Effect of Bacteria and Amoebae on Rhizosphere Phosphatase Activity

The contributions of various components of soil microflora and microfauna to rhizosphere phosphatase activity were determined with hydroponic cultures. Three treatments were employed: (i) plants alone (Bouteloua gracilis (H.B.K.) Lag. ex Steud.) (ii) plants plus bacteria (Pseudomonas sp.), and (iii) plants plus bacteria plus amoebae (Acanthamoeba sp.). No alkaline phosphatase was detected, but an appreciable amount of acid phosphatase activity (120 to 500 nmol of p-nitrophenylphosphate hydrolyzed per h per plant) was found in the root culture solutions. The presence of bacteria or bacteria and amoebae increased the amount of acid phosphatase in solution, and properties of additional activity were identical to properties of plant acid phosphatase. The presence of bacteria or bacteria and amoebae increased both solution and root phosphatase activities at most initial phosphate concentrations.     

Regulation of root development in <Emphasis Type="BoldItalic">Arabidopsis thaliana</Emphasis> by phytohormone-secreting epiphytic methylobacteria

In numerous experimental studies, seedlings of the model dicot Arabidopsis thaliana have been raised on sterile mineral salt agar. However, under natural conditions, no plant has ever grown in an environment without bacteria. Here, we document that germ-free (gnotobiotic) seedlings, raised on mineral salt agar without sucrose, develop very short root hairs. In the presence of a soil extract that contains naturally occurring microbes, root hair elongation is promoted; this effect can be mimicked by the addition of methylobacteria to germ-free seedlings. Using five different bacterial species (Methylobacterium mesophilicum, Methylobacterium extorquens, Methylobacterium oryzae, Methylobacterium podarium, and Methylobacterium radiotolerans), we show that, over 9 days of seedling development in a light-dark cycle, root development (hair elongation, length of the primary root, branching patterns) is regulated by these epiphytic microbes that occur in the rhizosphere of field-grown plants. In a sterile liquid culture test system, auxin (IAA) inhibited root growth with little effect on hair elongation and significantly stimulated hypocotyl enlargement. Cytokinins (trans-zeatin, kinetin) and ethylene (application of the precursor ACC) likewise exerted an inhibitory effect on root growth but, in contrast to IAA, drastically stimulated root hair elongation. Methylobacteria are phytosymbionts that produce/secrete cytokinins. We conclude that, under real-world conditions (soil), the provision of these phytohormones by methylobacteria (and other epiphytic microbes) regulates root development during seedling establishment.     

Long-distance signalling and a mutational analysis of branching in pea

Four ramosus mutants with increased branching at basal andaerial nodes have been used to investigate the genetic regulation of budoutgrowth in Pisum sativum L. (garden pea). Studies oflong-distance signalling, xylem sap cytokinin concentrations, shootauxin level, auxin transport and auxin response are discussed. A modelof branching control is presented that encompasses twograft-transmissible signals in addition to auxin and cytokinin. Mutantsrms1 through rms4 are not deficient in indole-3-aceticacid (IAA) or in the basipetal transport of this hormone. Three of thefour mutants, rms1, rms3 and rms4, have veryreduced cytokinin concentrations in xylem sap from roots. This reductionin xylem sap cytokinin concentration appears to be caused by a propertyof the shoot and may be part of a feedback mechanism induced by anaspect of bud outgrowth. The shoot-to-root feedback signal is unlikelyto be auxin itself, as auxin levels and transport are not correlatedwith xylem sap cytokinin concentrations in various intact and graftedmutant and wild-type plants. Rms1 and Rms2 act inshoot and rootstock to regulate the level or transport ofgraft-transmissible signals. Various grafting studies and double mutantanalyses have associated Rms2 with the regulation of theshoot-to-root feedback signal. Rms1 is associated with a secondunknown graft-transmissible signal that is postulated to move in thedirection of root-to-shoot. Exogenous auxin appears to interact withboth of the signals regulated by Rms1 and Rms2 in theinhibition of branching after decapitation. The action of Rms3and Rms4 is less apparent at this stage, although both appearto act largely in the shoot.     

Regulation of root morphogenesis in arbuscular mycorrhizae: what role do fungal exudates,phosphate, sugars and hormones play in lateral root formation?

Background

Arbuscular mycorrhizae (AMs) form a widespread root–fungus symbiosis that improves plant phosphate (Pi) acquisition and modifies the physiology and development of host plants. Increased branching is recognized as a general feature of AM roots, and has been interpreted as a means of increasing suitable sites for colonization. Fungal exudates, which are involved in the dialogue between AM fungi and their host during the pre-colonization phase, play a well-documented role in lateral root (LR) formation. In addition, the increased Pi content of AM plants, in relation to Pi-starved controls, as well as changes in the delivery of carbohydrates to the roots and modulation of phytohormone concentration, transport and sensitivity, are probably involved in increasing root system branching.

Scope

This review discusses the possible causes of increased branching in AM plants. The differential root responses to Pi, sugars and hormones of potential AM host species are also highlighted and discussed in comparison with those of the non-host Arabidopsis thaliana.

Conclusions

Fungal exudates are probably the main compounds regulating AM root morphogenesis during the first colonization steps, while a complex network of interactions governs root development in established AMs. Colonization and high Pi act synergistically to increase root branching, and sugar transport towards the arbusculated cells may contribute to LR formation. In addition, AM colonization and high Pi generally increase auxin and cytokinin and decrease ethylene and strigolactone levels. With the exception of cytokinins, which seem to regulate mainly the root:shoot biomass ratio, these hormones play a leading role in governing root morphogenesis, with strigolactones and ethylene blocking LR formation in the non-colonized, Pi-starved plants, and auxin inducing them in colonized plants, or in plants grown under high Pi conditions.     

Roles for Auxin, Cytokinin, and Strigolactone in Regulating Shoot Branching

Many processes have been described in the control of shoot branching. Apical dominance is defined as the control exerted by the shoot tip on the outgrowth of axillary buds, whereas correlative inhibition includes the suppression of growth by other growing buds or shoots. The level, signaling, and/or flow of the plant hormone auxin in stems and buds is thought to be involved in these processes. In addition, RAMOSUS (RMS) branching genes in pea (Pisum sativum) control the synthesis and perception of a long-distance inhibitory branching signal produced in the stem and roots, a strigolactone or product. Auxin treatment affects the expression of RMS genes, but it is unclear whether the RMS network can regulate branching independently of auxin. Here, we explore whether apical dominance and correlative inhibition show independent or additive effects in rms mutant plants. Bud outgrowth and branch lengths are enhanced in decapitated and stem-girdled rms mutants compared with intact control plants. This may relate to an RMS-independent induction of axillary bud outgrowth by these treatments. Correlative inhibition was also apparent in rms mutant plants, again indicating an RMS-independent component. Treatments giving reductions in RMS1 and RMS5 gene expression, auxin transport, and auxin level in the main stem were not always sufficient to promote bud outgrowth. We suggest that this may relate to a failure to induce the expression of cytokinin biosynthesis genes, which always correlated with bud outgrowth in our treatments. We present a new model that accounts for apical dominance, correlative inhibition, RMS gene action, and auxin and cytokinin and their interactions in controlling the progression of buds through different control points from dormancy to sustained growth.     

The influence of cytokinin-auxin cross-regulation on cell-fate determination in Arabidopsis thaliana root development

Root growth and development in Arabidopsis thaliana are sustained by a specialised zone termed the meristem, which contains a population of dividing and differentiating cells that are functionally analogous to a stem cell niche in animals. The hormones auxin and cytokinin control meristem size antagonistically. Local accumulation of auxin promotes cell division and the initiation of a lateral root primordium. By contrast, high cytokinin concentrations disrupt the regular pattern of divisions that characterises lateral root development, and promote differentiation. The way in which the hormones interact is controlled by a genetic regulatory network. In this paper, we propose a deterministic mathematical model to describe this network and present model simulations that reproduce the experimentally observed effects of cytokinin on the expression of auxin regulated genes. We show how auxin response genes and auxin efflux transporters may be affected by the presence of cytokinin. We also analyse and compare the responses of the hormones auxin and cytokinin to changes in their supply with the responses obtained by genetic mutations of SHY2, which encodes a protein that plays a key role in balancing cytokinin and auxin regulation of meristem size. We show that although shy2 mutations can qualitatively reproduce the effect of varying auxin and cytokinin supply on their response genes, some elements of the network respond differently to changes in hormonal supply and to genetic mutations, implying a different, general response of the network. We conclude that an analysis based on the ratio between these two hormones may be misleading and that a mathematical model can serve as a useful tool for stimulate further experimental work by predicting the response of the network to changes in hormone levels and to other genetic mutations.     

<Emphasis Type="Italic">Bacillus methylotrophicus</Emphasis> M4-96 isolated from maize (<Emphasis Type="Italic">Zea mays</Emphasis>) rhizoplane increases growth and auxin content in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> via emission of volatiles

Plant growth-promoting rhizobacteria stimulate plant growth and development via different mechanisms. In this study, we characterized the effect of volatiles from Bacillus methylotrophicus M4-96 isolated from the maize rhizosphere on root and shoot development, and auxin homeostasis in Arabidopsis thaliana. Phytostimulation occurred after 4 days of interaction between M4-96 and Arabidopsis grown on opposite sides of divided Petri plates, as revealed by enhanced primary root growth, root branching, leaf formation, and shoot biomass accumulation. Analysis of indole-3-acetic acid content revealed two- and threefold higher accumulation in the shoot and root of bacterized seedlings, respectively, compared to uninoculated plants, which was correlated with increased expression of the auxin response marker DR5::GUS. The auxin transport inhibitor 1-naphthylphthalamic acid inhibited primary root growth and lateral root formation in axenically grown seedlings and antagonized the plant growth-promoting effects of M4-96. Analysis of bacterial volatile compounds revealed the presence of four classes of compounds, including ten ketones, eight alcohols, one aldehyde, and two hydrocarbons. However, the abundance of ketones and alcohols represented 88.73 and 8.05%, respectively, of all airborne signals detected, with acetoin being the main compound produced. Application of acetoin had a different effect from application of volatiles, suggesting that either the entire pool or acetoin acting in concert with another unidentified compound underlies the strong phytostimulatory response. Taken together, our results show that B. methylotrophicus M4-96 generates bioactive volatiles that increase the active auxin pool of plants, stimulate the growth and formation of new organs, and reprogram root morphogenesis.     

Simulated herbivory effects on rhizosphere organisms in pea (Pisum sativum) depended on phosphate

The effects of simulated aboveground herbivory and phosphate addition to soil on rhizosphere organisms (arbuscular mychorrhiza (AM), Rhizobium spp., bacteria, protozoa and nematodes) were studied in a 2 by 2 factorial designed pot experiment with Pea plants (Pisum sativum). Measurements were performed on 24 day old plants that were still in the nutrient acquisition phase before flowering. AM colonization and bacterial feeding nematodes were stimulated by high simulated her- bivory especially when plants were phosphate deficient. Total number of nematodes was higher with phosphate deficiency. Furthermore, non-significant peak values in soil respiration, total number of nematodes, and bacterial number were observed in phosphate deficient plants with high simulated herbivory. In phosphate amended plants, fast-growing protozoa and bacterial feeding nematodes decreased at high simulated herbivory. These results support the hypothesis that the plant regulates abundances of both AM and free-living rhizosphere organisms and thereby the amount of plant-available nutrients, according to demand via root exudation. Rhizobium spp. was significantly stimulated by phosphate addition but not affected by simulated herbivory. Metabolites produced by rhizosphere bacteria from plants exposed to high simulated herbivory in phosphate amended soil stimulated seed performance. Possible interactions between protozoa and nematodes in relation to production and composition of bacteria in the rhizosphere are discussed.     

Soil Nitrogen Status Modifies Rice Root Response to Nematode-Bacteria Interactions in the Rhizosphere

It has been hypothesized that faunal activity in the rhizosphere influences root growth via an auxin-dependent pathway. In this study, two methods were used to adjust nematode and bacterial populations within experimental soils. One is “exclusion”, where soil mixed with pig manure was placed in two bags with different mesh sizes (1mm and 5μm diameter), and then surrounded by an outer layer of unamended soil resulting in soil with a greater populations of bacterial-feeding nematodes (1mm) and a control treatment (5μm). The second method is “inoculation”, whereby autoclaved soil was inoculated with bacteria (E. coli and Pseudomonas) and Nematodes (Cephalobus and C. elegans). In order to detect the changes in the rice’s perception of auxin under different nutrient and auxin conditions in the presence of soil bacterial-feeding nematodes, responses of soil chemistry (NH₄⁺, NO₃^- and indole acetic acid (IAA)), rice root growth and the expression of an auxin responsive gene GH3-2 were measured. Results showed that, under low soil nutrient conditions (exclusion), low NO₃^- correlated with increased root branching and IAA correlated with increased root elongation and GH3-2 expression. However, under high soil nutrient conditions (inoculation), a high NH₄⁺ to NO₃^- ratio promoted an increase in root surface area and there was an additional influence of NH₄⁺ and NO₃^- on GH3-2 expression. Thus it was concluded that soil bacterial-feeding nematodes influenced soil nutritional status and soil IAA content, promoting root growth via an auxin dependent pathway that was offset by soil nitrogen status.     

Stimulation of root hair elongation in Arabidopsis thaliana by low phosphorus availability

Low phosphorus availability stimulates root hair elongation in many plants, which may have adaptive significance in soil phosphorus acquisition. We investigated the effect of low phosphorus on the elongation of Arabidopsis thaliana root hairs. Arabidopsis thaliana plants were grown in plant culture containing high (1000 mmol m^?3) or low (1 mmol m^?3) phosphorus concentrations, and root hair elongation was analysed by image analysis. After 15d of growth, low-phosphorus plants developed root hairs averaging 0.9 mm in length while high-phosphorus plants of the same age developed root hairs averaging 0.3 mm in length. Increased root hair length in low-phosphorus plants was a result of both increased growth duration and increased growth rate. Root hair length decreased logarithmically in response to increasing phosphorus concentration. Local changes in phosphorus availability influenced root hair growth regardless of the phosphorus status of the plant. Low phosphorus stimulated root hair elongation in the hairless axr2 mutant, exogenously applied IAA stimulated root hair elongation in wild-type high-phosphorus plants and the auxin antagonist CM PA inhibited root hair elongation in low-phosphorus plants. These results indicate that auxin may be involved in the low-phosphorus response in root hairs.     

Rhizosphere Bacteria Enhance Selenium Accumulation and Volatilization by Indian Mustard

Indian mustard (Brassica juncea L.) accumulates high tissue Se concentrations and volatilizes Se in relatively nontoxic forms, such as dimethylselenide. This study showed that the presence of bacteria in the rhizosphere of Indian mustard was necessary to achieve the best rates of plant Se accumulation and volatilization of selenate. Experiments with the antibiotic ampicillin showed that bacteria facilitated 35% of plant Se volatilization and 70% of plant tissue accumulation. These results were confirmed by inoculating axenic plants with rhizosphere bacteria. Compared with axenic controls, plants inoculated with rhizosphere bacteria had 5-fold higher Se concentrations in roots (the site of volatilization) and 4-fold higher rates of Se volatilization. Plants with bacteria contained a heat-labile compound in their root exudate; when this compound was added to the rhizosphere of axenic plants, Se accumulation in plant tissues increased. Plants with bacteria had an increased root surface area compared with axenic plants; the increased area was unlikely to have caused their increased tissue Se accumulation because they did not accumulate more Se when supplied with selenite or selenomethionine. Rhizosphere bacteria also possibly increased plant Se volatilization because they enabled plants to overcome a rate-limiting step in the Se volatilization pathway, i.e. Se accumulation in plant tissues.     

Auxin influences strigolactones in pea mycorrhizal symbiosis

Hormone interactions are essential for the control of many developmental processes, including intracellular symbioses. The interaction between auxin and the new plant hormone strigolactone in the regulation of arbuscular mycorrhizal symbiosis was examined in one of the few auxin deficient mutants available in a mycorrhizal species, the auxin-deficient bsh mutant of pea (Pisum sativum). Mycorrhizal colonisation with the fungus Glomus intraradices was significantly reduced in the low auxin bsh mutant. The bsh mutant also exhibited a reduction in strigolactone exudation and the expression of a key strigolactone biosynthesis gene (PsCCD8). Strigolactone exudation was also reduced in wild type plants when the auxin content was reduced by stem girdling. Low strigolactone levels appear to be at least partially responsible for the reduced colonisation of the bsh mutant, as application of the synthetic strigolactone GR24 could partially rescue the mycorrhizal phenotype of bsh mutants. Data presented here indicates root auxin content was correlated with strigolactone exudation in both mutant and wild type plants. Mutant studies suggest that auxin may regulate early events in the formation of arbuscular mycorrhizal symbiosis by controlling strigolactone levels, both in the rhizosphere and possibly during early root colonisation.     

Auxin and cytokinin regulate each other's levels via a metabolic feedback loop

The hormones auxin and cytokinin are key regulators of plant growth and development. As they are active at minute concentrations and regulate dynamic processes, cell and tissue levels of the hormones are finely controlled developmentally, diurnally, and in response to environmental variables. This fine control, along with a regulation of the capacity to respond ensures that the appropriate type, duration and intensity of responses are elicited. We have recently discovered that cytokinin and auxin regulate the synthesis of each other, demonstrating a mechanism for mutual feed back and feed forward control of auxin and cytokinin levels. This regulatory loop could be important for many developmental processes in plants, i.e., in fine-tuning plant hormone levels in the developing meristems of the root and shoot apex. These findings could also give a molecular explanation for earlier observations of auxin and cytokinin effects on cell cultures,¹ where specific auxin and cytokinin ratios have been used to trigger different morphological events.Key words: auxin, cytokinin, biosynthesis, metabolism, signaling, root development, interactions     

Iron-Oxidizing Bacteria Are Associated with Ferric Hydroxide Precipitates (Fe-Plaque) on the Roots of Wetland Plants

The presence of Fe-oxidizing bacteria in the rhizosphere of four different species of wetland plants was investigated in a diverse wetland environment that had Fe(II) concentrations ranging from tens to hundreds of micromoles per liter and a pH range of 3.5 to 6.8. Enrichments for neutrophilic, putatively lithotrophic Fe-oxidizing bacteria were successful on roots from all four species; acidophilic Fe-oxidizing bacteria were enriched only on roots from plants whose root systems were exposed to soil solutions with a pH of <4. In Sagittaria australis there was a positive correlation (P < 0.01) between cell numbers and the total amount of Fe present; the same correlation was not found for Leersia oryzoides. These results present the first evidence for culturable Fe-oxidizing bacteria associated with Fe-plaque in the rhizosphere.     

Effects of therolABC,rolAB, and CaMV 35S-rolC genes on growth and nitrogen fixation inLotus corniculatus L.

Lotus corniculatus has been transformed byAgrobacterium tumefaciens harbouring therolABC, rolAB, and 35S-rolC constructs. Growth, shoot and root morphology, cytokinin and auxin concentrations in the shoots, chlorophyll content in the leaves, nodulation and nodule nitrogenase activity of clonal progenies of selected transformants, in which the transferredrol sequences were proved, were compared with control plants. The most striking alterations were observed in the 35S-rolC transformants (dwarfism, shoot and root branching, shorter internodes, delayed nodule formation, decreased nitrogenase activity, reduced content of leaf pigments) in comparison with either therolAB, rolABC, or controls. TherolAB androlABC transformants were very similar to the control plants in their morphology as well as in most other traits measured.     

Regeneration in Streptocarpus Leaf Discs and its Regulation by Temperature and Growth Substances

The formation of adventitious buds and roots in leaf discs of Streptocarpus x bybridus‘Constant Nymph’ were both stimulated by relatively low temperatures (12 and 18°C) applied to isolated discs or to the growing plants before leaf harvest. Auxins also promoted both bud and root formation, the optimum concentration for rooting always being one to two orders of magnitude higher than the optimum for budding. Cytokinins had only a small stimulatory effect on bud formation. At higher concentrations it was inhibitory and even counteracted the stimulatory effect of auxin on bud formation. As usual, root formation was inhibited by cytokinin. GA₃ inhibited both bud and root formation but the inhibition was reversible by auxin. In presence of optimum auxin levels abscisic acid enhanced bud formation. It had little effect on root formation except for an inhibition at high concentrations. The effects of exogenous auxin and cytokinin suggest that Streptocarpus leaves have a high and non-limiting level of endogenous cytokinin with auxin as the limiting factor for both root and bud formation. This would also explain the exceptionally high regeneration ability of this plant.