The Growth-Inhibitory Effect of Increased Planting Density Can Be Reduced by Abscisic Acid-Degrading Bacteria.

High-density planting can increase crop productivity per unit area of cultivated land. However, the application of this technology is limited by the inhibition of plant growth in the presence of neighbors, which is not only due to their competition for resources but is also caused by growth regulators. Specifically, the abscisic acid (ABA) accumulated in plants under increased density of planting has been shown to inhibit their growth. The goal of the present study was to test the hypothesis that bacteria capable of degrading ABA can reduce the growth inhibitory effect of competition among plants by reducing concentration of this hormone in plants and their environment. Lettuce plants were grown both individually and three per pot; the rhizosphere was inoculated with a strain of Pseudomonas plecoglossicida 2.4-D capable of degrading ABA. Plant growth was recorded in parallel with immunoassaying ABA concentration in the pots and plants. The presence of neighbors indeed inhibited the growth of non-inoculated lettuce plants. Bacterial inoculation positively affected the growth of grouped plants, reducing the negative effects of competition. The bacteria-induced increase in the mass of competing plants was greater than that in the single ones. ABA concentration was increased by the presence of neighbors both in soil and plant shoots associated with the inhibition of plant growth, but accumulation of this hormone as well as inhibition of the growth of grouped plants was prevented by bacteria. The results confirm the role of ABA in the response of plants to the presence of competitors as well as the possibility of reducing the negative effect of competition on plant productivity with the help of bacteria capable of degrading this hormone.

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.

Effect of Low Light Stress on Distribution of Auxin (Indole-3-acetic Acid) between Shoot and Roots and Development of Lateral Roots in Barley Plants.

Depending on their habitat conditions, plants can greatly change the growth rate of their roots. However, the mechanisms of such responses remain insufficiently clear. The influence of a low level of illumination on the content of endogenous auxins, their localization in leaves and transport from shoots to roots were studied and related to the lateral root branching of barley plants. Following two days' reduction in illumination, a 10-fold reduction in the emergence of lateral roots was found. Auxin (IAA, indole-3-acetic acid) content decreased by 84% in roots and by 30% in shoots, and immunolocalization revealed lowered IAA levels in phloem cells of leaf sections. The reduced content of IAA found in the plants under low light suggests an inhibition of production of this hormone under these conditions. At the same time, two-fold downregulation of the LAX3 gene expression, facilitating IAA influx into the cells, was detected in the roots, as well as a decline in auxin diffusion from shoots through the phloem by about 60%. It was suggested that the reduced emergence of lateral roots in barley under a low level of illumination was due to a disturbance of auxin transport through the phloem and down-regulation of the genes responsible for auxin transport in plant roots. The results confirm the importance of the long distance transport of auxins for the control of the growth of roots under conditions of low light. Further study of the mechanisms that control the transport of auxins from shoots to roots in other plant species is required.

Effect of ipt Gene Induction in Transgenic Tobacco Plants on Hydraulic Conductance, Formation of Apoplastic Barriers and Aquaporin Activity under Heat Shock.

Cytokinins are known to keep stomata open, which supports gas exchange and correlates with increased photosynthesis. However, keeping the stomata open can be detrimental if the increased transpiration is not compensated for by water supply to the shoots. In this study, we traced the effect of ipt (isopentenyl transferase) gene induction, which increases the concentration of cytokinins in transgenic tobacco plants, on transpiration and hydraulic conductivity. Since water flow depends on the conductivity of the apoplast, the deposition of lignin and suberin in the apoplast was studied by staining with berberine. The effect of an increased concentration of cytokinins on the flow of water through aquaporins (AQPs) was revealed by inhibition of AQPs with HgCl2. It was shown that an elevated concentration of cytokinins in ipt-transgenic plants increases hydraulic conductivity by enhancing the activity of aquaporins and reducing the formation of apoplastic barriers. The simultaneous effect of cytokinins on both stomatal and hydraulic conductivity makes it possible to coordinate the evaporation of water from leaves and its flow from roots to leaves, thereby maintaining the water balance and leaf hydration.

Effect of Salinity on Stomatal Conductance, Leaf Hydraulic Conductance, HvPIP2 Aquaporin, and Abscisic Acid Abundance in Barley Leaf Cells.

The stomatal closure of salt-stressed plants reduces transpiration bringing about the maintenance of plant tissue hydration. The aim of this work was to test for any involvement of aquaporins (AQPs) in stomatal closure under salinity. The changes in the level of aquaporins in the cells were detected with the help of an immunohistochemical technique using antibodies against HvPIP2;2. In parallel, leaf sections were stained for abscisic acid (ABA). The effects of salinity were compared to those of exogenously applied ABA on leaf HvPIP2;2 levels and the stomatal and leaf hydraulic conductance of barley plants. Salinity reduced the abundance of HvPIP2;2 in the cells of the mestome sheath due to it being the more likely hydraulic barrier due to the deposition of lignin, accompanied by a decline in the hydraulic conductivity, transpiration, and ABA accumulation. The effects of exogenous ABA differed from those of salinity. This hormone decreased transpiration but increased the shoot hydraulic conductivity and PIP2;2 abundance. The difference in the action of the exogenous hormone and salinity may be related to the difference in the ABA distribution between leaf cells, with the hormone accumulating mainly in the mesophyll of salt-stressed plants and in the cells of the bundle sheaths of ABA-treated plants. The obtained results suggest the following succession of events: salinity decreases water flow into the shoots due to the decreased abundance of PIP2;2 and hydraulic conductance, while the decline in leaf hydration leads to the production of ABA in the leaves and stomatal closure.

Association of Barley Root Elongation with ABA-Dependent Transport of Cytokinins from Roots and Shoots under Supra-Optimal Concentrations of Nitrates and Phosphates.

Changes in root elongation are important for the acquisition of mineral nutrients by plants. Plant hormones, cytokinins, and abscisic acid (ABA) and their interaction are important for the control of root elongation under changes in the availability of ions. However, their role in growth responses to supra-optimal concentrations of nitrates and phosphates has not been sufficiently studied and was addressed in the present research. Effects of supra-optimal concentrations of these ions on root elongation and distribution of cytokinins between roots and shoots were studied in ABA-deficient barley mutant Az34 and its parental variety, Steptoe. Cytokinin concentration in the cells of the growing root tips was analyzed with the help of an immunohistochemical technique. Increased concentrations of nitrates and phosphates led to the accumulation of ABA and cytokinins in the root tips, accompanied by a decline in shoot cytokinin content and inhibition of root elongation in Steptoe. Neither of the effects were detected in Az34, suggesting the importance of the ability of plants to accumulate ABA for the control of these responses. Since cytokinins are known to inhibit root elongation, the effect of supra-optimal concentration of nitrates and phosphates on root growth is likely to be due to the accumulation of cytokinins brought about by ABA-induced inhibition of cytokinin transport from roots to shoots.

Influence of Hydrocarbon-Oxidizing Bacteria on the Growth, Biochemical Characteristics, and Hormonal Status of Barley Plants and the Content of Petroleum Hydrocarbons in the Soil.

Much attention is paid to the relationship between bacteria and plants in the process of the bioremediation of oil-contaminated soils, but the effect of petroleum degrading bacteria that synthesize phytohormones on the content and distribution of these compounds in plants is poorly studied. The goal of the present field experiment was to study the effects of hydrocarbon-oxidizing bacteria that produce auxins on the growth, biochemical characteristics, and hormonal status of barley plants in the presence of oil, as well as assessing the effect of bacteria and plants separately and in association with the content of oil hydrocarbons in the soil. The treatment of plants with strains of Enterobacter sp. UOM 3 and Pseudomonas hunanensis IB C7 led to an increase in the length and mass of roots and shoots and the leaf surface index, and an improvement in some parameters of the elements of the crop structure, which were suppressed by the pollutant. The most noticeable effect of bacteria on the plant hormonal system was a decrease in the accumulation of abscisic acid. The data obtained indicate that the introduction of microorganisms weakened the negative effects on plants under abiotic stress caused by the presence of oil. Plant-bacteria associations were more effective in reducing the content of hydrocarbons in the soil and increasing its microbiological activity than when either organism was used individually.

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 Phosphate Shortage on Root Growth and Hormone Content of Barley Depend on Capacity of the Roots to Accumulate ABA.

Although changes in root architecture in response to the environment can optimize mineral and water nutrient uptake, mechanisms regulating these changes are not well-understood. We investigated whether P deprivation effects on root development are mediated by abscisic acid (ABA) and its interactions with other hormones. The ABA-deficient barley mutant Az34 and its wild-type (WT) were grown in P-deprived and P-replete conditions, and hormones were measured in whole roots and root tips. Although P deprivation decreased growth in shoot mass similarly in both genotypes, only the WT increased primary root length and number of lateral roots. The effect was accompanied by ABA accumulation in root tips, a response not seen in Az34. Increased ABA in P-deprived WT was accompanied by decreased concentrations of cytokinin, an inhibitor of root extension. Furthermore, P-deficiency in the WT increased auxin concentration in whole root systems in association with increased root branching. In the ABA-deficient mutant, P-starvation failed to stimulate root elongation or promote branching, and there was no decline in cytokinin and no increase in auxin. The results demonstrate ABA's ability to mediate in root growth responses to P starvation in barley, an effect linked to its effects on cytokinin and auxin concentrations.

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.

Capacity of Pseudomonas Strains to Degrade Hydrocarbons, Produce Auxins and Maintain Plant Growth under Normal Conditions and in the Presence of Petroleum Contaminants.

The phytoremediation of soil contaminated with petroleum oil products relies on co-operation between plants and rhizosphere bacteria, including the plant growth-promoting effect of the bacteria. We studied the capacity of strains of Pseudomonas, selected as oil degraders, to produce plant hormones and promote plant growth. Strains with intermediate auxin production were the most effective in stimulating the seedling growth of seven plant species under normal conditions. Bacterial seed treatment resulted in about a 1.6-fold increase in the weight of barley seedlings, with the increment being much lower in other plant species. The strains P. plecoglossicida 2.4-D and P. hunanensis IB C7, characterized by highly efficient oil degradation (about 70%) and stable intermediate in vitro auxin production in the presence of oil, were selected for further study with barley. These strains increased the seed germination percentage approximately two-fold under 5% oil concentration in the soil, while a positive effect on further seedling growth was significant when the oil concentration was raised to 8%. This resulted in a 1.3-1.7-fold increase in the seedling mass after 7 days of growth, depending on the bacterial strain. Thus, strains of oil-degrading bacteria selected for their intermediate and stable production of auxin were found to be effective ameliorators of plant growth inhibition resulting from petroleum stress.

Dependence of growth inhibiting action of increased planting density on capacity of lettuce plants to synthesize ABA.

Inhibition of lettuce plant growth under increased planting density was accompanied by accumulation of abscisic acid (ABA) in the shoots of competing plants. To check causal relationship between these responses we studied the effect of decreased synthesis of ABA on growth indexes and hormonal balance of lettuce plants under elevated density of their planting (one (single) or three (competing) plants per pot). Herbicide fluridone was used to inhibit ABA synthesis. Preliminary experiments with single plants showed that presence of fluridone in the soil solution at rather low concentration (0.001mg/L) did not affect either chlorophyll content or growth rate of shoots and roots during at least one week. Treatment of competing (grouped) plants with this concentration of fluridone prevented both accumulation of ABA and competition induced growth inhibition. These results confirm important role of this hormone in the growth inhibiting effect of increased planting density. Furthermore, as in the case of ABA, fluridone prevented allocation of indoleacetic acid (IAA) to the shoots of competing plants likely contributing to leveling off the increase in the ratio of leaf area to their mass that is characteristic effect of shading in the dense plant populations. The results suggest involvement of ABA in allocation of IAA in competing plants. Application of fluridone did not influence the concentration of cytokinins in the shoots, whose level was decreased by competition either in fluridone treated or control (untreated with fluridone) plants. Accumulation of ABA in the shoots of competing plants accompanied by inhibition of their growth and the absence of either accumulation of ABA or inhibition of their growth in fluridone treated grouped plants confirms importance of ABA synthesis for growth response to competition.

Exogenous methyl jasmonate regulates cytokinin content by modulating cytokinin oxidase activity in wheat seedlings under salinity.

The treatment of 4-days-old wheat seedlings with methyl jasmonate (MeJA) in concentration optimal for their growth (0.1 µM) resulted in a rapid transient almost two-fold increase in the level of cytokinins (CKs). MeJA-induced accumulation of CKs was due to inhibition of both cytokinin oxidase (CKX) (cytokinin oxidase/dehydrogenase, EC 1.5.99.12) gene expression and activity of this enzyme. Pretreatment of wheat seedlings with MeJA decreased the growth-retarding effect of sodium chloride salinity and accelerated growth recovery after withdrawal of NaCl from the incubation medium. We speculate that this protective effect of the hormone might be due to MeJA's ability to prevent the salinity-induced decline in CK concentration that was caused by inhibition of gene expression and activity of CKX in wheat seedlings. The data might indicate an important role for endogenous cytokinins in the implementation of growth-promoting and protective effects of exogenous MeJA application on wheat plants.

The effect of competition from neighbours on stomatal conductance in lettuce and tomato plants.

Competition decreased transpiration from young lettuce plants after 2 days, before any reductions in leaf area became apparent, and stomatal conductance (g(s) ) of lettuce and tomato plants was also reduced. Stomatal closure was not due to hydraulic signals or competition for nutrients, as soil water content, leaf water status and leaf nitrate concentrations were unaffected by neighbours. Competition-induced stomatal closure was absent in an abscisic acid (ABA)-deficient tomato mutant, flacca, indicating a fundamental involvement of ABA. Although tomato xylem sap ABA concentrations were unaffected by the presence of neighbours, ABA/pH-based stomatal modulation is still likely to underlie the response to competition, as soil and xylem sap alkalization was observed in competing plants. Competition also modulated leaf ethylene production, and treatment of lettuce plants with an ethylene perception inhibitor (1-methylcyclopropene) diminished the difference in g(s) between single and competing plants grown in a controlled environment room, but increased it in plants grown in the greenhouse: ethylene altered the extent of the stomatal response to competition. Effects of competition on g(s) are discussed in terms of the detection of the absence of neighbours: increases in g(s) and carbon fixation may allow faster initial space occupancy within an emerging community/crop.

Abscisic acid accumulation in the roots of nutrient-limited plants: its impact on the differential growth of roots and shoots.

We describe the involvement of abscisic acid (ABA) in the control of differential growth of roots and shoots of nutrient limited durum wheat plants. A ten-fold dilution of the optimal concentration of nutrient solution inhibited shoot growth, while root growth remained unchanged, resulting in a decreased shoot/root ratio. Addition of fluridone (inhibitor of ABA synthesis) prevented growth allocation in favour of the roots. This suggests the involvement of ABA in the redirecting of growth in favour of roots under limited nutrient supply. The ABA content was greater in shoots and growing apical root parts of starved plants than in nutrient sufficient plants. Accumulation of ABA in shoots of nutrient deficient plants was linked to a decrease in leaf turgor. Increased flow of ABA in the phloem apparently contributed to the accumulation of ABA in the apical part of the roots. Thus, partitioning of growth between roots and shoots of wheat plants limited in mineral nutrients appears to be modulated by accumulation of ABA in roots. This ABA may originate in the shoots, where its synthesis is stimulated by the loss of leaf turgor.