Influence of Plant Growth-Promoting Rhizobacteria on the Formation of Apoplastic Barriers and Uptake of Water and Potassium by Wheat Plants.

The formation of apoplastic barriers is important for controlling the uptake of water and ions by plants, thereby influencing plant growth. However, the effects of plant growth-promoting bacteria on the formation of apoplastic barriers, and the relationship between these effects and the ability of bacteria to influence the content of hormones in plants, have not been sufficiently studied. The content of cytokinins, auxins and potassium, characteristics of water relations, deposition of lignin and suberin and the formation of Casparian bands in the root endodermis of durum wheat (Triticum durum Desf.) plants were evaluated after the introduction of the cytokinin-producing bacterium Bacillus subtilis IB-22 or the auxin-producing bacterium Pseudomonas mandelii IB-Ki14 into their rhizosphere. The experiments were carried out in laboratory conditions in pots with agrochernozem at an optimal level of illumination and watering. Both strains increased shoot biomass, leaf area and chlorophyll content in leaves. Bacteria enhanced the formation of apoplastic barriers, which were most pronounced when plants were treated with P. mandelii IB-Ki14. At the same time, P. mandelii IB-Ki14 caused no decrease in the hydraulic conductivity, while inoculation with B. subtilis IB-22, increased hydraulic conductivity. Cell wall lignification reduced the potassium content in the roots, but did not affect its content in the shoots of plants inoculated with P. mandelii IB-Ki14. Inoculation with B. subtilis IB-22 did not change the potassium content in the roots, but increased it in the shoots.

Effects of a Pseudomonas Strain on the Lipid Transfer Proteins, Appoplast Barriers and Activity of Aquaporins Associated with Hydraulic Conductance of Pea Plants.

Lipid transfer proteins (LTPs) are known to be involved in suberin deposition in the Casparian bands of pea roots, thereby reinforcing apoplast barriers. Moreover, the Pseudomonas mandelii IB-Ki14 strain accelerated formation of the Casparian bands in wheat plants, although involvement of LTPs in the process was not studied. Here, we investigated the effects of P. mandelii IB-Ki14 on LTPs, formation of the Casparian bands, hydraulic conductance and activity of aquaporins (AQPs) in pea plants. RT PCR showed a 1.6-1.9-fold up-regulation of the PsLTP-coding genes and an increase in the abundance of LTP proteins in the phloem of pea roots induced by the treatment with P. mandelii IB-Ki14. The treatment was accompanied with increased deposition of suberin in the Casparian bands. Hydraulic conductance did not decrease in association with the bacterial treatment despite strengthening of the apoplast barriers. At the same time, the Fenton reagent, serving as an AQPs inhibitor, decreased hydraulic conductance to a greater extent in treated plants relative to the control group, indicating an increase in the AQP activity by the bacteria. We hypothesize that P. mandelii IB-Ki14 stimulates deposition of suberin, in the biosynthesis of which LTPs are involved, and increases aquaporin activity, which in turn prevents a decrease in hydraulic conductance due to formation of the apoplast barriers in pea roots.

The Effects of Rhizosphere Inoculation with Pseudomonas mandelii on Formation of Apoplast Barriers, HvPIP2 Aquaporins and Hydraulic Conductance of Barley.

Pseudomonas mandelii strain IB-Ki14 has recently been shown to strengthen the apoplastic barriers of salt-stressed plants, which prevents the entry of toxic sodium. It was of interest to find out whether the same effect manifests itself in the absence of salinity and how this affects the hydraulic conductivity of barley plants. Berberine staining confirmed that the bacterial treatment enhanced the deposition of lignin and suberin and formation of Casparian bands in the roots of barley plants. The calculation of hydraulic conductance by relating transpiration to leaf water potential showed that it did not decrease in bacteria-treated plants. We hypothesized that reduced apoplastic conductivity could be compensated by the higher conductivity of the water pathway across the membranes. This assumption was confirmed by the results of the immunolocalization of HvPIP2;5 aquaporins with specific antibodies, showing their increased abundance around the areas of the endodermis and exodermis of bacteria-treated plants. The immunolocalization with antibodies against auxins and abscisic acid revealed elevated levels of these hormones in the roots of plants treated with bacteria. This root accumulation of hormones is likely to be associated with the ability of Pseudomonas mandelii IB-Ki14 to synthesize these hormones. The involvement of abscisic acid in the control of aquaporin abundance and auxins-in the regulation of and formation of apoplast barriers-is discussed.

Effects of Phytohormone-Producing Rhizobacteria on Casparian Band Formation, Ion Homeostasis and Salt Tolerance of Durum Wheat.

Inoculation with plant growth-promoting rhizobacteria can increase plant salt resistance. We aimed to reveal bacterial effects on the formation of apoplastic barriers and hormone concentration in relation to maintaining ion homeostasis and growth of salt-stressed plants. The rhizosphere of a durum wheat variety was inoculated with cytokinin-producing Bacillus subtilis and auxin-producing Pseudomonas mandelii strains. Plant growth, deposition of lignin and suberin and concentrations of sodium, potassium, phosphorus and hormones were studied in the plants exposed to salinity. Accumulation of sodium inhibited plant growth accompanied by a decline in potassium in roots and phosphorus in shoots of the salt-stressed plants. Inoculation with both bacterial strains resulted in faster appearance of Casparian bands in root endodermis and an increased growth of salt-stressed plants. B. subtilis prevented the decline in both potassium and phosphorus concentrations and increased concentration of cytokinins in salt-stressed plants. P. mandelii decreased the level of sodium accumulation and increased the concentration of auxin. Growth promotion was greater in plants inoculated with B. subtilis. Increased ion homeostasis may be related to the capacity of bacteria to accelerate the formation of Casparian bands preventing uncontrolled diffusion of solutes through the apoplast. We discuss the relative impacts of the decline in Na accumulation and maintenance of K and P content for growth improvement of salt-stressed plants and their possible relation to the changes in hormone concentration in plants.

Growth-Promoting Effect of Rhizobacterium (Bacillus subtilis IB22) in Salt-Stressed Barley Depends on Abscisic Acid Accumulation in the Roots.

An ABA-deficient barley mutant (Az34) and its parental cultivar (Steptoe) were compared. Plants of salt-stressed Az34 (100 mmol m-3 NaCl for 10 days) grown in sand were 40% smaller than those of "Steptoe", exhibited a lower leaf relative water content and lower ABA concentrations. Rhizosphere inoculation with IB22 increased plant growth of both genotypes. IB22 inoculation raised ABA in roots of salt-stressed plants by supplying ABA exogenously and by up-regulating ABA synthesis gene HvNCED2 and down-regulating ABA catabolic gene HvCYP707A1. Inoculation partially compensated for the inherent ABA deficiency of the mutant. Transcript abundance of HvNCED2 and related HvNCED1 in the absence of inoculation was 10 times higher in roots than in shoots of both mutant and parent, indicating that ABA was mainly synthesized in roots. Under salt stress, accumulation of ABA in the roots of bacteria-treated plants was accompanied by a decline in shoot ABA suggesting bacterial inhibition of ABA transport from roots to shoots. ABA accumulation in the roots of bacteria-treated Az34 was accompanied by increased leaf hydration, the probable outcome of increased root hydraulic conductance. Thereby, we tested the hypothesis that the ability of rhizobacterium (Bacillus subtilis IB22) to modify responses of plants to salt stress depends on abscisic acid (ABA) accumulating in roots.

Effects of Association of Barley Plants with Hydrocarbon-Degrading Bacteria on the Content of Soluble Organic Compounds in Clean and Oil-Contaminated Sand.

Plant-bacteria consortia are more effective in bioremediation of petroleum contaminated soil than when either organism is used individually. The reason for this is that plant root exudates promote growth and activity of oil degrading bacteria. However, insufficient attention has been paid to the ability of bacteria to influence root exudation. Therefore, the influence of barley plants and/or bacterial inoculation (Pseudomonas hunanensis IB C7 and Enterobacter sp. UOM 3) on the content of organic acids, sugars and plant hormones in the eluate from clean and oil-polluted sand was studied separately or in combination. These strains are capable of oxidizing hydrocarbons and synthesizing auxins. Concentrations of organic acids and sugars were determined using capillary electrophoresis, and hormones by enzyme-linked immunosorbent assays. In the absence of plants, no sugars were detected in the sand, confirming that root exudates are their main source. Introducing bacteria into the sand increased total contents of organic compounds both in the presence and absence of oil. This increase could be related to the increase in auxin amounts in the sand eluate, as well as in plants. The results indicate that bacteria are able to increase the level of root exudation. Since auxins can promote root exudation, bacterial production of this hormone is likely responsible for increased concentrations of soluble organic compounds in the sand. Bacterial mediation of root exudation by affecting plant hormonal status should be considered when choosing microorganisms for phytoremediation.

Effects of Plant Growth Promoting Rhizobacteria on the Content of Abscisic Acid and Salt Resistance of Wheat Plants.

Although salinity inhibits plant growth, application of appropriate rhizosphere bacteria can diminish this negative effect. We studied one possible mechanism that may underlie this beneficial response. Wheat plants were inoculated with Bacillus subtilis IB-22 and Pseudomonas mandelii IB-Ki14 and their consequences for growth, water relations, and concentrations of the hormone abscisic acid (ABA) were followed in the presence of soil salinity. Salinity alone increased ABA concentration in wheat leaves and roots and this was associated with decreased stomatal conductance, but also with chlorophyll loss. Bacterial treatment raised ABA concentrations in roots, suppressed accumulation of leaf ABA, decreased chlorophyll loss, and promoted leaf area and transpiration. However, water balance was maintained due to increased water uptake by inoculated plants, brought about in part by a larger root system. The effect may be the outcome of ABA action since the hormone is known to maintain root extension in stressed plants. Root ABA concentration was highest in salt-stressed plants inoculated with B. subtilis and this contributed to greater root hydraulic conductivity. We conclude that bacteria can raise salt resistance in wheat by increasing root ABA, resulting in larger root systems that can also possess enhanced hydraulic conductivity thereby supporting better-hydrated leaves.

Rhizobacteria Inoculation Effects on Phytohormone Status of Potato Microclones Cultivated In Vitro under Osmotic Stress.

Water deficits inhibit plant growth and decrease crop productivity. Remedies are needed to counter this increasingly urgent problem in practical farming. One possible approach is to utilize rhizobacteria known to increase plant resistance to abiotic and other stresses. We therefore studied the effects of inoculating the culture medium of potato microplants grown in vitro with Azospirillum brasilense Sp245 or Ochrobactrum cytisi IPA7.2. Growth and hormone content of the plants were evaluated under stress-free conditions and under a water deficit imposed with polyethylene glycol (PEG 6000). Inoculation with either bacterium promoted the growth in terms of leaf mass accumulation. The effects were associated with increased concentrations of auxin and cytokinin hormones in the leaves and stems and with suppression of an increase in the leaf abscisic acid that PEG treatment otherwise promoted in the potato microplants. O. cytisi IPA7.2 had a greater growth-stimulating effect than A. brasilense Sp245 on stressed plants, while A. brasilense Sp245 was more effective in unstressed plants. The effects were likely to be the result of changes to the plant's hormonal balance brought about by the bacteria.

Phytohormone Mediation of Interactions Between Plants and Non-Symbiotic Growth Promoting Bacteria Under Edaphic Stresses.

The capacity of rhizoshere bacteria to influence plant hormonal status, by bacterial production or metabolism of hormones, is considered an important mechanism by which they promote plant growth, and productivity. Nevertheless, inoculating these bacteria into the plant rhizosphere may produce beneficial or detrimental results depending on bacterial effects on hormone composition and quantity in planta, and the environmental conditions under which the plants are growing. This review considers some effects of bacterial hormone production or metabolism on root growth and development and shoot physiological processes. We analyze how these changes in root and shoot growth and function help plants adapt to their growth conditions, especially as these change from optimal to stressful. Consistent effects are addressed, along with plant responses to specific environmental stresses: drought, salinity, and soil contamination (with petroleum in particular).

Study of cytokinin transport from shoots to roots of wheat plants is informed by a novel method of differential localization of free cytokinin bases or their ribosylated forms by means of their specific fixation.

The aim of the present report was to demonstrate how a novel approach for immunohistochemical localization of cytokinins in the leaf and particularly in the phloem may complement to the study of their long-distance transport. Different procedures of fixation were used to conjugate either cytokinin bases or their ribosides to proteins of cytoplasm to enable visualization and differential localization of these cytokinins in the leaf cells of wheat plants. In parallel to immunolocalization of cytokinins in the leaf cells, we immunoassayed distribution of free bases of cytokinins, their nucleotides and ribosides between roots and shoots of wheat plants as well as their presence in phloem sap after incubation of leaves in a solution supplemented with either trans-zeatin or isopentenyladenine. The obtained data show ribosylation of the zeatin applied to the leaves and its elevated level in the phloem sap supported by in vivo localization showing the presence of ribosylated forms of zeatin in leaf vessels. This suggests that conversion of zeatin to its riboside is important for the shoot-to-root transport of zeatin-type cytokinins in wheat. Exogenous isopentenyladenine was not modified, but diffused from the leaves as free base. These metabolic differences may not be universal and may depend on the plant species and age. Although the measurements of cytokinins in the phloem sap and root tissue is the most defining for determining cytokinin transport, study of immunolocalization of either free cytokinin bases or their ribosylated forms may be a valuable source of information for predicting their transport in the phloem and to the roots.

Cytokinin producing bacteria stimulate amino acid deposition by wheat roots.

Phytohormone production is one mechanism by which rhizobacteria can stimulate plant growth, but it is not clear whether the bacteria gain from this mechanism. The hypothesis that microbial-derived cytokinin phytohormones stimulate root exudation of amino acids was tested. The rhizosphere of wheat plants was drenched with the synthetic cytokinin trans-zeatin or inoculated with Bacillus subtilis IB-22 (which produces zeatin type cytokinins) or B. subtilis IB-21 (which failed to accumulate cytokinins). Growing plants in a split root system allowed spatial separation of zeatin application or rhizobacterial inoculation to one compartment and analyses of amino acid release from roots (rhizodeposition) into the other compartment (without either microbial inoculation or treatment with exogenous hormone). Supplying B. subtilis IB-22 or zeatin to either the whole root system or half of the roots increased concentrations of amino acids in the soil solution although the magnitude of the increase was greater when whole roots were treated. There was some similarity in amino acid concentrations induced by either bacterial or zeatin treatment. Thus B. subtilis IB-22 increased amino acid rhizodeposition, likely due to its ability to produce cytokinins. Furthermore, B. subtilis strain IB-21, which failed to accumulate cytokinins in culture media, did not significantly affect amino acid concentrations in the wheat rhizosphere. The ability of rhizobacteria to produce cytokinins and thereby stimulate rhizodeposition may be important in enhancing rhizobacterial colonization of the rhizoplane.

Accumulation of cytokinins in roots and their export to the shoots of durum wheat plants treated with the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP).

Cytokinin flow from roots to shoots can serve as a long-distance signal important for root-to-shoot communication. In the past, changes in cytokinin flow from roots to shoots have been mainly attributed to changes in the rate of synthesis or breakdown in the roots. The present research tested the possibility that active uptake of cytokinin by root cells may also influence its export to shoots. To this end, we collapsed the proton gradient across root membranes using the protonophore carbonyl cyanide m-chlorophenylhydrazone (CCCP) to inhibit secondary active uptake of exogenous and endogenous cytokinins. We report the impact of CCCP on cytokinin concentrations and delivery in xylem sap and on accumulation in shoots of 7-day-old wheat plants in the presence and absence of exogenous cytokinin applied as zeatin. Zeatin treatment increased the total accumulation of cytokinin in roots and shoots but the effect was smaller for the shoots. Immunohistochemical localization of cytokinins using zeatin-specific antibodies showed an increase in immunostaining of the cells adjacent to xylem in the roots of zeatin-treated plants. Inhibition of secondary active cytokinin uptake by CCCP application decreased cytokinin accumulation in root cells but increased both flow from the roots and accumulation in the shoots. The possible importance of secondary active uptake of cytokinins by root cells for the control of their export to the shoot is discussed.

Effect of partial root excision on shoot water relations.

Removing 4 out of 5 serminal roots from 7-day-old wheat seedlings arrested leaf elongation for 1.5 h. This effect can be explained by an initial decrease in foliar water content resulting from the smaller root surface area available for water uptake. Subsequently, leaf hydration increased with time and came to equal that of intact plants within 2 h. The rehydration was seemingly effected by an increasing conductivity of the one remaining root axis, since transpiration of the partially de-rooted plants did not fall below that of controls. With time, leaf elongation resumed, but at a slower rate than in intact plants. This slower growth may be attributed to a decrease in leaf extensibility since this was found to be reduced when measured by a counterweight technique involving linear displacement transducers. Loss of extensibility was associated with decreased IAA concentration in the leaf elongation zone.