Physiological,morphological, and growth responses ofPlatanus occidentalis seedlings to flooding

Summary Flooding ofPlatanus occidentalis L. seedlings for up to 40 days induced several changes including early stomatal closure, greatly accelerated ethylene production by stems, formation of hypertrophied lenticels and adventitious roots on submerged portions of stems, and marked growth inhibition. Poor adaptation ofPlatanus occidentalis seedlings to soil inundation was shown in stomatal closure during the entire flooding period, inhibition of root elongation and branching, and death of roots. Some adaptation to flooding was indicated by (1) production of hypertrophied lenticels which may assist in exchange of dissolved gases in flood water and in release of toxic compounds, and (2) production of adventitious roots on stems which may increase absorption of water. These adaptations appeared to be associated with greatly stimulated ethylene production in stems of flooded plants. The greater reduction of root growth over shoot growth in flooded seedlings will result in decreased drought tolerance after the flood waters recede. The generally low tolerance to flooding of seedlings of species that are widely rated as highly flood tolerant is emphasized.     

Interactions between ethylene, gibberellin and abscisic acid regulate emergence and growth rate of adventitious roots in deepwater rice

Growth of adventitious roots is induced in deepwater rice (Oryza sativa L.) when plants become submerged. Ethylene which accumulates in flooded plant parts is responsible for root growth induction. Gibberellin (GA) is ineffective on its own but acts in a synergistic manner together with ethylene to promote the number of penetrating roots and the growth rate of emerged roots. Studies with the GA biosynthesis inhibitor paclobutrazol revealed that root emergence was dependent on GA activity. Abscisic acid (ABA) acted as a competitive inhibitor of GA activity. Root growth rate on the other hand was dependent on GA concentration and ABA acted as a potent inhibitor possibly of GA but also of ethylene signaling. The results indicated that root emergence and elongation are distinct phases of adventitious root growth that are regulated through different networking between ethylene, GA and ABA signaling pathways. Adventitious root emergence must be coordinated with programmed death of epidermal cells which cover root primordia. Epidermal cell death is also controlled by ethylene, GA and ABA albeit with cell-type specific cross-talk. Different interactions between the same hormones may be a means to ensure proper timing of cell death and root emergence and to adjust the growth rate of emerged adventitious roots.     

Ethylene-Induced Activation of Xylanase in Adventitious Roots of Maize as a Response to the Stress Effect of Root Submersion

Submersion of roots of ten-day-old maize (Zea maysL.) seedlings was accompanied by a decrease in pO₂and an increase in pCO₂of the medium adjacent to the roots. These changes stimulated ethylene evolution in intact plants. Enhanced biosynthesis of ethylene was accompanied by xylanase activation in adventitious roots. As a result, an enhanced formation of aerenchyma was observed in the cortex of adventitious roots. Therefore, these processes resulted in the development of a ventilation system by which O₂can reach the root system exposed to hypoxia. The volume of aerenchyma was assessed by the volume of gas cavities (porosity). In contrast to the main root, the growth of adventitious roots was not inhibited under these conditions. Enlargement of the stem base and increase in the number of aerenchymatous adventitious roots facilitated the oxygen supply to the submerged organs of the plants.     

Temporal changes in the growth, saponin content and antioxidant defense in the adventitious roots of Panax ginseng subjected to nitric oxide elicitation

Nitric oxide (NO) is a diffusible, gaseous signaling molecule. In plants, NO influences growth and development, and it can also affect plant responses to various stresses. Because NO induces root differentiation and interacts with reactive oxygen species, we examined the temporal effect of NO elicitation on root growth, saponin accumulation and antioxidant defense responses in the adventitious roots of mountain ginseng (Panax ginseng). The observations revealed that NO is involved in root growth and saponin production. Elicitation with sodium nitroprusside (SNP) activated O₂ ⁻-generating NADPH oxidase (NOX) activity, which most probably subsequently enhanced growth of adventitious roots of mountain ginseng. A severe inhibition of NOX activity and decline in dry weight of SNP elicited adventitious roots in the presence of NOX inhibitor (diphenyl iodonium, DPI), which further supports involvement of NOX in root growth. Enhanced activities of antioxidant enzymes by SNP appear to be responsible for low H₂O₂, less lipid peroxidation, and modulation of ascorbate and non-protein thiol statuses in the adventitious roots of mountain ginseng. Dry mass, saponin content and NOX activity was related with NO content present in adventitious roots of mountain ginseng.     

Ethylene insensitivity impedes a subset of responses to phosphorus deficiency in tomato and petunia

The role of ethylene in growth and developmental responses to low phosphorus stress was evaluated using ethylene-insensitive 'Never-ripe' (Nr) tomato and etr1 petunia plants. Low phosphorus increased adventitious root formation in 'Pearson' (wild-type) tomato plants, but not in Nr, supporting a role for ethylene in adventitious root development and showing that ethylene is important for this aspect of phosphorus response. Low phosphorus reduced ethylene production by adventitious roots of both genotypes, suggesting that ethylene perception--not production--regulates carbon allocation to adventitious roots at the expense of other roots under low phosphorus stress. With the exception of its effect on adventitious rooting, Nr had positive effects on growth and biomass accumulation in tomato whereas etr1 tended to have negative effects on petunia. This was particularly evident during the recovery from transplanting, when the effective quantum yield of photosystem II of etr1 petunia grown with low phosphorus was significantly lower than 'Mitchell Diploid', suggesting that etr1 petunia plants may undergo more severe post-transplant stress at low phosphorus availability. Our results demonstrate that ethylene mediates adventitious root formation in response to phosphorus stress and plays an important role for quick recovery of plants exposed to multiple environmental stresses, i.e. transplanting and low phosphorus.     

Some Physiological Characteristics of the Ethylene-requiring Tomato Mutant Diageotropica

The diageotropica mutant of tomato (Lycopersicon esculentum Mill.) is shown to require exogenous ethylene for normal growth and development. This single gene mutant is characterized by unsupported horizontal growth of shoots and roots, dark green hyponastic leaf segments, thin rigid stems, and primary and adventitious roots which lack lateral roots. Experiments with growth regulators indicate that the mutant does not produce normal amounts of ethylene in response to auxin treatment. Tests with ethylene-producing compounds or ethylene precursors demonstrate that the mutant requires ethylene for normality. Ethylene concentrations as low as 0.005 microliters per liter are capable of completely normalizing mutant characteristics. This mutant with its isogenic parent variety, cv. VFN8, should be a suitable tool for investigating auxin-stimulated ethylene production and their interrelationship in the control of plant morphology and physiology.     

Effects of explant types and media salt strength on growth and secondary metabolite accumulation in adventitious roots of <Emphasis Type="Italic">Periploca sepium</Emphasis> Bunge

In order to study the capabilities of Periploca sepium adventitious root induction in different types of explants, we selected leaves, roots and stems with or without buds. The growth of adventitious roots and periplocin content in these roots were determined. In order to investigate the suitable media salt strength, we cultured the adventitious roots in different salt strength (0.25, 0.50, 1.0, 1.5, 2.0) of Murashige and Skoog (MS) media supplemented with 1 mg/l indole butyric acid (IBA) and 30 g/l sucrose. The results showed that both leaf and root explants were proven suitable for the adventitious root induction; however, the stems could hardly induce adventitious roots no matter whether the stems had buds or not. Further studies reported that adventitious root proliferation and periplocin production derived from root explants were higher than those derived from leaf explants. So the root explants were the optimum explants for adventitious root induction, growth and periplocin production. The salt strength experiment showed that with the increasing salt strength (1.0–2.0 MS), adventitious root growth decreased significantly, as well as periplocin content in comparison with lower (0.25–0.5 MS) salt strength media.     

Production of Biomass and Ginsenosides from Adventitious Roots of Panax ginseng in Bioreactor Cultures

Organic nutrients play a central role during Panax ginseng adventitious root culture in bioreactor systems. To understand how the nutrient elements were uptaken during the adventitious root growth as well as the production of biomass and natural ginsenosides, a biotechnological approach to identifying the nutritional physiology of ginseng in a commercial‐scale bioreactor was necessary. Normal MS medium nutrient in the bioreactor culture of adventitious roots resulted in slow growth, low biomass, and Rg and Rb ginsenoside contents. When the ginsenoside production increased to higher levels, a group of regulatory nutritional elements that have the potential to interact with biomass was identified. The effects of the salt strength of the medium, of macroelements, metal elements, the ammonia/nitrate ratio, sucrose concentration, and osmotic agents on the growth, the formation of biomass and the production of ginsenosides from adventitious roots were investigated. Appropriate conditions allowed for a maximum ginsenoide production of up to 12.42 [mg/g DW] to be obtained after 5 weeks of culture. The results demonstrated that the key organic nutrients can be regulated to improve the biomass and growth, and increase the ginsenoside yield in bioreactor cultures of P. ginseng adventitious roots.     

Decreased Ethylene Biosynthesis, and Induction of Aerenchyma, by Nitrogen- or Phosphate-Starvation in Adventitious Roots of Zea mays L

Plants of Zea mays L. cv TX5855 were grown in a complete, well oxygenated nutrient solution then subjected to nutrient starvation by omitting either nitrate and ammonium or phosphate from the solution. These treatments induced the formation of aerenchyma close to the apex of the adventitious roots that subsequently emerged from the base of the shoot, a response similar to that shown earlier to be induced by hypoxia. Compared with control plants supplied with all nutrients throughout, N- or P-starvation consistently depressed the rates of ethylene release by excised, 25 mm apical segments of adventitious roots. Some enzymes and substrates of the ethylene biosynthetic pathway were examined. The content of 1-amino cyclopropane-1-carboxylic acid (ACC) paralleled the differences in ethylene production rates, being depressed by N or P deficiency, while malonyl-ACC showed a similar trend. Activity of ACC synthase and of ethylene forming enzyme (g⁻¹ fresh weight) was also greater in control roots than in nutrient starved ones. These results indicate that much of the ethylene biosynthetic pathway is slowed under conditions of N- or P-starvation. Thus, by contrast to the effects of hypoxia, the induction of aerenchyma in roots of Zea mays by nutrient starvation is not related to an enhanced biosynthesis and/or accumulation of ethylene in the root tips.     

Role of auxin and its modulators in the adventitious rooting of <Emphasis Type="Italic">Eucalyptus</Emphasis> species differing in recalcitrance

It is well established that auxins play a central role in the determination of rooting capacity, which is essential for vegetative propagation. Recent studies with apple trees have pointed to significant effects of auxin stability, wound related phenolics and ethylene production in the control of adventitious rooting. In the present study, a comparative analysis of the adventitious rooting of microcuttings of Eucalyptus saligna (easy-to-root species) and Eucalyptus globulus (difficult-to-root species) was carried out with different types of auxins, light intensities, presence or absence of apical meristem, different concentrations of phenolic compounds and presence or absence of an ethylene action inhibitor. Parameters evaluated were the percent rooting, number of roots per rooted cutting, length of longest root and mean rooting time. Results showed that auxins of intermediate stability are more favorable to rooting (particularly for the recalcitrant species), higher light intensities in the presence of exogenous auxins promote the rooting response, the absence of meristematic apex or externally supplied phenolics are not limiting for the rooting induced by exogenous auxins, and ethylene appears to play a minor role in the development of adventitious roots in microcuttings of Eucalyptus, indicating that the rhizogenic response results from direct effect of auxins.     

Involvement of ethylene in the rooting of seedling shoot cultures of <Emphasis Type="Italic">Bixa orellana</Emphasis> L.

Using seedlings derived from the shoot apex of annatto (Bixa orellana L. cv. Bico-de-Pato) we observed the rooting frequency of B. orellana, the number and length of roots and the rate of ethylene production during 30 d in culture. The rhizogenesis response was affected by auxins (NAA or IBA) and by both the ethylene biosynthesis precursor 1-aminocyclopropane-1-carboxylic acid (ACC) and the inhibitor 2-aminoethoxyvinylglycine (AVG). Auxin supplementation to the medium resulted in root induction, ethylene production, and an undesirable callusing in the epidermal and cortical tissues. Irrespective of the presence of auxins, supplementing the medium with ACC promoted ethylene biosynthesis and callusing, which resulted in increased cell proliferation mainly in the cortical and vascular tissues, while the epidermis was mostly unaltered. In both ACC and auxin-supplemented medium, increased ethylene production and callusing occurred, suggesting a synergistic effect between these two responses. ACC was capable of inducing adventitious root formation, but the roots produced had a wrinkled appearance when compared to normal roots. Conversely, AVG reduced ethylene production and callusing, while the epidermis, cortex, and inner tissues remained unaltered, regardless of the presence of auxins. AVG was beneficial in these aspects, although its application led to a reduction in the number of roots and in the average root length. In conclusion, it was not possible to establish a direct relation between ethylene and rooting, but we hypothesize that, under the experimental conditions described, ethylene may enhance tissue sensitivity to auxin. However, ethylene did not seem essential to the rhizogenesis process in annatto.     

Responses of Melaleuca quinquenervia seedlings to flooding

Abstract Studies were conducted on effects of flooding for 15, 30, 60, and 90 days on morphological changes, stomatal aperture, water potential, and growth of seedlings of Melaleuca quinquenervia, a species often planted for reclamation of swamps. Flooding rapidly induced formation of many hair-like adventitious roots as well as a few thick adventitious roots that originated on the original root system. Some adventitious roots also formed on submerged portions of the stem. Melaleuca seedlings were very tolerant of flooding as shown by only slight reduction in dry weight increment of shoots after 30 days of flooding in stagnant water. Although flooding for 60 or 90 days significantly reduced dry weight increment of leaves, dry weight increment of roots was not inhibited by any flooding treatment, reflecting both degeneration of some of the original roots and compensatory growth of adventitious roots. On certain days flooding induced stomatal closure on both adaxial and abaxial leaf surfaces. Extensive production of adventitious roots and some stomatal reopening after a critical period of flooding appeared to be important factors in the flooding tolerance of Melaleuca and are consistent with its aggressiveness and vigorous growth on wet sites.     

Inhibition by silver ions of gas space (aerenchyma) formation in adventitious roots of Zea mays L. subjected to exogenous ethylene or to oxygen deficiency

We have studied the role of ethylene in accelerating the lytic formation of gas spaces (aerenchyma) in the cortex of adventitious roots of maize (Zea mays L.) growing in poorly aerated conditions. Such roots had previously been shown to contain increased concentrations of ethylene. Ten day-old maize plants bearing seminal roots and one whorl of emerging adventitious roots were grown in nutrient solution bubbled with air, ethylene in air (0.1 to 5.0 l l^–1), or allowed to become oxygen-deficient in nonaerated (but not completely anaerobic) solution. Additions of 0.1 l l^–1 ethylene or more promoted the formation of aerenchyma, with lysis of up to 47% of the cortical cells. The effects of non-aeration were similar to those of exogenous ethylene. When silver ions, an ethylene antagonist, were present at low, non-toxic concentrations (circa 0.6 M), aerenchyma formation was prevented in ethylene treated roots and in those exposed to oxygen deficiency. Silver ions also blocked the inhibiting effect of exogenous ethylene on root extension. By contrast, the suppression of aerenchyma formation by silver ions under oxygendeficient conditions was associated with a retardation of root extension, indicating the importance of aerenchyma for root growth in poorly aerated media. Rates of production of ethylene by excised roots were stimulated by a previous non-aeration treatment. The effectiveness of Ag⁺ in inhibiting equally the action on cortical cells of exogenous ethylene and of non-aeration, supports the view that gas space (aerenchyma) formation in adventitious roots adpted to oxygendeficient environments is mediated by increased concentrations of endogenous ethylene. The possibility that extra ethylene could arise from increased biosynthesis of a precursor in root tissues with a restricted oxygen supply is discussed.     

Contribution of adventitious vs initial roots to growth and physiology of black spruce seedlings

Black spruce (Picea mariana [Mill.] BSP) is a boreal tree species characterized by the formation of an adventitious root system. Unlike initial roots from seed germination, adventitious roots gradually appear above the root collar, until they constitute most of mature black spruce root system. Little is known about the physiological role they play and their influence on tree growth relative to initial roots. We hypothesized that adventitious roots present an advantage over initial roots in acquiring water and nutrients. To test this hypothesis, the absorptive capacities of the two root systems were explored in a controlled environment during one growing season. Black spruce seedlings were placed in a double‐pot system allowing irrigation (25 and 100% water container capacity) and fertilization (with or without fertilizer) inputs independent to initial and adventitious roots. After 14 weeks, growth parameters (height, diameter, biomass), physiology (net photosynthetic rate, stomatal conductance, shoot water potential) and nutrient content (N, P, K, Ca and Mg foliar content) were compared. Most measured parameters showed no difference for the same treatment on adventitious or initial roots, except for root biomass. Indeed, fertilized black spruce seedlings invested heavily in adventitious root production, twice as much as initial roots. This was also the case when adventitious roots alone were irrigated, while seedlings with adventitious roots subjected to low irrigation produced initial root biomass equivalent to that of adventitious roots. We conclude that black spruce seedlings perform equally well through adventitious and initial roots, but if resources are abundant, they strongly promote development of adventitious roots.     

Podophyllotoxin production via cell and adventitious root cultures of <Emphasis Type="Italic">Podophyllum peltatum</Emphasis>

Podophyllum peltatum is an important medicinal plant that produces podophyllotoxin (PTOX) with anti-cancer properties. We established the embryogenic cell and adventitious root culture systems in P. peltatum and analyzed PTOX production. For the growth of embryogenic cell clumps in shake flask culture, the most efficient concentration of 2,4-dichloroacetic acid (2,4-D) was 6.78 μM, and the growth of embryogenic cell clumps was 15.9-fold increased in Murashige and Skoog MS liquid medium with 6.78 μM 2,4-D after 3 wk of culture. To induce adventitious roots, half-strength MS medium showed the best results for adventitious root induction compared to full strength MS medium and MS medium lacking NH₄NO₃. Optimal indole-3-butyric acid concentration for adventitious root formation was 14.78 μM. In liquid medium, the frequency of adventitious root formation from root segments was 87.7% and the number of laterally formed adventitious roots was 22.3 per segment. PTOX production in embryogenic cells and adventitious roots was confirmed by liquid chromatography and electrospray ionization–tandem mass spectrometry analysis. High-performance liquid chromatography analysis revealed that adventitious roots contained higher PTOX than embryogenic cell clumps. Elicitor treatment (20 μM methyl jasmonate) strongly enhanced the production of PTOX in both embryogenic cell clumps and adventitious roots. This observation suggests that both embryogenic cell and adventitious root culture can be adopted to produce PTOX.     

Ethylene Controls Adventitious Root Initiation Sites in <Emphasis Type="Italic">Arabidopsis</Emphasis> Hypocotyls Independently of Strigolactones

Adventitious root formation is essential for cutting propagation of diverse species; however, until recently little was known about its regulation. Strigolactones and ethylene have both been shown to inhibit adventitious roots and it has been suggested that ethylene interacts with strigolactones in root hair elongation. We have investigated the interaction between strigolactones and ethylene in regulating adventitious root formation in intact seedlings of Arabidopsis thaliana. We used strigolactone mutants together with 1-aminocyclopropane-1-carboxylic acid (ACC) (ethylene precursor) treatments and ethylene mutants together with GR24 (strigolactone agonist) treatments. Importantly, we conducted a detailed mapping of adventitious root initiation along the hypocotyl and measured ethylene production in strigolactone mutants. ACC treatments resulted in a slight increase in adventitious root formation at low doses and a decrease at higher doses, in both wild-type and strigolactone mutants. Furthermore, the distribution of adventitious roots dramatically changed to the top third of the hypocotyl in a dose-dependent manner with ACC treatments in both wild-type and strigolactone mutants. The ethylene mutants all responded to treatments with GR24. Wild type and max4 (strigolactone-deficient mutant) produced the same amount of ethylene, while emanation from max2 (strigolactone-insensitive mutant) was lower. We conclude that strigolactones and ethylene act largely independently in regulating adventitious root formation with ethylene controlling the distribution of root initiation sites. This role for ethylene may have implications for flood response because both ethylene and adventitious root development are crucial for flood tolerance.     

Ethylene-induced activation of xylanase in adventitious roots of maize as a response to the stress effect of root submersion]

Submersion of roots of ten-day-old maize (Zea mays L.) seedlings was accompanied by a decrease in pO2 and an increase in pCO2 of the medium adjacent to roots. These changes stimulated ethylene evolution in intact plants. Enhanced biosynthesis of ethylene was accompanied by xylanase activation in adventitious roots. As a result, an enhanced formation of aerenchyma was observed in the cortex of adventitious roots. Therefore, these processes resulted in the development of a ventilation system by which O2 can reach the root system exposed to hypoxia. The volume of aerenchyma was assessed by the volume of gas cavities (porosity). In contrast to the main root, the growth of adventitious roots was not inhibited under these conditions. Enlargement of the stem base and increase in the number of aerenchymatous adventitious roots facilitated the oxygen supply to submerged organs of plants.     

Enhanced ethylene production by primary roots of Zea mays L. in response to sub-ambient partial pressures of oxygen

Ethylene production by primary roots of 72–h-old intact seedlings of Zea mays L. cv. LG11 was studied under ambient and sub-ambient oxygen partial pressures (pO₂) using a gas flow-through system linked to a photoacoustic laser detector. Despite precautions to minimize physical perturbation to seedlings while setting-up, ethylene production in air was faster during the first 6h than later, in association with a small temporary swelling of the roots. When roots were switched from air (20–8kPa O₂) to 3 or 5kPa O₂ after 6h, ethylene production increased within 2—3 h. When, the roots were returned to air 16 h later, ethylene production decreased within 2—3 h. The presence of 10kPa CO₂ did not interfere with the effect of 3kPa O₂. Transferring roots from air to 12–5kPa did not change ethylene production, while a reduction to 1 kPa O₂ induced a small increase. The extra ethylene formed in 3 and 5 kPa O₂ was associated with plagiotropism, swelling, root hair production, and after 72 h, increased amounts of intercellular space (aerenchyma) in the root cortex. Root extension was also slowed down, but the pattern of response to oxygen shortage did not always match that of ethylene production. On return to air, subsequent growth patterns became normal within a few hours. In the complete absence of oxygen, no ethylene production was detected, even when anaerobic roots were returned to air after 16 h.     

Endogenous ethylene requirement for adventitious root induction and growth in tomato cotyledons and lavandin microcuttings in vitro

The role of ethylene in the formation of adventitious roots in vitro was studied in tomato (Lycopersicon esculentum Mill. cv. UC 105) cotyledons and lavandin (Lavandula officinalis Chaix × Lavandula latifolia microshoots. Both systems were able to form roots on hormone-free medium evolving low amounts of ethylene. The addition of 20–50 M indole-3-acetic acid (IAA) inhibited root formation in tomato cotyledons while increasing ethylene production. Naphthaleneacetic acid (NAA, 3 M) stimulated root number in lavandin explants and induced a transient rise in ethylene evolution. Enhanced ethylene levels via the endogenous precursors 1-aminocyclopropane-1-carboxylic acid (ACC, 25–50 M) drastically impaired root regeneration and growth in tomato. In lavandin, 10 M ACC stimulated ethylene production and significantly inhibited the rooting percentage and root growth. Conversely, ACC enhanced the root number in the presence of NAA only. Severe inhibition of rooting was also caused by ethylene reduction via biosynthetic inhibitors, aminoethoxyvinylglycine (AVG, 5–10 M) in tomato, and salicylic acid (SA, 100 M) in lavandin. A strict requirement of endogenous ethylene for adventitious root induction and growth is thus suggested.Abbreviations LS Linsmaier and Skoog medium - BA N⁶-benzyladenine - NAA 1-naphthaleneacetic acid - IAA Indole-3-acetic acid - AVG Aminoethoxyvinylglycine - SA Salicylic acid - ACC 1-aminocyclopropane-1-carboxylic acid