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See detailWhat we have learned about the physiology of in vitro adventitious rooting of woody plants and how it relates to improvements in the practice
Kevers, Claire ULg; Hausman, Jean-françois; Faivre-Rampant, Odile et al

in Niemi, Karoliina (Ed.) Adventitious root formation of forest trees and horticultural plants - from genes to applications (2009)

Natural auxins and synthetic analogs are the most powerful exogenous stimulators of adventitious rooting of ligneous as well as for herbaceous cuttings. For several years we have investigated the ... [more ▼]

Natural auxins and synthetic analogs are the most powerful exogenous stimulators of adventitious rooting of ligneous as well as for herbaceous cuttings. For several years we have investigated the physiology of rooting, including the metabolism of endogenous auxins and their molecular mechanisms of action. Our work has resulted in discoveries leading to practical strategies allowing faster and improved rooting of cuttings from shoots raised in vitro, and subsequently resulting in roots better adapted for acclimatization and to overcome so-called (apparent) recalcitrance-to-rooting of some species. In this chapter we describe: (i) how variation in auxin treatments during rooting can be used to regulate the endogenous levels of auxins; (ii) the importance of not maintaining auxin treatment throughout the whole rooting process, thereby the endogenous auxin level itself not always being high; (iii) the division of the organogenetic process into at least three main rooting interdependent phases, i.e. induction, initiation, and expression, with different durations depending on the species and with different requirements for each phase, and (iv) how polyamines, even if less effective from outside, and their metabolism are as important as auxins in the rooting process. We will also describe how cross-talk between hormones, manipulations with related genes and mutants, and some similarities between the induction of rooting and evocation of flowering, offer new tools for further progress in the field. [less ▲]

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See detailEffect of NaCl and mannitol iso-osmotic stresses on proline and free polyamine levels in embryogenic Fraxinus angustifolia callus
Tonon, Giustino; Kevers, Claire ULg; Faivre-Rampant, Odile et al

in Journal of Plant Physiology (2004), 161(6), 701-708

With the aim to differentiate the ionic and osmotic components of salt stress, short and long-term changes in free polyamines and proline induced by iso-osmotic concentrations of NaCl (0.1 mol/L and 0 ... [more ▼]

With the aim to differentiate the ionic and osmotic components of salt stress, short and long-term changes in free polyamines and proline induced by iso-osmotic concentrations of NaCl (0.1 mol/L and 0.2mol/L) and mannitol (0.2mol/L and 0.4mol/L) were determined in Fraxinus angustifolia callus. The peculiarities of the short-term responses were: i) a very early (30 min) and temporary increase in Putrescine (Pu) and Spermine (Spm) as a consequence of salt treatment, and ii) a continuous accumulation of Spermidine (Spd) and Spm in response to mannitol. The changes of Proline (Pro) were quite limited both in the short and in the long term, and generally occurred later than Polyamine (PAs) changes took place, suggesting a regulatory mechanism of PAs metabolism on Pro biosynthesis. In the long-term, no drastic accumulations of Pro or PAs in response to NaCl and mannitol were observed, suggesting that their physiological role is unlikely to be that of osmo-compatible solutes in this plant system. The salt induced a higher callus growth inhibition effect than did mannitol and this inhibition was associated with the reduction of endogenous levels of PAs, especially Pu. However, while a diverging time course was observed under lethal salt concentration (0.2 mol/L NaCl), a high parallelism in the endogenous changes of Pro and Pu was observed under all non-lethal conditions (control - 0.2 and 0.4 mol/L mannitol - 0.1 mol/L NaCl). Therefore the synchronous changes of Pro and Pu can be considered as a physiological trait associated with cell survival. These results indicate a strong metabolic co-ordination between PAs and Pro pathways and suggest that the metabolic fluxes through these pathways start competing only when the stress level is high enough to be lethal for cells. [less ▲]

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See detailRooting blockage in the tobacco rac mutant occurs at the initiation phase, and induces diversion to xylem differentiation
Faivre-Rampant, Odile; D'Angeli, S.; Falasca, G. et al

in Plant Biosystems (2003), 137(2), 163-174

The rac mutant of Nicotiana tabacum L. cv. Xanthii is impaired in adventitious root formation. The objective of the present study was to determine whether or not the root induction phase occurs in the rac ... [more ▼]

The rac mutant of Nicotiana tabacum L. cv. Xanthii is impaired in adventitious root formation. The objective of the present study was to determine whether or not the root induction phase occurs in the rac mutant, and if so, to determine what causes the induced cells to become incapable of organising root primordia. To this end, rac and wild-type shoots were cultured in vitro for 7 days under conditions suitable for obtaining roots in the wild-type (i.e., exposure to 5 muM indole-3-butyric acid for 4 h, and then transfer to hormone-free medium), and then histologically and biochemically analysed during culture. The variations in peroxidase activity, and in cellular levels of auxins and polyamines revealed that the induction phase occurs in rac shoots, although it lasts longer than in the wild-type ones. Furthermore, both auxin and polyamines were consistently higher in rac shoots compared to the wild-type. After induction, auxin and putrescine levels abruptly decreased in the wild-type shoots, whereas they decreased much more slowly in the rac mutant. The histological analysis of the wild-type shoots showed that the abrupt decrease in auxin and polyamine levels were correlated with a normal initiation phase. In fact, wild-type shoots showed cell divisions in the procambium already at day 2, resulting in the formation of root primordia at day 4, and in root emergence between days 5 and 7. In rac shoots, despite the fact that the procambium cells were activated to undergo cell division, the initiation phase was highly perturbed, and the procambial cells developed tracheary elements instead of adventitious roots. The different morphogenic responses of the two genotypes are discussed in the light of the differences in auxin content after the induction phase. [less ▲]

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See detailChanging concepts in plant hormone action
Gaspar, Thomas ULg; Kevers, Claire ULg; Faivre-Rampant, Odile et al

in In Vitro Cellular & Developmental Biology-Plant (2003), 39(2, MAR-APR), 85-106

A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be ... [more ▼]

A plant hormone is not, in the classic animal sense, a chemical synthesized in one organ, transported to a second organ to exert a chemical action to control a physiological event. Any phytohormone can be synthesized everywhere and can influence different growth and development processes at different places. The concept of physiological activity under hormonal control cannot be dissociated from changes in concentrations at the site of action, from spatial differences and changes in the tissue's sensitivity to the compound, from its transport and its metabolism, from balances and interactions with the other phytohormones, or in their metabolic relationships, and in their signaling pathways as well. Secondary messengers are also involved. Hormonal involvement in physiological processes can appear through several distinct manifestations (as environmental sensors, homeostatic regulators and spatio-temporal synchronizers, resource allocators, biotime adjusters, etc.), dependent on or integrated with the primary biochemical pathways. The time has also passed for the hypothesized 'specific' developmental hormones, rhizocaline, caulocaline, and florigen: root, stem, and flower formation result from a sequential control of specific events at the right places through a coordinated control by electrical signals, the known phytohormones and nonspecific molecules of primary and secondary metabolism, and involve both cytoplasmic and apoplastic compartments. These contemporary views are examined in this review. [less ▲]

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See detailCuttings of the non-rooting rac tobacco mutant overaccumulate phenolic compounds
Faivre-Rampant, Odile; Charpentier, Jean Paul; Kevers, Claire ULg et al

in Functional Plant Biology (2002), 29(1), 63-71

The auxin and phenolic contents, as well as phenylalanine ammonia-lyase (PAL) activity, were determined in in vitro cultured shoots of the recalcitrant-to-root rac mutant of tobacco, and compared with ... [more ▼]

The auxin and phenolic contents, as well as phenylalanine ammonia-lyase (PAL) activity, were determined in in vitro cultured shoots of the recalcitrant-to-root rac mutant of tobacco, and compared with wild-type shoots. The mutant and wild-type shoots showed similar auxin changes during the culture cycle, but with higher contents for the mutant. A transient peak of auxin (corresponding to the achievement of the rooting inductive phase) occurred at day 14 in both types of shoots, but earlier in the basal parts of the wild-type stems. The rac shoots contained more phenolics, corresponding with an increased PAL activity. The most abundant phenolic compound found in the two types of tobacco was chlorogenic acid, which was more abundant in the rac shoots. Rutin was also detected at a higher concentration in the mutant shoots. Basal parts of wild-type shoots treated with 10(-3) chlorogenic acid reacted by accumulating auxins and, unlike untreated controls, did not form adventitious roots. The relationships between those biochemical analyses in relation to the growth limitation of the rac mutant, and the inhibition of its root development, are discussed. [less ▲]

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See detailAuxins in the biology of roots
Gaspar, Thomas ULg; Faivre-Rampant, Odile; Kevers, Claire ULg et al

in Waisel, Yoav; Eshel, Amram; Kafkafi, Uzi (Eds.) Auxins in the biology of roots (2002)

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See detailModified hormonal balance in rooting-recalcitrant rac mutant tobacco shoots
Faivre-Rampant, Odile; Kevers, Claire ULg; Dommes, Jacques ULg et al

in Plant Biosystems (2001), 135(1), 85-93

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the DS wild-type. The mutant shoots grew at a lower rate and did not root whatever ... [more ▼]

The rooting-recalcitrant rac tobacco mutant has been multiplied in vitro via outgrowth of axillary buds in parallel to the DS wild-type. The mutant shoots grew at a lower rate and did not root whatever the treatments, whereas the wild-type shoots rooted spontaneously during the culture cycle without auxin treatment. The mutant and wild-type shoots showed similar peroxidase variations along the culture cycle (21 days) but with higher levels of activity for the rac mutant: minimum peroxidase activity occurrey at day 14 in whole shoots of both tobacco genotypes, but already at day 7 in the basal parts of the stems (where roots appear) of the wild-type tobacco, while it was delayed in the mutant. Free and conjugated auxin and polyamine levels were also determined in whole shoots and basal parts of the stems. The rac mutant was characterised by higher auxin and polyamine contents. A peak of auxins and polyamines appeared at day 14 in the whole shoots whatever the tobacco genotype. This peak was delayed in the basal parts of the rac stems compared to the wild-type ones. The mutant shoots contained higher levels of benzyladenine and isopentenyladenosine at the end of the culture cycle, whereas zeatin riboside was more abundant in wild-type shoots. In response to increasing concentrations of indole-3-butyric acid (IBA) and 1-naphthaleneacetic acid (NAA), only the wild-type shoots responded by an increase in growth rate followed by inhibition at high concentrations. The rac shoot responses were very low or nonexistent. Peroxidase activity was also measured in E basal parts of tobacco stems grown in the presence of IBA. Results suggest growth inhibition related to auxin accumulation, possibly combined with elevated putrescine content. Second, rooting induction seems to take place in both tobacco genotypes; however, the process of root formation is blocked in the mutant. The lack of initiation and expression phases of rooting in relation to auxin content in the mutant is discussed. [less ▲]

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See detailIntegrating phytohormone metabolism and action with primary biochemical pathways. I. Interrelationships between auxins, cytokinins, ethylene and polyamines in growth and development processes
Gaspar, Thomas ULg; Kevers, Claire ULg; Hausman, Jean-François et al

in Greppin, Hubert; Penel, Claude; Broughton, Walter (Eds.) et al Integrated Plant Systems (2000)

The paper begins with a review of the phytohormone (plant hormone) concept vs the sense of the mammalian hormones. The term "plant growth regulators" including new naturally occurring substances is ... [more ▼]

The paper begins with a review of the phytohormone (plant hormone) concept vs the sense of the mammalian hormones. The term "plant growth regulators" including new naturally occurring substances is discussed in regard to their effects on both growth and development processes, and possible different mechanisms of action in two different physiological directions. Mutual interactions between auxins, cytokinins, ethylene and polyamines are examined in several growth and development processes, showing the complexity of approach of the hormonal actions in plants . The indissociability of some hormonal relationships, and their obligatory integration with primary biochemical pathways is finally shown. [less ▲]

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See detailIAA-oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro
Faivre-Rampant, Odile; Kevers, Claire ULg; Gaspar, Thomas ULg

in Plant Science (2000), 153(1), 73-80

The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, inc, of tobacco compared ... [more ▼]

The peroxidase and IAA-oxidase activities, the degree of auxin protection and the amount of soluble phenolics were determined in in vitro cultured shoots of a nonrooting mutant, inc, of tobacco compared to its wild homologue. The mutant and wild shoots showed similar peroxidase variations along the growth cycle of 21 days, but with higher levels of activity for the I ac mutant. During this growth cycle, the minimum of peroxidase activity occurred at day 14 for both tobacco whole shoots. However. this minimum of activity did not occur at the same day in the basal part of the stem, where roots may appear, of the two types of tobacco. Both mutant and wild whole shoots showed about the same IAA-oxidase activity in the fractions resulting from a gel filtration of the crude extracts through a sephadex G-100 column but differed in the degree of auxin protection. The rac shoots exhibited a very high level of auxin protectors of low molecular weight, among which chlorogenic acid. They were also characterized by eight to nine times higher level of soluble phenolics. The relationships between these biochemical aspects in relation to the absence of root formation in the me mutant are discussed. (C) 2000 Elsevier Science Ireland Ltd. All rights reserved. [less ▲]

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See detailThe recalcitrance to rooting of the micropropagated shoots of the rac tobacco mutant: Implications of polyamines and of the polyamine metabolism
Faivre-Rampant, Odile; Kevers, Claire ULg; Dommes, Jacques ULg et al

in Plant Physiology & Biochemistry (2000), 38(6), 441-448

Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine ... [more ▼]

Rooting of wild-type tobacco (Nicotiana tabacum cv. Xanthi) shoots raised in vitro was promoted by polyamines in the absence of any other growth regulator and was inhibited by two inhibitors of polyamine metabolism. The auxin insensitive and recalcitrant to rooting rac mutant shoots did not respond to the same treatments. The activities of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), diamine oxidase (DAO), polyamine oxidase (PAO) and transglutaminases (TGases), and the titres of free and conjugated polyamines were estimated in the whole shoots and the basal parts of the stems of both tobaccos in the course of multiplication in vitro. The rac shoots grew at a lower rate. The wild-type rooted from the 7th day without special treatment. During the second week of culture, the shoots of both tobaccos were actively growing and showed an increase in ADC, ODC, DAO, PAO and TGase activities. Afterwards all these activities declined. These changes were concomitant with an increase in the polyamine contents (free and conjugated). Biosynthesis and oxidation of polyamines apparently occurred simultaneously and seemed directly correlated. In the basal part of the mutant stems however, the accumulation of free and conjugated putrescine as well as the transient increase in biosynthetic enzyme activities were delayed compared to the wild-type. These results are discussed in relation to growth behaviour and to root formation. (C) 2000 Editions scientifiques et medicales Elsevier SAS. [less ▲]

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See detailPutrescine metabolism in a fully habituated nonorganogenic sugar beet callus and its relationship with growth
Kevers, Claire ULg; Bisbis, Badia; Faivre-Rampant, Odile et al

in Journal of Plant Physiology (1999), 154(4), 503-508

A fully-habituated and nonorganogenic (HNO) sugar beet callus was previously shown to overproduce polyamines, as compared with a normal (N) auxin- and cytokinin-dependent callus of the same strain ... [more ▼]

A fully-habituated and nonorganogenic (HNO) sugar beet callus was previously shown to overproduce polyamines, as compared with a normal (N) auxin- and cytokinin-dependent callus of the same strain. Because relationships were established between polyamine levels and metabolism with different growth and development processes, some key enzymes in the metabolic pathways of polyamines were investigated in the HNO callus, and their involvement in growth appraised. Putrescine was found to be the major free and conjugated polyamine in the HNO callus. It was biosynthesised preferentially via ornithine and ornithine decarboxylase (ODC), which is in agreement with the surplus of synthesised ornithine. Diamine (DAO) and polyamine (PAO)-oxidase activities were also highest in the HNO callus, as compared with the normal, with DAO being the more active. Transglutaminase activities (+/- Ca) were also higher in HNO than in normal callus. Addition of different polyamines or of inhibitors of their biosynthesis to the culture medium of the HNO callus modified the level of endogenous polyamines and affected callus growth. The results thus pointed out a higher polyamine metabolism, particularly of putrescine, in the actively growing auxin- and cytokinin-independent callus than in the normal one. They also provided evidence for the sensitivity of a habituated tissue type towards this class of growth regulators. [less ▲]

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See detailPeroxidase activity, ethylene production, lignification and growth limitation in shoots of a nonrooting mutant of tobacco
Faivre-Rampant, Odile; Kevers, Claire ULg; Bellini, Catherine et al

in Plant Physiology & Biochemistry (1998), 36(12), 873-877

The rooting recalcitrant rac Nicotiana tabacum cv Xanthi mutant has been multiplied in vitro under the form of shoots in parallel to wild-type. rac Shoots grew at a lower rate and did not root whatever ... [more ▼]

The rooting recalcitrant rac Nicotiana tabacum cv Xanthi mutant has been multiplied in vitro under the form of shoots in parallel to wild-type. rac Shoots grew at a lower rate and did not root whatever the treatments when compared to those of wild-type shoots. They were characterized by a higher lignin level, a higher total specific peroxidase activity with higher activity of both acidic and basic isoperoxidases (although missing and supernumerary isoenzymes were observed), and higher ethylene production. These observations might be causally related to growth inhibitions as similar incidences have been noted in different stress-induced growth limitation, through cell wall rigidification and auxin catabolism. The relationship between these aspects and rooting recalcitrance remains to be explored. (C) Elsevier, Paris. [less ▲]

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