Oligogalacturonides are able to induce flowers to form on tobacco explants

Oligogalacturonides (α-1,4-D-galactosyluronic acid oligomers) are fragments of the homogalacturonan component of the primary cell walls of higher plants. Treatment of cell walls with endopolygalacturonase (EPG) releases the polysaccharides rhamnogalacturonan-1 (RG-I) and rhamnogalacturonan-II (RG-II), and variously sized oligogalacturonide fragments of homogalacturonan. The EPG-released sycamore cell wall components are able to regulate several morphogenetic processes of tobacco thin-cell layer (TCL) explants. We followed one of these morphogenesis-regulating activities, namely, the induction of flower formation on TCLs, to purify the biologically active component from EPG-released material. Saponification of the methyl and acetyl esters of the EPG-released material did not reduce the flower-inducing activity. However, EPG treatment of the deesterified EPG-released material destroyed the flower-inducing activity, establishing that the active substance contained several, consecutive α-1,4-linked galactosyluronic acid residues that are required for the flower-inducing activity. The flower-inducing activity was purified and shown to be α-1,4-linked Oligogalacturonides with a degree of polymerization (DP) of 12-14, which exhibited half-maximum activity at approximately 0.4 μM. Smaller oligogalacturonides, RG-I and RG-II, did not, even at higher concentrations, induce flowers to form. The ability of oligogalacturonides to stimulate the formation of flowers on tobacco explants provides further evidence of the pleiotropic nature of this oligosaccharin.     

Oligogalacturonides enhance cytokinin-induced vegetative shoot formation in tobacco explants, inhibit polyamine biosynthetic gene expression, and promote long-term remobilisation of cell calcium

Long-sized oligogalacturonides (OGs) are cell wall fragments that induce defence and developmental responses. The Ca²⁺-dependent “egg-box” conformation is required for their activity, and polyamines may prevent them from adopting this conformation. Although OGs are known to inhibit auxin-induced growth processes, their effect on cytokinin-induced ones requires investigation. In the present work OGs were shown to promote cytokinin (benzyladenine, BA)-induced vegetative shoot formation from tobacco leaf explants, independent of the presence of CaCl₂ in the medium and of auxin (indoleacetic acid, IAA) supply. The effect of polyamines, putrescine (PU) and spermidine (SD) supplied with/without their biosynthetic inhibitors (DFMO, CHA) was also investigated, and showed that spermidine enhanced adventitious vegetative shoot formation, but only on medium containing Ca²⁺ and IAA. Treatments with inhibitors blocked this promotive effect. OGs did not alter free polyamine concentrations, but caused a moderate increase of conjugated ones, and exhibited an early inhibitory effect on polyamine biosynthetic gene expression. OGs, but not SD, caused long-term changes in calcium-associated epifluorescent signals in the cell walls, and, later, inside the cells of specific tissues. Electron microscopy analysis (ESI system) demonstrated that calcium accumulated in the cell walls and vacuoles of OG-cultured explants. The relationship between OGs, cytokinin, calcium, and polyamines in adventitious vegetative shoot formation is discussed.     

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     

Oligogalacturonides inhibit the induction of late but not of early auxin-responsive genes in tobacco

Oligogalacturonides (OGs) released from the plant cell wall regulate several defense responses, as well as various aspects of plant growth and development. In these latter effects, OGs exhibit auxin-antagonist activity. To shed light on the mechanism by which OGs antagonise auxin, we analysed the ability of these oligosaccharides to inhibit the activity of four auxin-up-regulated promoters [pGm-GH3 of soybean (Glycine max L. Merr.), pNt114 of tobacco (Nicotiana tabacum L.), and prolB and prolD of Agrobacterium rhizogenes] driving the expression of the beta-glucuronidase reporter gene (GUS) in transgenic tobacco seedlings. Our results indicate that OGs at submicromolar concentrations inhibit the activation by auxin of pNt114, prolB and prolD, but not that of pGm-GH3. Comparative analysis of the kinetics of activation of the four promoters in response to the hormone shows that, while pGm-GH3 is rapidly activated, the other three promoters exhibit a delayed activation, with a lag of at least 4 h before the appearance of GUS activity. The lack of effect of the OGs on early auxin-responsive genes was confirmed by RNA gel blot analysis of the tobacco genes Nt-GH3 and Nt-iaa2.3/2.5. Our results suggest that the auxin-antagonist action of OGs affects the expression of late but not of early auxin-responsive genes.     

Oligogalacturonides stimulate antioxidant system in alfalfa roots

Alfalfa (Medicago sativa L.) roots were treated with 50 and 100 μg cm⁻³ of oligogalacturonide (OGA) solutions with a degree of polymerization between 7 and 15. Changes in the activities of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), monodehydroascorbate reductase (MDHAR) and dehydroascorbate reductase (DHAR) as well as ascorbate (ASC) content were determined in crude extract of alfalfa roots after 30, 60 and 120 min of treatment. An increase in the SOD activity was observed in roots treated with 50 and 100 μg cm⁻³ OGA, which could be related to its O₂ ^·− scavenging function. As concern H₂O₂ scavenging, CAT activity was increased in the first 30 min by both OGA concentrations, while POX was a key enzyme at higher OGA concentration and treatment duration. ASC content firstly increased upon exposure to high OGA concentration, and then decreased after longer treatment while low OGA concentration had no effect on ASC content.     

Long-sized oligogalacturonides inhibit,whereas spermidine enhances,xylogenesis in tobacco leaf explants

Abstract

Long-sized oligogalacturonides (OGs) are plant cell wall fragments involved in defence responses and developmental processes. A hormone/OG interaction in the control of different organogenic processes is known. However, hormones also modulate polyamine (PA) effects on organogenesis. Furthermore, OGs are known to affect mitotic activity leading to specific morphogenic events, and PAs are known to affect mitotic activity leading to xylogenesis. Thus, it may be reasonable to assume that OGs and PAs affect mitotic activity in the same cell types, and in the same hormone-induced morphogenic processes, e.g., xylogenesis. To gain further insight into this aspect, the effects of OGs, and of putrescine (Put) and spermidine (Spd), on auxin (indoleacetic acid, IAA) plus benzyladenine (BA)-induced morphogenesis in tobacco leaf explants were investigated histologically. The effect of PA biosynthetic inhibitors in the culture medium was also monitored, as well as the combined application of the inhibitor with the corresponding PA. Results show that vascular mitoses consistently occurred in the control (IAA+BA-treated) explants, leading exclusively to xylogenic nodule formation. The application of OGs resulted in an inhibition of vascular mitoses, and into a strong reduction of vascular nodule formation. By contrast, Spd enhanced both vascular mitoses and nodule formation, and Put was less effective than Spd on both events. Taken together, the results reveal a new biological activity of OGs and Spd in morphogenesis, obtained under the same hormonal conditions, and in the same tissue (i.e., the vascular parenchyma), namely the inhibition of xylogenesis by OGs, and its promotion by Spd. The fact that the effects of Spd and OGs on this morphogenic event may involve a different relationship with auxin is discussed.     

Further experiments on spermidine-mediated floral-bud formation in thin-layer explants of Wisconsin 38 tobacco

We studied the effects of various polyamines on bud regeneration in thin-layer tissue explants of vegetative and floweringNicotiana tabacum L. cv. Wisconsin 38, in which application of exogenous spermidine (Spd) to vegetative cultures causes the initiation and development of some flower buds (Kaur-Sawhney et al. 1988 Planta173, 282). We now show that this effect is dependent on the time and duration of application, Spd being required from the start of the cultures for about three weeks. Neither putrescine nor spermine is effective in the concentration range tested. Spermidine cannot replace kinetin (N⁶-furfurylaminopurine) in cultures at the time of floral bud formation, but once the buds are initiated in the presence of kinetin, addition of Spd to the medium greatly increases the number of floral buds that develop into normal flowers. Addition of Spd to similar cultures derived from young, non-flowering plants did not cause the appearance of floral buds but rather induced a profusion of vegetative buds. These results indicate a morphogenetic role of Spd in bud differentiation. Dedicated to Professor Hans Mohr on the occasion of his 60th birthday     

Role of ethylene in auxin-induced flower bud formation in tobacco explants

The effect of ethytene on in vitro flower bud formation in thin-layer explants from tobacco pendicels ( Nicotiana tabacum L. cv. Samsun) was studied Endogenous ethylene production was stimulated by l-minocyclopropanc-l-carhoxylic acid (ACT), and inhibited by aminoethoxyviny lglycine (AVG). resulting in higher and lower ethylene accumulation. respectively. In the presence of an elevated ethylene concentration, the number of flower buds formed after 7 days of culture in explants was increased, compared with the control. Treatment with AVG or with AgNO₃ which blocks ethylene action resulted in decreased bud numbers after 7 days of culture. A different effect of ethylene was visible after 14 days of culture, when regeneration was complete. Treatment with AgNO₃ led to more bud regeneration, and increasing ethylene concentrations to lower bud numbers. The endogenous production of ethylene was enhanced by high concentrations of 1-naphthaleneacetic acid (NAA).
The inhibitory effect of applied ethylene was almost 100% in explants cultured at low concentrations of NAA (below 1 μ M ). but hardly visible at high concentrations (4.5 μ M ). As a consequence, the optimal NAA concentration shifted to a higher value in the presence of ethylene. These results are interpreted as a reduction in tissue sensitivity to auxin and in regenerative capability by ethylene. The effect of ethylene on auxin action is not exerted at the level of hormone concentration. Neither NAA uptake nor conversion to conjugates was effected by ethylene.     

A plasma membrane-bound enzyme of tobacco roots catalyses the formation of nitric oxide from nitrite

Purified plasma membranes (PMs) of tobacco (Nicotiana tabacum L. cv. Samsun) roots exhibited a nitrite-reducing enzyme activity that resulted in nitric oxide (NO) formation. This enzyme activity was not detected in soluble protein fractions or in PM vesicles of leaves. At the pH optimum of pH 6.0, nitrite was reduced to NO with reduced cytochrome c as electron donor at a rate comparable to the nitrate-reducing activity of root-specific succinate-dependent PM-bound nitrate reductase (PM-NR). The hitherto unknown PM-bound nitrite: NO-reductase (NI-NOR) was insensitive to cyanide and anti-NR IgG and thereby proven to be different from PM-NR. Furthermore, PM-NR and NI-NOR were separated by gel-filtration chromatography and apparent molecular masses of 310 kDa for NI-NOR and 200 kDa for PM-NR were estimated. The PM-associated NI-NOR may reduce the apoplastic nitrite produced by PM-NR in vivo and may play a role in nitrate signalling via NO formation. Received: 8 May 2000 / Accepted: 24 August 2000     

In vitro formation and development of floral buds on tobacco stem explants: effects of kinetin and other factors

Stem segments were excised from plants of Wisconsin 38 tobacco (Nicotiana tabacum L.) in three regions differing in their distance below the inflorescence. They were cultured in vitro in 8- or 16-hr days. After 8 weeks, floral and vegetative buds were counted, and extent of floral development was assessed. Kinetin at 10(-5)m inhibited formation and development of floral buds regardless of indoleacetic acid concentration. Supplied at this concentration with adequate auxin, kinetin stimulated vegetative bud formation and may have caused floral bud abortion. Indoleacetic acid (>/= 10(-6)m) inhibited vegetative and floral bud formation when supplied with low kinetin concentration (/= 10(-6)m), it inhibited floral bud formation and stimulated vegetative bud formation. More floral buds were formed in 16-hr days than in 8-hr days. Few formed on explants other than those derived from the region nearest the inflorescence regardless of other treatment.     

Uptake and metabolism of NAA and BAP in explants of tobacco in relation toin vitro flower bud formation

In vitro flower bud initiation and development depend on the presence of two hormones in the culture medium—auxin (NAA) and cytokinin (BAP). The uptake of both NAA and BAP by the explants was shown to be proportional to the concentrations supplied in the medium over a period of 4 days after the onset of culture. However, when supplied at equal concentrations for 24 h, the NAA uptake was up to 10-fold higher than the BAP uptake. Both hormones are rapidly metabolized by the explants. Nevertheless, the concentrations of free hormones inside the explants appeared to be high and in the case of NAA exceeded the concentration in the medium by more than 1 order of magnitude within 24 h. Apparently flower bud initiation in tobacco explants requires relatively high concentrations free NAA and BAP in the tissue maintained by a continuous supply in the medium. There are at present no indications that the products of hormone metabolism are directly involved in bud formation.     

Induction of indoleacetic Acid synthetases in tobacco pith explants

Formation of indoleacetic acid synthetases in tobacco pith explants was determined by following the growth of tissue cultures under conditions of indole-3-acetic acid (IAA) deprivation and by measuring the enzymatic conversion of tryptophan to IAA in the cultures. The pith explants obtained from the parent plant (Nicotiana glauca) and from basal regions of the tumor-prone hybrid (N. glauca × N. langsdorffii) both show a requirement for exogenous IAA for growth initiation in culture. The parent pith requires the constant presence of added IAA for continued growth, but hybrid pith, after initial treatment with IAA, will grow without further additions. IAA synthetases are detected in the cell homogenates of hybrid pith explants cultured with either continuous or initial IAA addition. These observations indicate that IAA may induce its own production. In contrast, IAA synthetases are not found in the parent pith under comparable culture conditions. Besides IAA, nonhormonal compounds such as indole and tryptophan are also capable of stimulating growth of hybrid pith, possibly through the induction of IAA synthetases needed for IAA formation. Indole and tryptophan are, however, inactive in growth promotion of the parent pith. These results suggest that the genomic expression of IAA synthetase formation is more stringently controlled in N. glauca than in the tumorprone hybrid.     

Spermidine and flower-bud differentiation in thin-layer explants of tobacco

Three lines of evidence indicate a connection between high spermidine levels and floral initiation in thin-layer tissue cultures of Wisconsin-38 tobacco (Nicotiana tabacum L.). (1) Spermidine levels are much higher in floral buds than in vegetative buds. (2) Inhibition of spermidine synthesis by cyclohexylamine prevents the rise in spermidine titer, inhibits floral initiation and promotes the formation of vegetative buds instead. (3) Application of exogenous spermidine causes floral initiation in cultures which would otherwise form vegetative buds.     

烟草幼苗根系分泌自毒物质种类及PAEs对根系抗氧化性能的影响

采用GC-MS技术鉴定水培烟草Burley及K326在幼苗期不同生长阶段的根系分泌物;并用不同浓度邻苯二甲酸二丁酯(DBP)、邻苯二甲酸二异辛酯(DIOP)溶液浇灌盆栽烟草幼苗,研究其根系抗氧化性能变化。结果如下:(1)Burley根系分泌物主要有3类化合物,其中自毒物质邻苯二甲酸酯(PAEs)在二叶龄期、四叶龄期、六叶龄期的相对含量分别为7.6%、0.3%、未检出;而K326根系分泌物主要有9类化合物,PAEs在二叶龄期、四叶龄期、六叶龄期的相对含量分别为35.6%、51.3%、2.2%。(2)浓度高于0.1 mmol/L的PAEs使根中超氧阴离子自由基产生的速率显著(P0.05)增加;随着DIOP及DBP浓度的增加,超氧化物歧化酶、过氧化氢酶活性增加,在0.5 mmol/L时达到最大,然后随着处理浓度的增加而下降。丙二醛的浓度随着这两种PAEs处理浓度的增加而增大。结果表明:烟草根系分泌的自毒物质PAEs达到0.5 mmol/L时,能降低根系的抗氧化性能,造成根尖细胞膜系统的氧化损伤,引起根吸收功能等一系列生理生化变化,并最终表现出自毒作用。     

Nicotinic acid decarboxylation in tobacco roots

An enzyme system which catalyzes the O₂ dependent release of ¹⁴CO₂ from nicotinic acid-7-¹⁴C is present in tobacco roots. The enzyme is not present in tobacco leaves and stems. The nature of reaction catalyzed and its tissue distribution suggest that this enzyme may be involved in nicotine biosynthesis.     

Orientation of cellulose microfibrils in cortical cells of tobacco explants

The deposition of nascent cellulose microfibrils (CMFs) was studied in the walls of cortical cells in explants of Nicotiana tabacum L. flower stalks. In freshly cut explants the CMFs were deposited in two distinct and alternating orientations — all given with respect to the longitudinal axis of the cell —, at 75° and 115°, in a left-handed (S-helix) and right-handed (Z-helix) form, respectively. The CMFs deposited in these orientations did not form uninterrupted layers, but sheets in which both orientations were present. After explantation, the synthesis of CMFs and their deposition in bundles continued. New orientations occurred within 6 h. After 6 h a new sheet was deposited, with orientations of 15° (S-helix) and 165° (Z-helix). The changes could be seen as sudden bends in individual CMFs or in small bundles of CMFs. In the next stage, more CMFs were deposited with these new orientations and the bundles became larger. New orientations arose by a shift towards more longitudinal directions, starting from either the S-helix or the Z-helix form. It was only after an almost longitudinal orientation was reached that the CMFs were deposited in two opposing directions again and a new sheet was formed. Neither colchicine nor cremart influenced the changes in CMF deposition. It is concluded that microtubules do not control CMF deposition in cortical cells of tobacco explants; control of CMF deposition and microtubule orientation occurs by factors related to cell polarity.Abbreviations CMF cellulose microfibril - MT microtubule We thank Professor M.M.A. Sassen and Dr. G.W.M. Barendse (Department of Experimental Botany, University of Nijmegen, Nijmegen, The Netherlands) for helpful discussions and Mrs. A. Kemp for her assistance in the ethylene experiments.     

Reorganization of cortical microtubules during cell differentiation in tobacco explants

Summary Using indirect immunofluorescence on polyethylene glycol embedded material, the organization of cortical microtubules (MTs) has been studied in explants ofNicotiana tabacum. Within 6 hours after explantation the orientation of the cortical MTs shifts from transverse to longitudinal to the long axis of the cell in all cells. This change of direction is followed by further shifts that occur only locally and predict the orientation of future cell divisions. These reorientations are independent of the formation of protrusions and buds that will develop in the explants (after 4–7 days) and they represent a stage of de-differentiation of the explants. After two days of culturing clusters of cells can be recognized, at the proximal side of the explants, with randomly oriented cortical MTs. These regions represent the origin of the protrusions from which floral buds will develop. The formation of these clusters represent the first signs of re-differentiation and formation of new polar axes in the explants. The cells thus show a very early commitment (within 2 days) as to their differentiation.Abbreviations BAP benzyl-amino-purine - DMSO dimethylsulfoxid - EGTA ethylene glycol bis(2-aminoethylether)-N,N,N,N-tetraacetic acid - GA glutaraldehyde - MTs microtubules - MTOCs microtubule organizing centres - NAA -naphthalene acetic acid - PEG polyethylene glycol - PFA paraformaldehyde - PPBs preprophase bands