Oligogalacturonides inhibit the formation of roots on tobacco explants

α-1,4-Oligogalacturonides with degrees of polymerization (DPs) ranging from 6 to 18 or 2 to 8 were added to tobacco leaf explants and root formation was evaluated after 15 days of incubation. Auxin-induced formation of roots was inhibited by oligogalacturonides with DPs 6–18 but not by the oligogalacturonides with DPs 2–8. The inhibition of root formation by the larger oligogalacturonides was prevented by increasing the amount of auxin present in the medium. Oligogalacturonides (DPs 6–18) also inhibited root formation when added to tobacco thin cell-layer (TCL) explants in a medium that is known to induce the formation of roots. The addition of size-homogeneous oligogalacturonides, to either tobacco leaf explants or TCLs, established that oligogalacturonides with DPs between 10 and 14 were most active in inhibiting the formation of roots. These data suggest that oligogalacturonides of the same size as those known to elicit plant defense responses, and to affect floral development and membrane functions, also inhibit the induction of root morphogenesis in tobacco.     

INVESTIGATIONS ON THE ROOT FORMATION IN THE TISSUES OF HELIANTHUS TUBEROSUS CULTURED IN VITRO

Excised rhizome fragments of Helianthus tuberosus ‘Violet de Rennes’ produce roots in vitro; the sequence of morphogenetic phenomena is as follows: proliferation, differentiation of phloem and tracheids, organization of cambiums, and formation of root primordia from cambiums. This rhizogenesis is conditioned by five limiting factors: mineral salts, sugar, auxin, temperature, and light. The optimum level of each of these factors was determined and in certain cases also the threshold of action. For white light the beginning was about 1/1000 lux. These five limiting factors do not represent the same site of action. The mineral salts, especially nitrogen and calcium, have their effect when the cambiums have been organized. The sugars act before the formation of cambiums. Light operates principally upon cambium and this action is independent of photosynthesis. It was not possible to determine whether the auxin has a preferential site of action. A temperature higher than 15 C is essential to obtain the formation of cambiums but further production of roots is possible at 15 C or even less. The variations in temperature more or less around the optimum stimulate rhizogenesis. The supra-optimal temperatures mainly have their effect before the formation of cambiums and infra-optimal temperatures after. The effect of light can be exhibited at low temperature. Gibberellic acid in association with auxin favors root formation in the dark whereas in light it exhibits inhibiting properties. Rhizogenetic potentialities of tissues are reduced when these are cultured in the dark in presence of auxin. On the other hand, if auxin is applied after the isolation of explants, rhizogenesis is stimulated and the tissues are able to produce roots even in the dark. The action of light and of auxin can be dissociated; when the explants having absorbed a small quantity of auxin are transferred to a medium without auxin for 60 days in the dark, they conserve the rhizogenetic potential which is expressible on transferring them to light. Finally, grafting experiments suggested that light could induce the formation of a rhizogenetic factor in the tissues which is transmittable from cell to cell. These experiments have definitely established that the induction of root formation is governed by several factors which have complex interactions.     

Cytokinin and Ethylene Affect Auxin Transport-Dependent Rhizogenesis in Hypocotyls of Common Ice Plant (<Emphasis Type="Italic">Mesembryanthemum crystallinum</Emphasis> L<Emphasis Type="Italic">.</Emphasis>)

Hypocotyl explants of Mesembryanthemum crystallinum regenerated roots when cultured vertically with either the apical end (AE) or basal end (BE) in media containing indole-3-acetic acid (IAA). IAA alone induced roots regularly from the basal end of the explants, either from the cut surface immersed in the medium or from the opposite side. The inhibitors of auxin efflux carriers, α-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), inhibited rhizogenesis only from AE-cultured explants, indicating the role of polar auxin transport in root regeneration in this system. Cytokinin (zeatin, kinetin, BAP) added to auxin-containing medium reduced rhizogenesis from the explants maintained with BE and AE and additionally changed the IAA-induced pattern of rooting in AE-cultured explants by favoring rooting from the apical end and middle part of the hypocotyl with its concomitant reduction from the basal end. The addition of kinetin did not influence the content of IAA in the explants maintained with AE, suggesting that the cytokinin effect on root patterning was not dependent on auxin biosynthesis. Kinetin, however, strongly enhanced ethylene production. The importance of ethylene in regulating PAT-dependent rhizogenesis was tested by using an ethylene antagonist AgNO₃, an inhibitor of ethylene synthesis aminoethoxyvinylglycine (AVG), and a precursor of ethylene, 1-aminocyclopropane-1-carboxylic acid (ACC). AgNO₃ applied together with IAA or with IAA and kinetin strongly reduced the production of ethylene, inhibited rhizogenesis, and induced nonregenerative callus from BE, suggesting the need for ethylene signaling to elicit the rhizogenic action of auxin. A reduction of rhizogenesis and decrease of ethylene biosynthesis was also caused by AVG. In addition, AVG at 10 μM reversed the effect of cytokinin on root patterning, resulting in roots emerging only from BE on the medium with IAA and kinetin. Conversely, ACC at 200 μM markedly enhanced the production of ethylene and partly mimicked the effect of cytokinin when applied with IAA alone, thus confirming that in cultured hypocotyls of ice plant, cytokinin affects IAA-induced rhizogenesis through an ethylene-dependent pathway.     

Hormonal control of root development on epiphyllous plantlets of Bryophyllum (Kalanchoe) marnierianum: role of auxin and ethylene

Epiphyllous plantlets develop on leaves of Bryophyllum marnierianum when they are excised from the plant. Shortly after leaf excision, plantlet shoots develop from primordia located near the leaf margin. After the shoots have enlarged for several days, roots appear at their base. In this investigation, factors regulating plantlet root development were studied. The auxin transport inhibitor 2,3,5-triiodobenzoic acid (TIBA) abolished root formation without markedly affecting shoot growth. This suggested that auxin transport from the plantlet shoot induces root development. Excision of plantlet apical buds inhibits root development. Application of indole-3-acetic acid (IAA) in lanolin at the site of the apical buds restores root outgrowth. Naphthalene acetic acid (NAA), a synthetic auxin, reverses TIBA inhibition of plantlet root emergence on leaf explants. Both of these observations support the hypothesis that auxin, produced by the plantlet, induces root development. Exogenous ethylene causes precocious root development several days before that of a control without hormone. Ethylene treatment cannot bypass the TIBA block of root formation. Therefore, ethylene does not act downstream of auxin in root induction. However, ethylene amplifies the effects of low concentrations of NAA, which in the absence of ethylene do not induce roots. Ag(2)S(2)O(3), an ethylene blocker, and CoCl(2), an ethylene synthesis inhibitor, do not abolish plantlet root development. It is therefore unlikely that ethylene is essential for root formation. Taken together, the experiments suggest that roots develop when auxin transport from the shoot reaches a certain threshold. Ethylene may augment this effect by lowering the threshold and may come into play when the parent leaf senesces.     

Acides-aminés et amines libres ďexplants foliaires de Nicotiana tabacum cultivés in vitro sur des milieux induisant la rhizogenèse ou la caulogenèse

Free amino acids and amines in leaf explants of Nicotiana tabacum cultivated in vitro on media inducing rhizogenesis or caulogenesis.
Foliar explants of Nicotiana tabacum cv. Xanthi n.c. were cultivated on three different media: (1) a basal medium without hormone, so that no differentiation occurred in the explants; (2) with auxin added; and (3) with auxin plus cytokinin added, where the additions (2) and (3) promote rhizogenesis and caulogenesis, respectively. The content of free amino acids and amines of the three kinds of explants were investigated. In the two media lacking cytokinin, the explants contained great amounts of five amino acids (asparagine, glutamine, proline, glutamic acid and histidine) and of one aromatic amine, tyramine. In the cytokinin containing medium, only two amines accumulated in the explants: one aliphatic polyamine (putrescine) and one aromatic amine (phenethylamine). The increase in amino acids began immediately on the first days of culture. It was related neither to a more active proteolysis nor to the breaking of the correlations from the mother plant. It was induced by the addition of nutritional elements into the medium. On the other hand, the accumulation of aromatic amines occurred after a few days of culture and was transitory. A decrease was observed after the first emergence of new organs. The relation between the accumulations of these aromatic compounds and formation of roots or shoots is discussed.     

Plant Immunity Induced by Oligogalacturonides Alters Root Growth in a Process Involving Flavonoid Accumulation in Arabidopsis thaliana

Plants adapt to challenging environmental factors by modulating morphogenetic processes. Although it has been speculated that activation of defense responses against pathogens leads to plant growth adjustment, little is known about developmental and architectural responses to defense stimulators. In this report we evaluated the activity of oligogalacturonides (OGs), a class of molecules directly involved in plant immunity, to modulate root system architecture in Arabidopsis thaliana. We show that OGs induce PAD3 expression and camalexin synthesis, two well-known markers of defense responses. These effects were related to primary root growth inhibition and increased lateral root and root hair formation, which are reminiscent of altered auxin responses. Cellular analysis showed that the effect of these compounds on primary root growth was due to changes in cell elongation and increased flavonoid accumulation at the root elongation region. Moreover, the observations that similar changes in primary root growth were induced by naphthylphthalamic acid supply and that auxin- or flavonoid-related mutants tir1, doc1, pgp1, pgp4, pgp19, and tt4-1 show differential responses to primary root growth inhibition by OGs suggest that auxin homeostasis plays a role in the oligogalacturonide-induced alteration of root cell patterning. Our results suggest that OGs might play a dual function in adaptation of plants to pathogen challenge by inducing defense responses and plant architecture adjustment.     

杨树试管苗嫩茎生根过程中内源IAA的免疫金银定位

生长素类物质在木本植物生根过程中发挥重要作用。杨树生根与生长素的关系及生根过程中内源激素的变化已有大量报道,而生根过程中生长素的组织定位分析则尚未见报道。该文应用免疫化学分析方法对741杨（Populus alba×（P.davidiana×P.simonii）×P.tomentosa）嫩茎生根过程中内源IAA在组织中的分布进行了研究。结果显示,741杨的嫩茎在无外源激素的1/2MS培养基上诱导10天后可生根,14天后生根率达100%。诱导前,嫩茎基部组织中几乎没有IAA信号;诱导8天后,嫩茎基部维管组织中有大量的IAA积累,而且中部的维管组织中也有明显的IAA信号（主要分布在韧皮部和维管形成层）;10天后,形成不定根原基,此时IAA主要分布在根原基;12天后,根原基分化成不定根并突破表皮,IAA在不定根中的分布主要集中在根尖和中柱。该文对741杨的嫩茎生根过程中IAA的组织分布特点及运输途径进行了讨论。     

Oligogalacturonides stimulate pericycle cell wall thickening and cell divisions leading to stoma formation in tobacco leaf explants

Novel developmental events induced by micromolar concentrations of oligogalacturonides (OGs) in tobacco leaf explants cultured in vitro are described. Oligogalacturonides induced acceleration and synchronization of the mitotic activity of guard-cell precursors in the epidermis. In explants cultured for 24 h in the presence of OGs, the number of stomatal mitoses was higher than that observed in explants cultured in the absence of OGs; however, at the end of the culture period the density of mature stomata did not vary upon OG treatment. The OG-induced activation of stomatal mitosis was reduced by exogenously added indole-3-acetic acid (IAA). Oligogalacturonides also enhanced mean wall thickness, mainly due to cellulose deposition, of foliar pericycle cells, as well as the number of extra-thick-walled pericycle cells; the pericycle thus formed a sheath surrounding phloem and xylem. Indole-3-acetic acid decreased the number of extra-thick-walled cells forming in the presence of OGs but did not influence wall thickness. Moreover, OGs inhibited the stimulation of mitotic activity of phloem parenchyma cells (vascular mitoses) induced by auxin, leading to a nearly complete inhibition of IAA-induced formation of callus and of meristemoids of indirect origin. Instead, OGs did not influence mitotic activity occurring in the absence of auxin. All in all, our results provide further evidence of the pleiotropic role exerted on plant development by these oligosaccharins, and of the antagonism between auxin and OGs. Received: 4 March 1997 / Accepted: 15 July 1997     

Galacturonic acid-induced changes in strawberry plant development <Emphasis Type="Italic">in vitro</Emphasis>

The oligogalacturonides derived from trifluoroacetic acid hydrolysis and their monomer galacturonic acid were tested for their effect on development of strawberry explants (Fragaria × ananassa) in vitro. The addition of oligogalacturonides or galacturonic acid in the plant tissue culture medium at concentrations of 0.1 or 1.0 mM increased shoot and leaf number in the presence of added benzyl adenine. The response to oligogalacturonides or galacturonic acid was maintained in the absence of benzyl adenine but was lessened. Conversely, root elongation was increased in the absence of benzyl adenine. A combined analysis of these experiments also indicated the increase in shoot or leaf number. High-performance anion exchange chromatography of the bioassayed oligogalacturonide samples using a pulsed amperometric detector revealed that the sample consisted of galactouronides with a degree of polymerization from 1 to 5 and the proportion of the monomer was very high (94%).     

Expression of rolB in tobacco flowers affects the coordinated processes of anther dehiscence and style elongation

The effect of auxin on stamen and pistil development in tobacco flowers was investigated by means of the localized expression of rolB (root loci B), an Agrobacterium oncogene that increases auxin sensitivity in a cell-autonomous fashion. When rolB is driven by the promoter of the meiosis-specific Arabidopsis gene DMC1 (disrupted meiotic cDNA 1), expression occurs earlier in male than in female developing organs, resulting in a delay in anther dehiscence with respect to normal timing of pistil development. As a consequence of this developmental uncoupling, self-pollination is prevented in pDMC1:rolB plants. Histological analysis of pDMC1:GFP plants indicates that in tobacco, this promoter is active not only in meiocytes but also in somatic tissues of the anther. In contrast, simultaneous expression of rolB in anther and pistil somatic tissues, achieved by expressing a construct containing rolB under the control of the promoter of the petunia gene FBP7 (floral binding protein 7), results in a concomitant delay of both anther dehiscence and pistil development without affecting self-pollination of the plants. Analysis of plants harboring the pFBP7:GUS construct shows that in tobacco, this promoter is active not only in the ovules, as described for petunia, but also in pistil and anther somatic tissues involved in the dehiscence program. The delay in anther dehiscence and pistil development could be phenocopied by exogenous application of auxin. Jasmonic acid (JA) could not rescue the delay in anther dehiscence. These results suggest that auxin plays a key role in the timing of anther dehiscence, the dehiscence program is controlled by the somatic tissues of the anther, and auxin also regulates pistil development.     

Root meristems in Medicago truncatula tissue culture arise from vascular-derived procambial-like cells in a process regulated by ethylene

Leaf explants of Medicago truncatula were used to investigate the origins of auxin-induced root formation. On the application of auxin there is some callus formation (not the massive amount that occurs in response to auxin plus cytokinin) and roots appear shortly after the first visible callus. Histological examination reveals morphologically distinctive sheets of callus cells that emanate from the veins of the leaf explants and, within this cell type, root primordia are produced as well as some vascular tissue cells. What is suggested is that the vein-derived cells (VDCs) are procambial-like and function as pluripotent stem cells with a propensity to form root meristems or vascular tissues in response to added auxin. The development of root primordia from these pluripotent cells was clearly up-regulated by the use of the sickle (skl) mutant, which is a mutant impaired in ethylene signal transduction while the wild type and the sunn mutant, defective in auxin polar transport, produced similar numbers of roots. The skl mutant in generating many more roots concomitantly formed fewer vascular tissues. The root meristems differentiate similarly to normal roots producing a central cylinder of vascular tissue, which connects with the leaf explant veins. The VDCs appear to be derived from the cells of or near the phloem. The leaf observations suggest that a pool of stem cells exist in vascular tissue that, in combination with auxin and perhaps other factors, drive a diversity of plant development outcomes that is species specific. The way auxin interacts with other hormones is a key factor in determining the stem cell fate. The histological data in this study also assist in the interpretation of the molecular analysis of auxin-induced root formation in cultured leaves of M. truncatula.     

Aucsia gene silencing causes parthenocarpic fruit development in tomato

In angiosperms, auxin phytohormones play a crucial regulatory role in fruit initiation. The expression of auxin biosynthesis genes in ovules and placenta results in uncoupling of tomato (Solanum lycopersicum) fruit development from fertilization with production of parthenocarpic fruits. We have identified two newly described genes, named Aucsia genes, which are differentially expressed in auxin-synthesis (DefH9-iaaM) parthenocarpic tomato flower buds. The two tomato Aucsia genes encode 53-amino-acid-long peptides. We show, by RNA interference-mediated gene suppression, that Aucsia genes are involved in both reproductive and vegetative plant development. Aucsia-silenced tomato plants exhibited auxin-related phenotypes such as parthenocarpic fruit development, leaf fusions, and reflexed leaves. Auxin-induced rhizogenesis in cotyledon explants and polar auxin transport in roots were reduced in Aucsia-silenced plants compared with wild-type plants. In addition, Aucsia-silenced plants showed an increased sensitivity to 1-naphthylphthalamic acid, an inhibitor of polar auxin transport. We further prove that total indole-3-acetic acid content was increased in preanthesis Aucsia-silenced flower buds. Thus, the data presented demonstrate that Aucsia genes encode a novel family of plant peptides that control fruit initiation and affect other auxin-related biological processes in tomato. Aucsia homologous genes are present in both chlorophytes and streptophytes, and the encoded peptides are distinguished by a 16-amino-acid-long (PYSGXSTLALVARXSA) AUCSIA motif, a lysine-rich carboxyl-terminal region, and a conserved tyrosine-based endocytic sorting motif.     

Callogenesis and rhizogenesis in date palm leaf segments: are there similarities between the two auxin-induced pathways?

In date palm (Phoenix dactylifera L. cv. Ahmar, Arecaceae), as for many monocotyledons, callogenesis is a prerequisite for the initiation of somatic embryogenesis, and requires the presence of auxin in the medium. Immature leaf explants were cultivated in medium supplemented with either 1 or 54 μM 1-naphtaleneacetic acid in order to induce either rhizogenesis or callogenesis. Histological studies performed throughout the culture period established that precocious cell reactivation is similar in both morphogenetic pathways. Early cytological modifications are associated with cell reactivation and are observed in the pluripotent cells of perivascular sheaths. Divergence between the callogenesis and rhizogenesis pathways is observed later, during the subsequent determination and morphological differentiation phases. We established that in date palm, the rhizogenesis and callogenesis pathways are initiated from the same cell type, the ultimate developmental fate depending upon auxin concentration.     

The protein encoded by the rolB plant oncogene hydrolyses indole glucosides.

The rolB gene of Agrobacterium rhizogenes, whose expression stimulates the formation of roots by transformed plant tissues and other growth alterations in transgenic plants, codes for a beta-glucosidase able to hydrolyse indole-beta-glucosides. Indeed, we show that extracts of bacteria and/or plant tissue expressing the rolB protein hydrolyse indoxyl-beta-glucoside (plant indican). Because of the structural similarity between indoxyl-beta-glucoside and indole-3-acetyl-beta-glucoside (IAA-beta-glucoside), we propose that the physiological and developmental alterations in transgenic plants expressing the rolB gene could be the result of an increased intracellular auxin activity caused by the release of active auxins from inactive beta-glucosides. Thus two of the oncogenes carried by the T-DNA of the plant pathogen Agrobacterium rhizogenes (rolB and rolC) perturb plant growth and development by coding for beta-glucosidases with distinct specificities. Whereas the rolC beta-glucosidase releases cytokinins from their glucoside conjugates, the rolB encoded protein hydrolyses indole-beta-glucosides. The combined action of these two genes therefore is expected to modulate the intracellular concentration of two of the main growth factors active in plants.     

Biologically Active Oligosaccharides from Pectins of Pisum sativum L. Seedlings Affecting Root Generation

Two physiologically active oligosaccharide fractions were isolated from pectin of Pisum sativum L. cell wall after its partial acid hydrolysis. These fractions displayed stimulating and inhibiting effects on root formation in thin-layer explants. The subsequent separation of these fractions by gel permeation and anion-exchange chromatography resulted in fractions with effective concentrations two orders of magnitude lower than the concentrations of the initial fractions. The resulting oligosaccharides displayed their effect on the earliest stage of the rhizogenesis associated with formation of root primordias. The rhizogenesis-inhibiting fraction suppressed cell division by 30-50%. The stimulating fraction mainly contained fragments of xyloglucan and galactan, and the inhibiting fraction contained fragments of xyloglucan, galactan, and arabinan. The polymerization degrees of the stimulating and of the inhibiting oligosaccharides were 10-11 and 5-6, respectively.     

Tiba inhibition of <Emphasis Type="Italic">in vitro</Emphasis> organogenesis in excised tobacco leaf explants

Summary Triiodobenzoic acid (TIBA), an anti-auxin, was found to inhibit both shoot and root formation in cultured excised leaf explants of tobacco (Nicotiana tabacum L.). The shoot formation (SF) medium used required only exogenous cytokinin (N⁶-benzyladenine) and the root formation (RF) medium required both auxin (indole-3-butyric acid) and cytokinin (kinetin). By transferring the explants from SF or RF media to SF or RF media with TIBA (4.0×10⁻⁵ M), respectively or vice versa, at different times in culture, it was found that TIBA inhibition was at the time of meristemoid formation and after determination of organogenesis. This indicates that TIBA interfered with endogenous auxin involvement in organized cell division.     

In vitro direct rhizogenesis from Gerbera jamesonii Bolus leaf

The present report describes an original protocol for in vitro direct induction of roots from leaf explants of gerbera for the first time. Since gerbera has immense potential as a premium cut-flower, the major attempts were made on in vitro mass propagation chiefly through in vitro multiple shoot proliferation or callus regeneration. Nevertheless, rhizogenesis could be impending an unattempted method with its yet-to-be known advantages. In our study, the optimum conditions for direct root induction from leaf explants were assessed employing tissue culture technique. Leaves were inoculated to MS medium containing no or variable auxin sources and concentrations namely, 2,4-dichlorophenoxyacetic acid, indole-3-acetic acid (IAA), indole-3-butyric acid or α-naphthaleneacetic acid for root induction. It was evident that the maximum root induction (with a frequency of 92.6 %) occurred on MS media fortified with 1.5 mg l^?1 IAA, wherein root induction was observed as early as 11 days of culture and an average of ~19 roots with ~13 mm length was obtained from 4 cm² leaf segment after 45 days of culture. Stereo microscopic observation revealed the induction of roots and gradual developmental stages of rhizogenesis. The efficiency of direct root induction without any interim growth stages (such as, callus or shoots) in our study offers a reproducible system that could provide a model protocol for more comprehensive developmental studies on root growth.