Pleiotropic effects of tobacco-mosaic-virus movement protein on carbon metabolism in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L. cv. Xanthi) plants expressing wild-type or mutant forms of the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to study the effects of the TMV-MP on carbon metabolism in source leaves. Fully expanded source leaves of transgenic plants expressing the TMV-MP were found to retain more newly fixed ¹⁴C compared with control plants. Analysis of ¹⁴C-export from young leaves of TMV-MP plants, where the MP is yet to influence plasmodesmal size exclusion limit, indicated a similar pattern, in that daytime ¹⁴C export was slower in TMV-MP plants as compared to equivalent-aged leaves on control plants. Pulse-chase experiments were used to monitor radioactivity present in the different carbohydrate fractions, at specified intervals following ¹⁴CO₂ labeling. These studies established that the-TMV-MP can cause a significant adjustment in short-term ¹⁴-C-photosynthate storage and export. That these effects of the TMV-MP on carbon metabolism and phloem function were not attributable to the effect of this protein on plasmodesmal size exclusion limits, per se, was established using transgenic tobacco plants expressing temperature-sensitive and C-terminal deletion mutant forms of the TMV-MP. Collectively, these studies establish the pleiotropic nature of the TMV-MP in transgenic tobacco, and the results are discussed in terms of potential sites of interaction between the TMV-MP and endogenous processes involved in regulating carbon metabolism and export.Abbreviations MP movement protein - SEL size exclusion limit - TMV tobacco mosaic virus - ts temperature sensitive This work was supported by United State-Israel Binational Agricultural Research Development Fund grant No. 90-00070 (S.W. and W.J.L). We thank Roger N. Beachy for generously providing some of the transgenic plant lines employed in this study. This paper is a contribution from the Uri Kinamon Laboratory. A.A.O. was supported by a scholarship from the Kinamon Foundation.     

Chronic boron or copper deficiency induces limb teratogenesis in Xenopus

Sets of adult male and female Xenopus laevis were administered a boron-deficient (−B) diet under low-boron culture conditions, a boron-supplemented (+B) diet under ambient boron culture conditions, a copper-deficient (−Cu) diet under low-copper culture conditions, or a copper-supplemented (+Cu) diet under ambient copper culture conditions, for 120 d. Adults from each group were subsequently bred, and the progeny were cultured and bred. Results from these studies indicated that although pronounced effects on adult reproduction and early embryo-larval development were noted in the −B F₁ generation, no effects on limb development were observed. No significant effects on reproduction, early embryogenesis, or limb development were noted in the +B group, irrespective of generation. Highly specific forelimb and hindlimb defects, including axial flexures resulting in crossed limbs and reduction deficits, were observed in −B F₂ larvae, but not in the +B F₂ larvae. As was noted in the boron-deficiency studies, significant effects on reproduction and early embryo development were observed in the −Cu F₁ generation, but not in the +Cu F₁ generation. Unlike the effects associated with boron deficiency, maldevelopment of the hindlimbs (32 responders, n=40) was found in the F₁ generation.     

Influence of the tobacco mosaic virus 30-kDa movement protein on carbon metabolism and photosynthate partitioning in transgenic tobacco plants

Transgenic tobacco (Nicotiana tabacum L.) plants expressing the 30-kDa movement protein of tobacco mosaic virus (TMV-MP) were employed to investigate the influence of a localized change in mesophyll-bundle sheath plasmodesmal size exclusion limit on photosynthetic performance and on carbon metabolism and allocation. Under conditions of saturating irradiance, tobacco plants expressing the TMV-MP were found to have higher photosynthetic CO₂-response curves compared with vector control plants. However, this difference was significant only in the presence of elevated CO₂ levels. Photosynthetic measurements made in the green-house, under endogenous growth conditions, revealed that there was little difference between TMV-MP-expressing and control tobacco plants. However, analysis of carbon metabolites within source leaves where a TMV-MP-induced increase in plasmodesmal size exclusion limit had recently taken place established that the levels of sucrose, glucose, fructose and starch were considerably elevated above those present in equivalent control leaves. Although expression of the TMV-MP did not alter total plant biomass, it reduced carbon allocation to the lower region of the stem and roots. This difference in biomass distribution was clearly evident in the lower root-to-shoot ratios for the TMV-MP transgenic plants. Microinjection (dye-coupling) studies established that the TMV-MP-associated reduction in photosynthate delivery (allocation) to the roots was not due to a direct effect on root cortical plasmodesmata. Rather, this change appeared to result from an alteration in phloem transport from young source leaves in which the TMV-MP had yet to exert its influence over plasmodesmal size exclusion limits. These results are discussed in terms of the rate-limiting steps involved in sucrose movement into the phloem.Abbreviations PFD photon flux density - SEL size exclusion limit - TMV-MP tobacco mosaic virus movement protein This work was supported by National Science Foundation grant No. DCB-9016756 (W.J.L.) and United States-Israel Binational Science Foundation grant No. 90-00070 (S.W. and W.J.L.). Special thanks are due to Bryce Falk for the use of pathogen-free green-house space at the University of California, Davis, Plant Pathology Greenhouse Facility, and to Robert Pearcy, for the use of his gas-exchange system. R.J.H. was on sabbatical leave from the University of Rhode Island, Kingston, RI.     

Uptake,accumulation and metabolism of auxins in tobacco leaf protoplasts

Uptake and metabolism of exogenous naphthalene-1-acetic acid (NAA) and indole-3-acetic acid (IAA) have been studied in tobacco (Nicotiana tabacum L. cv. Xanthi) mesophyll protoplasts. Both auxins entered protoplasts by diffusion under the action of the transmembrane pH gradient without any detectable participation of an influx carrier. Molecules were accumulated by an anion-trapping mechanism and most of them were metabolized within hours, essentially as glucose-ester and amino-acid conjugates. Protoplasts were equipped with a functional auxin-efflux carrier as evidenced by the inhibitory effect of naphthylphtalamic acid on IAA efflux. Basically, similar mechanisms of NAA and IAA uptake occurred in protoplasts. However, the two auxins differed in their levels of accumulation, due to different membrane-transport characteristics, and the nature of the metabolites produced. This shows the need to estimate the accumulation and the metabolism of auxins when analyzing their effects in a given cell system. The internal auxin concentration could be modulated by changing the transmembrane pH gradient, giving an interesting perspective for discriminating between the effects of intra- and extracellular auxin on physiological processes.Abbreviations BA benzoic acid - C_i/C_e accumulation ratio of auxin - IAAasp N-[3-indolylacetyl]-dl-aspartic acid - NAA naphthalene-1-acetic acid - NAAasp N-[1-naphthylacetyl]-l-aspartic acid - NPA N-1-naphthylphthalamic acid The authors thank Dr. M. Caboche (I.N.R.A, Versailles, France) for his generous gifts of some amide derivatives of 1-NAA, Mr. P. Varennes and Dr. B. Das (I.C.S.N., C.N.R.S., Gif-sur-Yvette, France) for recording and interpreting the mass spectra of NAA glucose ester, and Prof. P. Manigault (Institut des Sciences Végétales, Gif-sur-Yvette) for microscopy measurements of protoplast dimensions. This work was supported by funds from the C.N.R.S, I.N.R.A, and E.E.C.     

Phenylalanine ammonia-lyase levels in protoplasts isolated from hypersensitive tobacco pre-infected with tobacco mosaic virus

Leaves of tobacco varieties carrying the N gene for hypersensitiviy react to tobacco mosaic virus (TMV) infection by forming necrotic lesions and by localizing the virus in the vicinity of these lesions. These changes are accompanied in the host by an increased metabolic activity, in particular by an increased production of phenolic compounds derived from phenylalanine. Necrogenesis apparently destroys cells which have become heavily infected despite this strong defense reaction. However, it has been demonstrated previously (Otsuki et al., 1972) that protoplasts derived from leaves which normally respond in vivo to virus inoculation by forming necrotic local lesions, show no such response when inoculated in vitro. In the present study we have investigated the effect of pre-infecting hypersensitive leaves with TMV on the production or the non-production of the factor(s) of necrosis at the level of either protoplasts or mesophyll cells isolated from these preinfected leaves. Phenylalanine ammonia-lyase (PAL), whose rate of synthesis has been shown (Duchesne et al., 1977) to increase in stimulated cells of infected leaves, was used as a biochemical marker in the search for the stimulus preceding necrogenesis. We found that this stimulus concerning PAL activity was never elicited in either protoplasts or mesophyll cells which were prepared just before the appearance of necrotic local lesions. This result did not depend on the conditions of pre-infection or on the methods used to isolate the protoplasts or mesophyll cells. We also assayed samples derived from pre-infected leaves that were already carrying local lesions, i.e., in which the stimulus and necrogenesis were already operating: not only did the isolated protoplasts and mesophyll cells not sustain the stimulus concerning PAL activity, but the stimulated enzyme activity decreased abruptly and, in most of the experiments, had disappeared within the time necessary for maceration. Evidence is presented showing that the non-elicitation or the abrupt decrease of stimulated PAL activity could not result from a selection of unstimulated cells or from a preferential destruction of stimulated cells during maceration of the leaves.Our results support the view that hypertonic osmotic pressure is responsible for the non-occurence of the hypersensitive response by acting according to one or both of the following processes: it suppresses the contacts through plasmodesmata between neighboring cells and, hence, it also suppresses the cell-to-cell diffusion of the factor(s) eliciting the stimulus; and/or since hypertonic osmotic pressure causes striking differences between leaf cells and protoplasts in total RNA and protein synthesis, these differences might include the suppression of synthesis of the elicitor of hypersensitivity.Abbreviations OMT O-methyltransferase - PAL phenylalanine ammonia-lyase - TMV Tobacco mosaic virus     

Hormonal control of tobacco protoplast nucleic acid metabolism during in vitro culture

Tobacco mesophyll protoplasts cultivated in vitro do not synthesize a measurable quantity of chloroplastic ribosomal RNA, but actively synthesize cytoplasmic ribosomal RNA, polyadenylated RNA, and proteins. These syntheses are essentially independent of the presence of hormones in the culture medium and are thus related to the ageing phenomenon induced by isolation from the plant and in-vitro culture. At all stages of culture and in all culture media, protoplasts incorporate low levels of thymidine into their DNA. However, the incorporation of considerable quantities of thymidine, indicative of the S phase, only takes place after 25–30 h and requires the presence of auxin and cytokinin.Abbreviations 6-BA 6-benzyladenine - 2,4-D 2,4 dichlorophenoxyacetic acid - DPC diethylpyrocarbonate - OD optical density; oligo-dT cellulose-oligothymidylic acid-cellulose - poly A⁺ RNA polyadenylated RNA - poly A^- RNA non-polyadenylated RNA - tRNA ribosomal RNA - SDS sodium dodecyl sulphate - TCA trichloroacetic acid - Tris buffer Tris (hydroxymethyl)aminomethane - tRNA transfer RNA     

Changes in protein synthesis during the initial stage of life of tobacco protoplasts

The biosynthesis of Fraction I protein in isolated protoplasts is compared with that in the plant. Radioactive precursors were incorporated into isolated protoplasts (in vitro labeling) and into leaves, from which the protoplasts were isolated later (in situ labeling). The biosynthesis of Fraction I protein stopped almost completely as soon as the protoplasts were incubated in the culture medium.     

Nitrogen-induced boron deficiency in lucerne

Summary In a glasshouse experiment with a boron deficient soil the application of nitrogen was found to decrease the boron concentration and boron uptake by lucerne (Medicago sativa). Without added boron, nitrogen applications killed the lucerne, probably by inducing severe boron deficiency. With added boron, the lowest rate of nitrogen application increased lucerne yield but further additions depressed yields. The effect was due to a physiological interaction rather than an effect of the nitrogen on the availability of the boron in the soil.     

Changes in polypeptide patterns in tobacco roots colonized by two Glomus species

Changes in gene expression were studied during the establishment of arbuscular mycorrhizal symbiosis in tobacco roots from an amphidiploid hybrid Nicotiana glutinosa x N. debneyi. Polypeptide patterns from control roots and from roots infected by Glomus mosseae or G. intraradices were resolved by two-dimensional polyacrylamide gel electrophoresis and followed in a time-course analysis. Arbuscular mycorrhizal infection led to significant modifications in polypeptide patterns with: (a) decreased amounts of some polypeptides, (b) increased accumulation of others, and (c) appearance of newly-induced polypeptides. Comparisons made during infection development by the two Glomus species demonstrated that protein modifications changed in relation to the mycorrhizal state of the tobacco roots.     

Effects of boron deficiency in cell suspension cultures of Populus alba L.

Cell suspension cultures of Populus alba L. (original cells) require at least 10 M boron for appropriate growth. Using original cells we established a cell line, T-5B, which can grow in a medium containing low levels of boron (5 M). The level of boron localized in the cell walls of T-5B cells was one-half that found in the cell walls of original cells maintained in medium containing 100 M boron, and the level of the rhamnogalacturonan II dimer, cross-linked by a borate ester, also decreased in the former. The sugar composition of whole cell walls of the T-5B cell line was similar that of the original cells, however pectic polysaccharides composed of arabinose or galacturonic acid were easily extracted from T-5B cell walls with 50 mM trans-1,2-cyclohexanediamine-N,N,N,N-tetraacetic acid. Our results suggest that boron deficiency causes a weakening of the interaction among pectic polysaccharides due to a decrease in boron-rhamnogalacturonanII cross-linkage.     

Inorganic pyrophosphate content and metabolites in potato and tobacco plants expressing E. coli pyrophosphatase in their cytosol

Metabolite levels and carbohydrates were investigated in the leaves of tobacco (Nicotiana tabacum L.) and leaves and tubers of potato (Solanum tuberosum L.) plants which had been transformed with pyrophosphatase from Escherichia coli. In tobacco the leaves contained two- to threefold less pyrophosphate than controls and showed a large increase in UDP-glucose, relative to hexose phosphate. There was a large accumulation of sucrose, hexoses and starch, but the soluble sugars increased more than starch. Growth of the stem and roots was inhibited and starch, sucrose and hexoses accumulated. In potato, the leaves contained two- to threefold less pyrophosphate and an increased UDP-glucose/ hexose-phosphate ratio. Sucrose increased and starch decreased. The plants produced a larger number of smaller tubers which contained more sucrose and less starch. The tubers contained threefold higher UDP-glucose, threefold lower hexose-phosphates, glycerate-3-phosphate and phosphoenolpyruvate, and up to sixfold more fructose-2,6-bisphosphatase than the wild-type tubers. It is concluded that removal of pyrophosphate from the cytosol inhibits plant growth. It is discussed how these results provide evidence that sucrose mobilisation via sucrose synthase provides one key site at which pyrophosphate is needed for plant growth, but is certainly not the only site at which pyrophosphate plays a crucial role.Abbreviations Fru2,6bisP fructose-2,6-bisphosphate - Fru6P fructose 6-phosphate - FW fresh weight - Glc1P glucose-1-phosphate - Glc6P glucose-6-phosphate - PEP phosphoenolpyruvate - 3PGA glycerate-3-phosphate - PFK phosphofructokinase - PFP pyrophosphate: fructose-6-phosphate phosphotransferase - Pi inorganic phosphate - PPi inorganic pyrophosphate - UDPGlc UDP-glucose This research was supported by the Deutsche Forschungsgemein-Schaft (SFB 137) and Sandoz AG (T.J., M.H., M.S.) and by the Bundesminister für Forschung und Technologie (U.S., L.W.).     

Stimulation by abscisic acid of RNA synthesis in discs from healthy and tobacco mosaic virus-infected tobacco leaves

Uptake of abscisic acid from the culture medium by discs of healthy and tobacco mosaic virus-infected tobacco leaves was measured. Small (two to five-fold) increases in abscisic acid concentration in discs caused increases in rates of [³H]uridine and [³H]adenine incorporation into total nucleic acid, virus RNA and host ribosomal RNA. Net accumulation of virus RNA was also enhanced by abscisic acid. This evidence for stimulation of RNA synthesis is compared with previous reports showing inhibition of RNA synthesis in other tissues. It is suggested that the increase in endogenous abscisic acid caused by tobacco mosaic virus infection may be at least partly responsible for observed increases in rates of RNA synthesis after infection.Abbreviations ABA abscisic acid - TMV tobacco mosaic virus     

Trypsin inhibitor activity in mature tobacco and tomato plants is mainly induced locally in response to insect attack,wounding and virus infection

Wounding of plants by insects is often mimicked in the laboratory by mechanical means such as cutting or crushing, and has not been compared directly with other forms of biotic stress such as virus infection. To compare the response of plants to these types of biotic and abiotic stress, trypsin inhibitor (TI) activity induced locally and systemically in mature tobacco (Nicotiana tabacum L.) and tomato (Lycopersicon esculentum L.) plants was followed for 12 days. In tobacco, cutting, crushing and insect feeding all induced comparable levels of TI activity of approx. 5 nmol·(mg leaf protein)^?1 in wounded leaves, while tobacco mosaic virus (TMV) infection of tobacco induced 10-fold lower amounts in the infected leaves. In tomato, feeding by insects also led to the induction of a level of TI activity of 5 nmol·(mg leaf protein)^?1. In contrast, both cutting and crushing of tomato leaves induced 10-fold higher amounts. These data show that biotic stress, in the form of insect feeding and TMV infection, and abiotic stress, in the form of wounding, have different effects on local levels of induced TI activity in mature tobacco and tomato plants. Irrespective of the type of wounding, in neither tobacco nor tomato could systemic induction of TI activity be observed in nearby unwounded leaves, which suggests that systemic induction of TI activity in mature tobacco and tomato plants is different from systemic TI induction in seedlings. Wounding of tobacco leaves, however, did increase the responsiveness to wounding elsewhere in the plant, as measured by an increased induction of TI activity.     

Boron deficiency inPinus radiata D. Don and the effect of applied boron on height growth and nutrient uptake

Summary Severe dieback observed inP. radiata D. Don plantations during dry years in Southeastern Australia was found to be due to boron deficiency. Borax applied in spring (August 1982) at rates of 50, 100 and 150 kg ha^–1 resulted in a marked increase in foliar B and an alleviation of B deficiency symptoms. During the dry summer following the application of borax no further dieback was observed and height growth of fertilized trees was significantly improved. Correction of B deficiency enhanced the uptake of P by the trees.