and ROS to levels near those in NbG6PDH-P2-silenced plants. Similarly, silencing NbG6PDH-P2 and NbNADK1 resulted in high susceptibility to P. infestans. These results suggest that NADPH produced by the P2 isoform of G6PDH in chloroplasts is responsible for HR cell death and ROS production mediated by RBOH and that NbNADK1 is involved in this pathway.     

Detection of bacterial aggregation in tobacco cell suspensions treated with pathogenic bacteria

Previous studies of this model system involving plant cell suspensions inoculated with bacteria, have documented that interactions with incompatible pathogens, which cause a hypersensitive response on whole plants, will cause a transient increase in oxygen uptake 2–4 h after inoculation. The initial objective of this study was to determine whether this oxygen uptake burst was a result of increased bacterial multiplication, possibly due to nutrient leakage from plant cells. The adaptation of flow cytometry and the use of fluorescent nucleic acid stains provided the precision needed to monitor bacterial concentrations in tobacco suspension cells inoculated with pathogenic and non-pathogenic Pseudomonas species. Surprisingly, there was a transient decrease in the planktonic, or free-living, bacteria in cell suspensions inoculated with isolate Pseudomonas syringae pv. syringae WT (HR+), an incompatible pathogen of tobacco. This decrease in planktonic numbers was followed by an apparent increase in bacterial multiplication. Examination of the samples with fluorescent microscopy revealed the formation of bacterial aggregates in the extracellular fluid of the Pss WT (HR+) inoculated plant cells. The size of the aggregates increased at the onset of the oxygen uptake response, and contained increasing numbers of bacterial cells. These aggregated bacterial cells appear to be removed along with plant cells, as a result of filtration during sample preparation, causing the apparent decrease in planktonic bacteria detected by flow cytometry. This bacterial aggregation was also observed with the compatible Pseudomonas tabaci pathogen, which does not induce a noticeable oxygen uptake burst. No aggregation was observed with suspension inoculated with Pseudomonas fluorescens, a saprophyte, or Pss B7 (HR−), a Tn5 mutant of P. s. syringae. This aggregation response was rapid, once initiated, and appeared similar to reports of adhesion involving Hrp pili.     

利用HYDRUS-2D模拟膜下滴灌玉米农田深层土壤水分动态与根系吸水

河套灌区农田地下水埋深普遍较浅且年内波动较大,明确不同膜下滴灌条件下深层土壤水分对根区的补给作用及作物根系吸水的响应差异有利于膜下滴灌技术的完善和推广。本研究开展了连续2 a(2017—2018年)的春玉米田间试验,设置3个膜下滴灌灌溉水平,分别控制土壤基质势下限为-10 kPa(S1)、-30 kPa(S3)和-50 kPa(S5)。利用HYDRUS-2D模型模拟0～120 cm深度土壤含水量、根层下边界(100 cm深度处)水分通量和作物根系吸水速率。结果表明,经过率定后的HYDRUS-2D模型对0～120 cm深度土壤含水量模拟结果的根均方差(RMSE)和决定系数(R~2)分别为0.039～0.042 cm~3·cm~(-3)和0.78～0.73,模拟结果可靠。膜下滴灌农田100 cm和120 cm深度处土壤含水量较高且处理间差异不大,说明不同滴灌条件对于100 cm以下深层土壤含水量影响较小;但不同处理显著影响根区下边界的水分通量和根系吸水速率。基质势下限控制水平越低,深层土壤水分对于根区的补给量(毛管上升)越大,S1、S3、S5生育期内累积补给量在31.9～49.6 mm之间。S5处理根系吸水速率较低,根系吸水受到显著抑制,从而造成作物生长指标和产量显著低于S1和S3处理(P0.05);而S1和S3之间籽粒产量差异不显著。综上,在本研究所设置的3个滴灌处理中,S3生育期内灌溉定额为240～300 mm,既较S1显著减少灌水量、提高水分利用效率,又具有较好的根系活力,有效利用深层土壤水分,因此建议该地区春玉米膜下滴灌的灌水下限为-30 kPa。     

Mechanism of cellular absorption of imidazolinones in soybean (Glycine max) leaf discs

The mechanism of uptake of imazapyr, imazethapyr, and imazaquin into soybean leaf discs was determined. Uptake into the leaf discs was linear with respect to external concentration from 10^?8 to 10^?3 M. Metabolic inhibitors and uncouplers (DNP, FCCP, CCCP, NaN₃, NaCN, DCMU) decreased uptake 58 to 85 %. Uptake was also sensitive to temperature, with maximum uptake occurring at 23°C. The Q₁₀ for uptake between 13 and 23°C was 1-7. The uptake of all 3 imidazolinones was sensitive to the pH of the uptake solution. Maximum uptake occurred at pH 4. However, there were differences in the rate of uptake among the three herbicides. Imazaquin was absorbed the most rapidly followed by imazethapyr and then imazapyr. These differences in uptake reflected the differences in the lipophilicity of the chemicals at pH 4 and 7. The apparent K_ow of imazaquin, imazethapyr and imazapyr at pH 4 was 7-7, 1-4, and 0-1, respectively, and at pH 7 was 0-04, 0-02, and 0-004, respectively. Based on these results the mechanism of uptake is probably best explained by ion trapping.     

Use and misuse of calorimetry with special reference to muscle physiology

Calorimetric experiments, on their own, can be extremely dangerous to biologists, for there are usually several possible, but alternative interpretations. The simplest interpretation is often not correct. The best way to use biological calorimetry on its own is to indicate good targets for biochemical investigations of a tissue. Even when biochemical investigations are carried out in parallel with calorimetry, dangers remain. In particular it is easy to assume that the heat observed is necessarily derived from the biochemical process measured. The way to avoid this error is to make an energy balance study. In most cases such a study requires calorimetric measurements on cell-free systems of known composition to establish ΔH values for in vivo conditions. Used in this way biological calorimetry is much more than a mere indicator that something is happening; it can lead to the most important statement about what is happening: that we do not yet understand it!