Identification of superoxide dismutase isoenzymes in tobacco pollen

We investigated the possible existence of superoxide dismutase (SOD; EC 1.15.1.1) isoenzymes in the pollen of Nicotiana tabacum (Petit Havana SR-1 cultivar). To detect SOD activity, crude extracts from tobacco pollen were subjected to native polyacrylamide gel electrophoresis followed by staining with nitroblue tetra-zolium (NBT). The presence of six SOD isoenzymes was detected in tobacco pollen. Treatment with SOD inhibitors indicated the presence of one manganese SOD (Mn SOD), five copper-zinc SOD (Cu/Zn SOD) isoenzymes, and the absence of iron SOD (Fe SOD).     

Effects of Cadmium on Antioxidant Enzyme Activities in Sugar Cane

Sugar cane (Saccharum officinarum L. cv. Copersucar SP80-3280) seedlings were grown in nutrient solution with varying concentrations (0, 2 and 5 mM) of cadmium chloride for 96 h. Leaves were analysed for catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Although a clear effect of CdCl₂ on plant growth was observed, the activity of SOD was not altered significantly. However, the CAT activity decreased as the concentration of CdCl₂ increased. GR exhibits a significant increase in activity at 2 and 5 mM CdCl₂. CAT and SOD isoenzymes were further characterised by analysis in non-denaturing PAGE. Activity staining for SOD revealed up to seven isoenzymes in untreated control and 2 mM CdCl₂ treated plants, corresponding to Cu/Zn-SOD isoenzymes. At 5 mM CdCl₂, only six Cu/Zn-SOD isoenzymes were observed. No Fe-SOD and Mn-SOD isoenzymes were detected. For CAT, one band of activity was observed.     

Localization of a 170 kDa myosin heavy chain in plant cells

Summary A polyclonal antibody directed against a 170 kDa myosin heavy chain from lily pollen tubes was employed to (a) assess the cellular distribution of the polypeptide using immunofluorescence methods, and (b) ascertain if similar polypeptides are present in pollen tubes and somatic cells of other species. Fluorescence is associated with particles of various size as well as an amorphous component, and is concentrated in the apical cytoplasm of lily and tobacco pollen tubes. Apical fluorescence is more extensive in lily than in tobacco, which may be related to different streaming patterns and apical zonation seen at the ultrastructural level. In suspension cells of tobacco andArabidopsis, fluorescence is concentrated around the nuclei. Dual localizations indicate that anti-myosin fluorescence may be associated with the presence of actin. Little or no staining was seen in controls consisting of either pre-immune serum or mono-specific IgG that had been preadsorbed with the 170 kDa polypeptide. Immunoblots show that a 170 kDa immunoreactive polypeptide is present in pollen tubes of tobacco andTradescantia virginiana in addition to lily, and in suspension culture cells of tobacco andArabidopsis and extracts of wholeArabidopsis seedlings. Our results show that a conserved 170 kDa myosin heavy chain is present in a variety of monocot and dicot cells. They are also consistent with the presence of multiple myosins in plants in general and pollen tubes in particular.Abbreviations BSA bovine serum albumin - IgG immunoglobulin G - Mf microfilament - Mt microtubule - PBS phosphate-buffered saline - PME 50 mM Pipes, 5mM EGTA - 2mM MgSO₄, pH6.9.     

Superoxide dismutase in Scenedesmus obliquus

In heterotrophically grown Scenedesmus obliquus, the specific activity of superoxide dismutase (SOD; EC 1.15.1.1) declined when glucose was abundant, increased as it was depleated, and remained steady at a high level when it was absent. Transition to autotrophic growth produced only a small (20% over 5 d) increase in specific activity above the values obtained in dark-grown cells after glucose and starch-reserve depletion. This small, but consistent, increase did, however, parallel a similar increase in photosynthetic capacity. Polyacrylamide-gel electrophoresis showed the existence of nine isoenzymes of SOD. The three major and one of the minor isoenzymes were present in all extracts while three minor isoenzymes were found only in autotrophically grown cells and two only in heterotrophically grown cells. Characterization studies indicated that two of the major isoenzymes are dimeric FeSODs the other is a tetrameric MnSOD, and of the minor isoenzymes, two are dimeric FeSODs and four are dimeric MnSODs.Abbreviation SOD superoxide dismutase     

Enhanced tolerances of transgenic tobacco plants expressing both superoxide dismutase and ascorbate peroxidase in chloroplasts against methyl viologen-mediated oxidative stress

In order to better understand the role of antioxidant enzymes in plant stress protection mechanisms, transgenic tobacco (Nicotiana tabacum cv. Xanthi) plants were developed that overexpress both superoxide dismutase (SOD) and ascorbate peroxidase (APX) in chloroplasts. These plants were evaluated for protection against methyl viologen (MV, paraquat)‐mediated oxidative damage both in leaf discs and whole plants. Transgenic plants that express either chloroplast‐targeted CuZnSOD (C) or MnSOD (M) and APX (A) were developed (referred to as CA plants and AM plants, respectively). These plant lines were crossed to produce plants that express all three transgenes (CMA plants and AMC plants). These plants had higher total APX and SOD activities than non‐transgenic (NT) plants and exhibit novel APX and SOD isoenzymes not detected in NT plants. As expected, transgenic plants that expressed single SODs showed levels of protection from MV that were only slightly improved compared to NT plants. The expression of either SOD isoform along with APX led to increased protection while expression of both SODs and APX provided the highest levels of protection against membrane damage in leaf discs and visual symptoms in whole plants.     

Molecular cloning and characterization of profilin from tobacco (Nicotiana tabacum): increased profilin expression during pollen maturation

Profilin has recently been identified as an actin-binding protein in higher plants. A cDNA coding for tobacco profilin, which shared an average sequence identity of 75% with other plant profilins, was isolated from a tobacco pollen cDNA library by antibody screening. Tobacco profilin was expressed in Escherichia coli and purified by affinity to poly-(L-proline) Sepharose. A rabbit antiserum was raised against recombinant tobacco profilin and used to estimate the amount of profilin expressed in different tobacco tissues. Profilin can be detected in different somatic tissues, but the expression is 50–100 fold higher in mature pollen. Immunofluorescence and confocal laser scanning microscopy showed a homogeneous distribution of profilin in the cytoplasm of in vitro cultured pollen grains and pollen tubes of tobacco whereas some growing pollen tubes were stained more intensively a their tip. A possible role of pollen profilin as a developmentally upregulated microfilament precursor in mature pollen is discussed.     

Molecular characterization of profilin isoforms from tobacco (Nicotiana tabacum) pollen

Profilins are actin-binding proteins in eukaryotes which participate in the phosphoinositide pathway via binding to PIP₂. Using polyclonal rabbit sera raised against plant profilins, the occurrence of several profilin isoforms is demonstrated in two-dimensionally analyzed tobacco pollen extracts. The cDNAs coding for two novel tobacco profilin isoforms (ntPro2, ntPro3) were isolated from a pollen cDNA library by antibody screening. When the cDNA and deduced amino acid sequences of the two isoforms were compared with a previously isolated tobacco pollen profilin cl)NA (ntPro1), significant differences were noted in the non-coding regions, whereas the coding sequences, in particular the functional domains, showed little variation. The cDNAs coding for the three tobacco profilin isoforms were expressed inEscherichia coli and shown to bind comparably to different anti-profilin antisera. The high degree of similarity among the different tobacco pollen profilin isoforms points to functional equivalence. Assuming that the presence of profilin is indispensable to the control of the large amounts of actin present in pollen, the occurrence of different profilin isoforms in pollen is interpreted to represent a protective mechanism against loss of profilin functions.     

Comparison of antioxidant responses to cadmium and lead in Bruguiera gymnorrhiza seedlings

Seedlings of mangrove plant Bruguiera gymnorrhiza cultured in sand with Hoagland’s nutrient solution were treated with 1 to 30 mM Cd(NO₃)₂ or Pb(NO₃)₂ for 2 months. In all Cd/Pb treatments, the malondialdehyde content increased while the chlorophyll content declined. Peroxidase (POD) and superoxide dismutase (SOD) activities in roots increased at moderate Cd/Pb concentrations (1–10 mM), whereas decreased at higher concentrations (20–30 mM). Catalase (CAT) activity in roots was inhibited by 1–10 mM Cd but enhanced by 1–10 mM Pb. The activities of POD, SOD and CAT in leaves were less affected by Cd and Pb than in roots. A new SOD and three CAT isoenzymes were induced by Pb. In contrast, no additional SOD and CAT isoenzymes were induced by Cd.     

Identification and immunolocalization of superoxide dismutase isoenzymes of olive pollen

Gametophytic tissues of plants are an area largely neglected in the broad literature on free radical processes in plants. In order to study the mechanisms of protection against oxidative stress in pollen, the presence of the key antioxidative enzyme superoxide dismutase (SOD; EC 1.15.1.1) was investigated. Crude extracts of olive tree ( Olea europaea L.) pollen were subjected to native PAGE in 10% polyacrylamide gels. The SOD activity staining of gels showed the presence of four isoenzymes. All the SODS were completely inhibited by 2 m M KCN and 5 m M H₂O₂, and therefore belong to the family of CuZn‐SODS. Isoelectric focusing (pH 3.5‐7) of crude extracts and further detection of SOD activity allowed determination of isoelectric points for the four isoforms, namely 4.60, 4.78, 5.08 and 5.22. The cross‐reactivity of pollen extracts with a polyclonal antibody to cytosolic CuZn‐SOD from spinach leaves was assayed by western blotting. After SDS‐PAGE and immunoblotting, a major polypeptide band of about 16.5 kDa was detected, which is characteristic of the subunit of most CuZn‐SODS. Immunocytochemical studies at TEM level using the same antiserum showed that CuZn‐SOD was localized in the cytoplasm of both vegetative and generative cells, and also in material adhered to the pollen wall. The olive pollen CuZn‐SODS could function in the protection against oxidative stress during pollen development.     

Modulation of expression of SOD isoenzymes in mud crab (Scylla serrata): effects of inhibitors,salinity and season

Presence of several isoenzymes of superoxide dismutase (SOD) were demonstrated in tissues (abdominal muscle: 7 number, hepatopancreas: 13 number and gills: 7 number) of mud crabs (Scylla serrata) by employing specific staining of the enzyme in native-PAGE. SOD isoenzymes in tissues of mud crab were found to be thermolabile. The intensity of a major SOD band in tissues of crabs was reduced by the treatment of H₂O₂ or chloroform:ethanol. KCN treatment resulted in splitting of that major SOD band into two or more distinct bands. SDS treatment resulted in disruption of SOD bands. A sex-specific SOD isoenzyme band of higher molecular weight was observed in gills and muscle in winter and summer seasons, respectively. The observed different SOD isoenzyme pattern in tissues at altered salinities and seasons suggests separate tissue-specific antioxidant adaptation strategies of crabs against abiotic factors.     

Antisense transgenesis of tobacco with a flax pectin methylesterase affects pollen ornamentation

Summary. Antisense transgenesis of tobacco (Nicotiana tabacum) with a partial flax (Linum usitatissimum L.) pectin methylesterase (Lupme3) cDNA sequence yielded plants with altered pollen content. Moreover, the characteristically sculptured cell wall surrounding the pollen grains was modified in transgenic tobacco plants: the wavy ornamentation was dramatically reduced, suggesting the involvement of the demethylation of pectin in the pollen cell wall-specific structure. Germination of pollen was decreased and the pollen tube surface aspect was also different in transgenic plants.Correspondence and reprints: Laboratoire de Biotechnologies et Physiologie Végétales, Faculté des Sciences, Université de Picardie Jules Verne, 33 rue Saint-Leu, 80039 Amiens Cedex, France.     

Peroxidase Activity and Isoperoxidase Pattern in Tobacco Leaves Infected with Tobacco Necrosis Virus and Other Viruses Inducing Necrotic and Non-Necrotic Alterations

The level of peroxidase activity was greatly enhanced in tobacco leaves infected by tobacco necrosis virus (TNV) and other viruses which induce necrotic symptoms (TMV, ToMV and PVY^N). The intensity was related to the age of the leaves infected: absent or neglible in mature leaves and very pronounced in young growing infected leaves. On the contrary, changes in peroxidase activity were negligible when the infection was provoked by viruses which do not produce necrotic reactions (TMV and PVY^O). Analysis of the peroxidase isoenzymes, pattern in tobacco leaves infected by TNV and other necrosis-inducing viruses revealed in all cases, a slight increase in anionic (pl 3.5–3.7) and a considerable increase in moderately anionic isoenzymes particularly the pl 4.6 isoenzyme which in TNV and PVY^N-infected leaves reached levels up to 21 and 72 times the healthy control values. A considerable increase in the cationic (pl9.3–8.8) isoenzymes and the appearance of one moderately cationic isoenzyme (pl 8.2) was also detected. In leaf extracts from-virus-infected tobacco leaves with nonnecrotic response, no, or negligible alterations on the isoenzyme pattern were detected. However, infection by a fungal parasite (Erisyphe cichoracearum), which established a fully compatible, non-necrotic, interaction with tobacco leaves, like the necrosis-inducing viruses, changed the isoperoxidase pattern. The data suggest the necrotic alterations and associated changes in the peroxidase activity and isoperoxidase pattern in virus-infected leaves are not clearly related.     

21-kDa polypeptide,a low-molecular-weight cyclophilin,is released from pollen of higher plants into the extracellular medium in vitro

Calcium ions play a key role in the elongation and orientation of pollen tubes. We found that significant amounts of 21-kDa polypeptide were specifically released into the extracellular medium when pollen grains of lily, Lilium longiflorum Thunb., were incubated in the presence of EGTA or at low concentrations of Ca²⁺. This phenomenon was also dependent on pH and on the concentrations of MgCl₂ in the medium; the release of 21-kDa polypeptide from pollen was suppressed by increasing the MgCl₂ concentration and by lowering pH. Germination of pollen grains was inhibited in the medium into which the 21-kDa polypeptide had been released. This inhibition was irreversible; germination did not occur on transfer of the pollen grains into basal culture medium. Immuno-electron microscopy using an antibody against 21-kDa polypeptide showed that this polypeptide was present in the cytoplasm, vegetative nucleus and generative cell. When the pollen was treated with a medium containing EGTA, the density of 21-kDa polypeptide in the cytoplasm significantly decreased, but its density in vegetative nuclei and the generative cell did not, suggesting that only cytoplasmic 21-kDa polypeptide was released into the extracellular medium. The 21-kDa polypeptide was also present in the pollen of other higher-plant species, such as Tradescantia virginiana L., Nicotiana tabacum L. (angiosperms), and Cryptomeria japonica D. Don. (gymnosperm), and was also released into the medium in the presence of EGTA. In the case of C. japonica, however, it was released from pollen at alkaline pH above 8.5. The expression of 21-kDa polypeptide was not pollen-specific, because 21-kDa components immunoreactive with the anti-21-kDa polypeptide serum also existed in vegetative organs and cells of lily or tobacco. However, the 21-kDa polypeptide was not released into the extracellular medium from cultured tobacco BY-2 cells, even in the presence of EGTA. Amino acid sequences of two peptide fragments derived from 21-kDa polypeptide matched well those of low-molecular-weight cyclophilin (CyP). The antiserum against 21-kDa polypeptide recognized the CyP A from calf thymus and that in A431 carcinoma cells. The 21-kDa polypeptide fraction purified from lily pollen possessed peptidyl-prolyl cis-trans isomerase activity, which was suppressed by cyclosporin A (CsA), an inhibitor of enzyme activities of CyPs. From these results, we concluded that the 21-kDa polypeptide is a low-molecular-weight CyP. The present study showed that CyP in the pollen of higher plants is released into the extracellular matrix under unfavorable conditions.Abbreviations CaM Calmodulin - CBB Coomassie-brilliant-blue - CsA Cyclosporin A - CyP Cyclophilin     

Localization of a kinesin-like protein in generative cells of tobacco

Summary We have obtained immunofluorescence and immunoblot evidence for the presence of kinesin-like protein (KLP) in pollen tubes of tobacco using an antibody generated against peptides encoded by theKATA gene ofArabidopsis. This antibody recognizes an M_r 140,000 polypeptide inArabidopsis seedlings, and stains the mitotic apparatus in this species as well as in tobacco suspension cells. In tobacco pollen tubes prepared for dual immunofluorescence localizations of KLP and -tubulin, the antibody binds transiently to microtubule (Mt) bundles and the nucleus in premitotic generative cells; it then stains the developing mitotic apparatus as the nuclear envelope breaks down. By metaphase, fluorescence is located over kinetochore fibers and associated Mts. Localization of KLP is concentrated in the midzone during anaphase, and by early cytokinesis, it closely brackets the cell plate. Phragmoplast fluorescence then spreads along the phragmoplast distal to the cell plate. Punctate staining is also detected along vegetative Mts. No KLP localization is seen in pollen tubes treated with antibody after it had been preadsorbed to the antigenic peptides. The antibody recognizes an M_r 110,000 polypeptide in extracts of tobacco pollen tubes, and a polypeptide of somewhat lower M_r inTradescantia pollen tubes. Our results show that KLP(s) related to KatAp are present in tobacco generative cells and may play roles in the organization and/or operation of the mitotic apparatus and phragmoplast.Abbreviations KLP kinesin-like protein - Mt microtubule - MA mitotic apparatus Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

Structural and functional characterization of a pollen-specific promoter <Emphasis Type="Italic">NTPp13</Emphasis> in tobacco

A novel 407 bp nucleotide sequence NTPp13 was isolated from tobacco (Nicotiana tabacum L.) by PCR, its structure and function were characterized. The NTPp13 sequence was highly homologous with the pollen-specific expression promoter Zm13 from maize (Zea mays L.) and contained some key motifs which controlled pollen-specific expression. The NTPp13 was fused to the β-glucuronidase (GUS) reporter gene and transferred into tobacco. Analysis of the transgenic plants revealed that this putative promoter fragment was sufficient to direct GUS expression specifically in the anther, exactly in the pollen and pollen tube, and that GUS activity reached the maximum at the stage of pollen grain began to separate. Further study showed that the expression of NTPp13 sequence at pollen was stable at the range of temperature measured. These data suggested that the NTPp13 sequence was likely the essential element of promoter region of an unknown pollen-specific gene from tobacco.     

Cytokinin-controlled Indoleacetic Acid Oxidase Isoenzymes in Tobacco Callus Cultures

Indoleacetic acid oxidase in tobacco callus tissues (Nicotiana tabacum L., cultivar White Gold) was resolved into seven anionic isoenzymes by polyacrylamide gel disc electrophoresis. Different concentrations of kinetin and zeatin in the presence of indoleacetic acid affected the level of this enzyme, particularly two fast-moving isoenzymes, A₅ and A₆. The optimal concentration of kinetin was 0.2 μm; increasing concentrations above this level progressively lowered the total activity of indoleacetic acid oxidase and repressed the development of isoenzymes A₅ and A₆. Actinomycin D and cycloheximide inhibited the development of these two isoenzymes under the influence of 0.2 μm kinetin, suggesting a requirement for RNA and protein synthesis. The cytokinin-promoted indoleacetic acid oxidase isoenzymes A₅ and A₆ increased with time and paralleled the dry weight increase of tobacco callus tissues, but the total activity of indoleacetic acid oxidase per unit dry weight of tobacco callus varied with time depending on the stage of plant growth.     

Effects of pollen-synthesized green fluorescent protein on pollen grain fitness

Genetically engineered pollen with a visible marker gene could be useful to monitor the movement of transgenic pollen provided there are no negative physiological or fitness effects of expressing such a gene. In this study, we measured the fitness of Nicotiana tabacum cv. Xanthi pollen expressing the marker gene green fluorescent protein (GFP). Average pollen tube germination frequencies and pollen tube growth rates in vitro were measured in three different types of plants: (1) plants producing GFP in pollen cells only (LAT59-GFP), (2) plants synthesizing GFP under the control of a constitutive promoter (CaMV 35S) in which no GFP was produced in pollen, and (3) non-transgenic plants. Pollen synthesizing the GFP protein did not differ significantly in average pollen germination frequencies from pollen without GFP (P=0.65). Average pollen tube growth rates over a 5-h period did not differ significantly between transgenic and non-transgenic types (R²=0.89, 0.98, and 0.95, respectively, for GFP-tagged, 35S-GFP, and wild type). Overall, GFP expression in pollen grains of tobacco was not found to have an effect on pollen fitness under the controlled experimental conditions of this study.     

Pine pollen dehiscence relative to thrips population dynamics

A two‐part study related tree pollen dehiscence and thrips population dynamics in the field, and directly evaluated effects of pine pollen deposition on Frankliniella spp. (Thysanoptera: Thripidae) reproduction on tobacco, Nicotiana tabacum L. (Solanaceae), under laboratory conditions. Ambient pollen and thrips were monitored in Tift County, GA, USA, from early spring to late summer/early fall from 2005 to 2008. Correlation analyses were conducted using weekly means of thrips collected on sticky traps compared to weekly pollen counts from a Burkard air sampler and pollen deposition sheets. There were significant positive correlations between pollen and thrips counts on later dates, suggesting that if a relationship exists between pine pollen dehiscence and thrips population dynamics, it will be delayed. Over all years combined, the first spring time peak in Frankliniella fusca (Hinds) counts on sticky traps occurred at 227 accumulated degree days (dd) after the first peak in pine pollen, approximately the dd required to complete a single F. fusca generation. Two subsequent thrips peaks occurred at approximately one and two F. fusca generations following the first peak. After 10 weeks following the final peak in pollen, there appears to be no more effect of pollen on thrips population dynamics. A tobacco leaf, lightly dusted with Slash pine [Pinus elliottii Engelmann (Pinaceae)] pollen to mimic natural deposition, showed a significant increase in the number of offspring produced per Frankliniella occidentalis (Pergande) and F. fusca female. The total offspring produced increased five‐fold in F. fusca and 22‐fold in F. occidentalis in 15 days on the pollen‐treated leaves over the untreated leaves.