Stanislav Yu. Veselov

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.

Common and specific responses to availability of mineral nutrients and water

Changes in resource (mineral nutrients and water) availability, due to their heterogeneous distribution in space and time, affect plant development. Plants need to sense these changes to optimize growth and biomass allocation by integrating root and shoot growth. Since a limited supply of water or nutrients can elicit similar physiological responses (the relative activation of root growth at the expense of shoot growth), similar underlying mechanisms may affect perception and acquisition of either nutrients or water. This review compares root and shoot responses to availability of different macronutrients and water. Attention is given to the roles of root-to-shoot signalling and shoot-to-root signalling, with regard to coordinating changes in root and shoot growth and development. Involvement of plant hormones in regulating physiological responses such as stomatal and hydraulic conductance is revealed by measuring the effects of resource availability on phytohormone concentrations in roots and shoots, and their flow between roots and shoots in xylem and phloem saps. More specific evidence can be obtained by measuring the physiological responses of genotypes with altered hormone responses or concentrations. We discuss the similarity and diversity of changes in shoot growth, allocation to root growth, and root architecture under changes in water, nitrate, and phosphorus availability, and the possible involvement of abscisic acid, indole-acetic acid, and cytokinin in their regulation. A better understanding of these mechanisms may contribute to better crop management for efficient use of these resources and to selecting crops for improved performance under suboptimal soil conditions.

ABA mediation of shoot cytokinin oxidase activity: assessing its impacts on cytokinin status and biomass allocation of nutrient-deprived durum wheat

Although nutrient deprivation alters the concentrations of several plant hormones, the role of each in decreasing shoot-to-root ratio is not clear. A 10-fold dilution of the nutrient concentration supplied to hydroponically-grown 7-day-old durum wheat (Triticum turgidum L. ssp. durum Desf.) plants decreased shoot growth, shoot-to-root ratio and shoot and root cytokinin concentrations, increased shoot ABA concentration and shoot cytokinin oxidase activity, but had no effect on xylem sap ABA and cytokinin concentrations. Nutrient deprivation also increased xylem concentrations of conjugated ABA. The role of ABA in these responses was addressed by adding 11.4 µm ABA to the nutrient solution of well fertilised plants, or 1.2 mm fluridone (an inhibitor of ABA biosynthesis) to the nutrient solution of nutrient-deprived plants. The former induced similar changes in shoot-to-root ratio (by inhibiting shoot growth), shoot ABA concentration, shoot and root cytokinin concentrations and shoot cytokinin oxidase activity as nutrient deprivation. Conversely, fluridone addition to nutrient-deprived plants restored shoot-to-root ratio (by inhibiting root growth), shoot ABA concentration, shoot and root cytokinin concentrations to levels similar to well fertilised plants. Although root growth maintenance during nutrient deprivation depends on a threshold ABA concentration, shoot growth inhibition is independent of shoot ABA status. Although fluridone decreased shoot cytokinin oxidase activity of nutrient-deprived plants, it was still 1.7-fold greater than well fertilised plants, implying that nutrient deprivation could also activate shoot cytokinin oxidase independently of ABA. These data question the root signal basis of cytokinin action, but demonstrate that changes in ABA status can regulate shoot cytokinin concentrations via altering their metabolism.

Effect on shoot water relations, and cytokinin and abscisic acid levels of inducing expression of a gene coding for isopentenyltransferase in roots of transgenic tobacco plants

Heat shock (HS) at 40 degrees C was given to the root system of Nicotiana tabacum wild type (WT) and to HSIPT transgenic plants transformed with the bacterial cytokinin biosynthesis gene isopentenyltransferase (ipt) cloned behind the heat shock 70 promoter from Drosophila melanogaster. HS increased cytokinin concentrations in roots and leaves of transgenic plants. The effect was smaller in WT plants and restricted to upper leaves. HS also increased the activity of the cytokinin-degrading enzyme cytokinin oxidase in leaves of transgenic plants. This suggests that increases in cytokinin concentration induced by HS were lessened but not eliminated by increases in cytokinin oxidase. Elevated levels of zeatin riboside (the main transportable form of cytokinin) were also found in the HS-treated roots. It is proposed that increases in leaves were the outcome of increased transport of this hormone from roots in the transpiration stream. In conjunction with increased leaf cytokinin concentration, HS treatment to the roots increased stomatal conductivity and transpiration in both transgenic and WT plants. Subsequently, increased transpiration depressed leaf relative water content. This, in turn, raised leaf abscisic acid (ABA) concentrations, resulting in stomatal closure. It is concluded that the preceding increases in leaf cytokinin concentration, stomatal opening, and faster transpiration resulting from the localized induction of ip gene expression in roots strengthens the concept of cytokinin involvement in root to shoot signalling.

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.

Rapid changes in root HvPIP2;2 aquaporins abundance and ABA concentration are required to enhance root hydraulic conductivity and maintain leaf water potential in response to increased evaporative demand

To address the involvement of abscisic acid (ABA) in regulating transpiration and root hydraulic conductivity (LpRoot) and their relative importance for maintaining leaf hydration, the ABA-deficient barley mutant Az34 and its parental wild-type (WT) genotype (cv. Steptoe) were grown in hydroponics and exposed to changes in atmospheric vapour pressure deficit (VPD) imposed by air warming. WT plants were capable of maintaining leaf water potential (ψL) that was likely due to increased LpRoot enabling higher water flow from the roots, which increased in response to air warming. The increased LpRoot and immunostaining for HvPIP2;2 aquaporins (AQPs) correlated with increased root ABA content of WT plants when exposed to increased air temperature. The failure of Az34 to maintain ψL during air warming may be due to lower LpRoot than WT plants, and an inability to respond to changes in air temperature. The correlation between root ABA content and LpRoot was further supported by increased root hydraulic conductivity in both genotypes when treated with exogenous ABA (10-5 M). Thus the ability of the root system to rapidly regulate ABA levels (and thence aquaporin abundance and hydraulic conductivity) seems important to maintain leaf hydration.

Effect of Competition and Treatment with Inhibitor of Ethylene Perception on Growth and Hormone Content of Lettuce Plants

We elucidated the effect of increased planting density (single and grouped competing plants) on concentrations of auxin, abscisic acid, and cytokinins in normal lettuce plants and in those with ethylene perception inhibited by 1-methylcyclopropene (1-MCP). An attempt was made to relate the changes in hormone concentration induced by competition and inhibition of ethylene sensitivity to growth responses of lettuce planting. The results showed changes in concentrations of auxins, cytokinins, and ABA in the response of lettuce to crowding. Accumulation of ABA in shoots was likely to contribute to inhibition of transpiration of the plants grown in the presence of neighbors. This assumption was supported by the results of application of an inhibitor of ABA synthesis (fluridone and carotenoid biosynthesis herbicide) resulting in increased transpiration of grouped, but not single plants. Increased planting density led to the decline in root auxins paralleled by inhibition of root growth. This effect was likely to be due to decreased auxin transport to the roots from the shoots suggested by accumulation of auxins in the shoots and inhibition of root growth by application of the auxin transport inhibitor [N-(1-naphtyl)phtalamic acid (NPA)]. Importance of the changes in hormone concentrations was confirmed by data showing that disturbance of auxin and cytokinin distribution detected in MCP-treated plants was accompanied by corresponding modification of the growth response.

Effect of auxin producing and phosphate solubilizing bacteria on mobility of soil phosphorus, growth rate, and P acquisition by wheat plants

Microorganisms capable of mobilizing phosphate promote plant growth, this activity being frequently accompanied by production of plant hormones auxins. However, the extent of contribution of these characteristics to promotion of plant growth remains unclear. Paenibacillus illinoisensis IB 1087 and Pseudomonas extremaustralis IB-Ki-13-1A strains were selected for their capacity to mobilize phosphates and to synthesize auxins in vitro. The effects of inoculating these bacteria on the content of mobile phosphorus in the soil as well as on the phosphorus and hormone content in wheat plants were studied and the observed responses were related to the changes in plant growth. Inoculation of bacteria into the soil increased P concentration in the plants suggesting their increased capacity for the efficient acquisition of phosphorus compounds, while concentration of mobile phosphorus in the soil was increased by its inoculation with bacteria only in the absence of plants. The treatment increased plants mass (to greater extent in the case of P. illinoisensis) in accordance with the increased level of auxins in the treated plant. Increased mass accumulation did not correlate with the potential ability of bacteria strains for production of auxins or phosphate mobilization in vitro. Our data indicate importance of increased auxin content in the plants for the stimulation of root growth and capacity for P uptake as influenced by growth-promoting bacteria.

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.

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.