Effects of Ca2+ channel blockers and protein kinase/phosphatase inhibitors on growth and anthraquinone production in Rubia cordifolia callus cultures transformed by the rolB and rolC genesBulgakov, V.; Tchernoded, G.; Mischenko, N.; Shkryl, Y.; Glazunov, V.; Fedoreyev, S.; Zhuravlev, Y.
doi: 10.1007/s00425-003-0996-5pmid: 14520561
The transformation of Rubia cordifolia L. cells by the 35S-rolB and 35S-rolC genes of Agrobacterium rhizogenes caused a growth inhibition of the resulting cultures and an induction of the biosynthesis of anthraquinone-type phytoalexins. Inhibitor studies revealed a striking difference between the rolC- and rolB-gene-transformed cultures in their sensitivity to verapamil, an L-type Ca2+ channel blocker. The rolC culture possessed a 2-fold lowered resistance to the inhibitor than the normal culture, while the rolB culture was 4-fold more resistant to the treatment. Additionally, growth of the rolC culture was totally inhibited when the culture was grown in Ca2+-free medium, whereas growth of the rolB culture was reduced by less than half. We interpreted these results as evidence for a lack of calcium homeostasis in both transgenic cultures. Anthraquinone (AQ) production was not inhibited in the normal or transformed cultures by the Ca2+ channel blockers verapamil and LaCl3, or by diphenylene iodonium, an inhibitor of NADPH oxidase, or by the protein kinase inhibitor staurosporine. These results indicate that the induction of AQ production in non-transgenic and transgenic cultures does not proceed through the activation of the common Ca2+-dependent NADPH oxidase pathway that mediates signal transduction between an elicitor–receptor complex via transcriptional activation of defense genes. Okadaic acid and cantharidin, inhibitors of protein phosphatases 1 and 2A, caused an increase in AQ production in transgenic cultures. Okadaic acid stimulated AQ accumulation in the non-transformed culture, whereas cantharidin had no effect. These results show that different phosphatases are involved in AQ synthesis in normal and transgenic cultures of R. cordifolia.
Generation of cyanogen-free transgenic cassavaSiritunga, Dimuth; Sayre, Richard
doi: 10.1007/s00425-003-1005-8pmid: 14520563
Cassava (Manihot esculenta Crantz.) is the major source of calories for subsistence farmers in sub-Saharan Africa. Cassava, however, contains potentially toxic levels of the cyanogenic glucoside, linamarin. The cyanogen content of cassava foods can be reduced to safe levels by maceration, soaking, rinsing and baking; however, short-cut processing techniques can yield toxic food products. Our objective was to eliminate cyanogens from cassava so as to eliminate the need for food processing. To achieve this goal we generated transgenic acyanogenic cassava plants in which the expression of the cytochrome P450 genes (CYP79D1 and CYP79D2), that catalyze the first-dedicated step in linamarin synthesis, was inhibited. Using a leaf-specific promoter to drive the antisense expression of the CYP79D1/CYP79D2 genes we observed up to a 94% reduction in leaf linamarin content associated with an inhibition of CYP79D1 and CYP79D2 expression. Importantly, the linamarin content of roots also was reduced by 99% in transgenic plants having between 60 and 94% reduction in leaf linamarin content. Analysis of CYP79D1/CYP79D2 transcript levels in transgenic roots indicated they were unchanged relative to wild-type plants. These results suggest that linamarin is transported from leaves to roots and that a threshold level of leaf linamarin production is required for transport.
Sugarbeet (Beta vulgaris L.): shoot regeneration from callus and callus protoplastsDovzhenko, Alexander; Koop, Hans-Ulrich
doi: 10.1007/s00425-003-1006-7pmid: 14520564
The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.
Aerenchyma formation in roots of maize during sulphate starvationBouranis, D.; Chorianopoulou, S.; Siyiannis, V.; Protonotarios, V.; Hawkesford, M.
doi: 10.1007/s00425-003-1007-6pmid: 12728316
Young maize (Zea mays L., Poaceae) plants were grown in a complete, well-oxygenated nutrient solution and then deprived of their external source of sulphate. This treatment induced the formation of aerenchyma in roots. In addition to the effect of sulphate starvation on root anatomy, the presence and location of superoxide anions and hydrogen peroxide, and changes in calcium and pH were examined. By day 6 of sulphate deprivation, aerenchyma started to form in the roots of plants and the first aerenchymatous spaces were apparent in the middle of the cortex. S-starvation also induced thickening of the cell walls of the endodermis. Active oxygen species appeared in groups of intact mid-cortex cells. Formation of superoxide anion and hydrogen peroxide was found in degenerating cells of the mid-cortex. Very few nuclei in the cortex of S-starved roots fluoresced, being shrunken and near to the cell wall. By day 12 of S-deprivation, a fully developed aerenchyma was apparent and there were only a few 'chains' of cells bridging hypodermis to endodermis and stele of roots. Cell walls of endodermis of S-starved roots increased 68% in thickness. Intensive fluorescence in the cell walls of the endodermal, hypodermal and to a lesser extent of epidermal cells was observed due to the formation of active oxygen species, while there was no fluorescence in the cortical cells. There was a higher Ca concentration in the cells walls of the endodermis and epidermis, compared to the rest of the S-starved root tissues. A higher pH was observed, mainly in the cell walls of the hypodermis and to a lesser extent in the cell walls of the endodermis. Superoxide anion and hydrogen peroxide was found in degenerating cells of the root cortex. There was no fluorescence of nuclei in the cortex of S-starved roots.
Type-h thioredoxins accumulate in the nucleus of developing wheat seed tissues suffering oxidative stressSerrato, Antonio; Cejudo, Francisco
doi: 10.1007/s00425-003-1009-4pmid: 14520565
Thioredoxin h (Trxh) proteins are ubiquitous in all wheat organs, but show the highest accumulation in mature seeds. This distribution suggests the expression of Trxh during seed development. In the present study, we have analyzed the pattern of Trxh expression in developing wheat (Triticum aestivum L.) seeds. Northern blot analysis detected a single band at any stage of development, which corresponded to the expression of at least two genes, TrxhA and TrxhB, as shown by competitive reverse transcription–polymerase chain reaction experiments. The analysis of the content of Trxh polypeptides showed the highest content in the embryo. The spatial pattern of accumulation of these proteins was established by immunocytological techniques. At early stages of development Trxh proteins localized to maternal tissues (nucellus projection cells and pedicel), the route of transport of nutrients to the developing endosperm. In the endosperm, Trxh proteins accumulated at a high level in the aleurone layer. At later stages of development, during seed maturation, Trxh proteins localized predominantly to the nucleus of both aleurone and scutellum cells, a feature exclusive of these seed tissues. The nuclear localization of Trxh proteins was associated with oxidative stress in these tissues, as shown by in situ staining of superoxide radicals in developing and germinating seeds.
The involvement of Ca2+ gradients, Ca2+ fluxes, and CaM kinaseII in polarization and germination of Silvetia compressa zygotesPu, Rongsun; Robinson, Kenneth
doi: 10.1007/s00425-003-1012-9pmid: 14520567
Previous work has shown that distinct Ca2+ gradients precede and predict the loci of germination of the zygotes of the brown alga, Silvetia compressa (J. Agardh) E. Serrão, T.O. Cho, S.M. Boo et Brawley, that are polarized by unilateral blue light. We show here that dark-grown S. compressa zygotes also form cytosolic Ca2+ gradients prior to germination and then germinate from the site of elevated Ca2+. In no case did germination occur without a prior formation of a Ca2+ gradient. Using the self-referencing Ca2+-selective probe, we measured highly localized influx of Ca2+ during photopolarization, indicating that extracellular stores supply at least some of the Ca2+ needed to construct a gradient. Finally, we find that germination was inhibited by a bath-applied inhibitor of calcium/calmodulin-dependent kinase II (CaM kinase II), KN-93 (but not by its inactive analog, KN-92), and by an injected inhibitory peptide for the kinase. KN-93 did not interfere with the photopolarization of the zygotes, consistent with the view that calmodulin is not involved in the initial response to light. The KN-93 results indicate that the requirement for active CaM kinase II for germination ends about 2 h before overt germination. We conclude that Ca2+ gradients, generated in part by localized calcium entry from the seawater, are an essential part of the process of polarity development and expression in these cells, regardless of the nature of the external cue that directs the orientation of the axis. Calmodulin and CaM kinase II are involved in interpreting (but not in establishing) the calcium gradient, allowing germination to occur at the site of elevated calcium, but CaM kinase II appears not to be involved in the initiation of germination.
Lithium treatment induces a hypersensitive-like response in tobaccoNaranjo, Miguel; Romero, Carlos; Bellés, José; Montesinos, Consuelo; Vicente, Oscar; Serrano, Ramón
doi: 10.1007/s00425-003-1017-4pmid: 14520568
Treatment of tobacco (Nicotiana tabacum L.) plants with lithium induces the formation of necrotic lesions and leaf curling as in the case of incompatible pathogen interactions. Further similarities at the molecular level include accumulation of ethylene and of salicylic and gentisic acids, and induced expression of pathogenesis-related PR-P, PR5 and PR1 genes. With the exception of PR1 induction, lithium produced the same effects in transgenic tobacco plants that do not accumulate salicylate because of overexpression of the bacterial hydroxylase gene nahG. On the other hand, inhibition of ethylene biosynthesis with aminoethoxyvinylglycine prevented lithium-induced cell death and PR5 expression. These results suggest that lithium triggers a hypersensitive-like response where ethylene signalling is essential.
Extensibility of isolated cell walls in the giant tip-growing cells of the xanthophycean alga Vaucheria terrestrisMine, Ichiro; Okuda, Kazuo
doi: 10.1007/s00425-003-0993-8pmid: 14520569
Apical cell wall fragments isolated from the giant-cellular xanthophycean alga Vaucheria terrestris sensu Götz were inflated with silicone oil by applying internal pressure ranging from 0.1 to 0.7 MPa, and the time-course of cell wall deformation was recorded and analyzed by videomicroscopy. Cell wall extensibility in the tip-growing region was estimated by the pressure required for cell wall extension, the amount of total extension until cell wall rupture and the rate of cell wall extension. Apical cell walls exhibited gradual extension, or creep, during inflation, which was eventually followed by rupture at the apical portion, whereas no appreciable extension was found in the cylindrical basal portion of the cell wall fragment. Besides the largest extension observed around the tip, substantial extension was also observed along the subapical region of the cell wall. The wall extensibility was dependent on the buffer pH used for infiltration before inflation. The optimum pH for the extension was about 8.0, but the cell wall was much less extensible after infiltration with an acidic buffer. Cell wall extensibility was dependent on the pH of the buffer used before inflation, regardless of that used in the previous infiltration. Moreover, pretreatment of the cell wall with a protease caused considerable loosening of cell walls, but affected the pH dependence of cell wall extensibility little. These results indicate that the extensibility of the cell walls in the giant tip-growing cells of the alga is distinct from that of plant cells that exhibit "acid growth" in its dependence on environmental pH and the role of cell wall proteins.