Role of cytokinin and auxin in shaping root architecture: regulating vascular differentiation, lateral root initiation, root apical dominance and root gravitropism.

BACKGROUND AND AIMS: Development and architecture of plant roots are regulated by phytohormones. Cytokinin (CK), synthesized in the root cap, promotes cytokinesis, vascular cambium sensitivity, vascular differentiation and root apical dominance. Auxin (indole-3-acetic acid, IAA), produced in young shoot organs, promotes root development and induces vascular differentiation. Both IAA and CK regulate root gravitropism. The aims of this study were to analyse the hormonal mechanisms that induce the root's primary vascular system, explain how differentiating-protoxylem vessels promote lateral root initiation, propose the concept of CK-dependent root apical dominance, and visualize the CK and IAA regulation of root gravitropiosm. KEY ISSUES: The hormonal analysis and proposed mechanisms yield new insights and extend previous concepts: how the radial pattern of the root protoxylem vs. protophloem strands is induced by alternating polar streams of high IAA vs. low IAA concentrations, respectively; how differentiating-protoxylem vessel elements stimulate lateral root initiation by auxin-ethylene-auxin signalling; and how root apical dominance is regulated by the root-cap-synthesized CK, which gives priority to the primary root in competition with its own lateral roots. CONCLUSIONS: CK and IAA are key hormones that regulate root development, its vascular differentiation and root gravitropism; these two hormones, together with ethylene, regulate lateral root initiation.

Immunolocalization of plasma-membrane H+-ATPase and tonoplast-type pyrophosphatase in the plasma membrane of the sieve element-companion cell complex in the stem of Ricinus communis L.

Plasma-membrane-located primary pumps were investigated in the sieve element (SE)-companion cell complex in the transport phloem of 2-week-old stems of Ricinus communis L. and, for comparison, in stems of Cucurbita pepo L. and in the secondary phloem of Agrobacterium tumefaciens-induced crown galls as a typical sink tissue. The plasma-membrane (PM) H+-ATPase and the tonoplast-type pyrophosphatase (PPase) were immunolocalized by epifluorescence and confocal laser scanning microscopy (CLSM) upon single or double labeling with specific monoclonal and polyclonal antibodies. Quantitative fluorescence evaluation by CLSM revealed both pumps in one membrane, the sieve-element PM. Different PM H+-ATPase antibody clones, raised against the PM H+-ATPase of Zea mays coleoptiles, induced in mouse and produced in mouse hybridoma cells, discriminated between different phloem cell types. Clones 30D5C4 and 44B8A1 labeled sieve elements and clone 46E5B11D5 labeled companion cells, indicating the existence of different phloem PM H+-ATPase isoforms. The results are discussed in terms of energization of SE transporters for retrieval of leaking sucrose, K+ and amino acids, as one of the unknown roles of ATP found in SEs. The function of the PPase could be related to phloem sucrose metabolism in support of ATP-requiring processes.

Vascularization is a general requirement for growth of plant and animal tumours.

Solid-tumour growth in animals as in humans depends on angiogenesis. Tumours that fail to induce the formation of new blood vessels do not enlarge beyond a few millimetres in diameter. Plant tumours induced by Agrobacterium tumefaciens can reach diameters of more than 100 mm, thus raising the question of how they are sufficiently supplied with nutrients and water. Until recently, these rapidly growing tumours were considered unorganized or partly organized masses. However, in analogy to animal and human tumours, growth of leaf and stem tumours depends on neovascularization. Plant tumour cells induce the formation of a sophisticated vascular network consisting of water-conducting vessels and assimilate-transporting sieve elements. Similar to animal and human tumours that overexpress angiogenic growth factors, plant tumours overexpress the T-DNA-encoded vascularization-promoting growth factors auxin and cytokinin upon AGROBACTERIUM: infection. High auxin levels induce ethylene emission from the tumours, which has a strong impact on tumour and host stem, as well as on root structure and function. Ethylene apparently stimulates abscisic acid synthesis in the leaves above the tumour, which reduces transpiration and thus protects the host plant from rapid wilting. Hence, for the elucidation of phytohormone-dependent vascular development in plants, such tumours are regarded as an excellent model system. The comparison of analogous requirement of neovascularization for tumour growth in plants, as in animals and humans, is discussed in terms of interdisciplinary strategies of possible prevention and therapy.

Electrical Membrane Properties of Leaves, Roots, and Single Root Cap Cells of Susceptible Avena sativa: Effect of Victorin C.

The effect of the purified host-selective toxin victorin C, a cyclized penta peptide, was compared to that of CCCP and vanadate on membrane functions of susceptible leaves, roots, and single root cap cells of Avena sativa with conventional electrophysiology. The plasmalemma depolarized irreversibly by about 80 millivolts and to below the diffusion potential within 1 hour. Concentrations as low as 12.5 picomolar were effective in the susceptible but not the resistant cultivar. Electrical membrane potential difference changes were independent of pH and could not be prevented by fusicoccin or Ca(2+). Membranes began to depolarize after a lag phase that never was shorter than 6.5 minutes, even with concentrations as high as 1.25 micromolar. Membrane depolarization was accompanied by a distinct decrease in specific membrane resistance from 4.5 to 1.0 ohm times square meter on average. These changes were followed by K(+) and Cl(-) efflux and extracellular alkalinization. ATP level and O(2) uptake did not decrease within 2 hours. It is concluded that the victorin-induced deleterious membrane alterations are not caused by direct interaction with the plasmalemma H(+)-ATPase, K(+) channels, lipid structure, nor energy metabolism, but they seem to be triggered by a cascade of events leading to an unspecific increase in membrane permeability.

Extra- and Intracellular pH and Membrane Potential Changes Induced by K, Cl, H(2)PO(4), and NO(3) Uptake and Fusicoccin in Root Hairs of Limnobium stoloniferum.

Short-term ion uptake into roots of Limnobium stoloniferum was followed extracellularly with ion selective macroelectrodes. Cytosolic or vacuolar pH, together with the electrical membrane potential, was recorded with microelectrodes both located in the same young root hair. At the onset of chloride, phosphate, and nitrate uptake the membrane potential transiently decreased by 50 to 100 millivolts. During Cl(-) and H(2)PO(4) (-) uptake cytosolic pH decreased by 0.2 to 0.3 pH units. Nitrate induced cytosolic alkalinization by 0.19 pH units, indicating rapid reduction. The extracellular medium alkalinized when anion uptake exceeded K(+) uptake. During fusicoccin-dependent plasmalemma hyperpolarization, extracellular and cytosolic pH remained rather constant. Upon K(+) absorption, FC intensified extracellular acidification and intracellular alkalinization (from 0.31 to 0.4 pH units). In the presence of Cl(-) FC induced intracellular acidification. Since H(+) fluxes per se do not change the pH, recorded pH changes only result from fluxes of the stronger ions. The extra- and intracellular pH changes, together with membrane depolarization, exclude mechanisms as K(+)/A(-) symport or HCO(3) (-)/A(-) antiport for anion uptake. Though not suitable to reveal the actual H(+)/A(-) stoichiometry, the results are consistent with an H(+)/A(-) cotransport mechanism.

Ion fluxes and pH changes induced by trans-plasmalemma electron transfer and fusicoccin in Lemna gibba L. (strain G1).

During the reduction of extracellular [Fe(CN)6](3-) at the plasmalemma of intact, K(+)-starved Lemna gibba L. fronds, the external medium was acidified and K(+) released, in the absence of inhibitors with rates of 10 e(-)/8.5 H(+)/1.5 K(+) (µmol·(g FW)(-1)·(-1)). In K(+) plants the larger K(+) efflux caused a lag phase in extracellular acidification and a change in rates to 10 e(-)/6 H(+)/4 K(+) and in the presence of CN(-)+salicylhydroxamic acid at pH 5 to 5.2 e(-)/0 H(+)/6.6 K(+). The e(-) transfer was accompanied by a membrane depolarization of up to 100 mV and a cytosolic acidification of about 0.6 pH units, but only in K(+) plants, where the extracellular acidification was smaller. These results indicate that a stimulation of the plasmalemma H(+)-ATPase may be triggered either by a cytosolic acidification or by a strong membrane depolarization. It is concluded that the redox system catalyses only uncoupled e(-) transfer without H(+) transfer across the plasmalemma. The obligatory, but secondary charge compensation is partially achieved by the rapid K(+) release upon membrane depolarization and partially by the activity of the plasma membrane H(+)-ATPase, but not by an e(-)/anion exchange. The extracellular acidification during [Fe(CN)6](3-) reduction is generated by the conversion of a strong trivalent into a strong tetravalent anion. This acidification is caused by changes in the concentration ratio of strong cations to strong anions. Efflux of K(+) and not the production of organic acids or NAD(P)H oxidation is the chemical cause of the measurable cytosolic acidification. Extracellular acidification was inversely correlated with intracellular acidification. Similarly, fusicoccin-induced pH changes were correlated with changes in the strong-ion concentration difference. Extracellular ± FC-dependent acidification and intracellular alkalinization of up to 0.6 pH units were strongly dependent on K(+) fluxes. The ferricyanide-triggered trans-plasmalemma electron-transfer system is an example of how measurable pH changes are the consequence and not the cause of charge-transfer-induced changes in strong-ion fluxes.

Uptake of Acidic and Basic Sugar Derivatives in Lemna gibba G1.

The uptake of acidic and basic sugar derivatives in Lemna gibba L. was studied. Uronic acids applied to the experimental solution (50 millimolar) induced a small decrease of the membrane potential (10 +/- 1 millivolt galacturonic acid, and 20 +/- 4 millivolt glucuronic acid). After incubation of the plants in a 0.1 millimolar solution of these substrates, no decrease in the concentration of reducing groups in the external solution was detected. Respiration increased by 31% with 50 millimolar galacturonic acid, whereas no effect was found with the same concentration of glucuronic acid. Glucosamine caused a considerable concentration-dependent membrane depolarization. ((14)C)glucosamine uptake followed Michaelis-Menten kinetics together with a linear component. Influx of this substrate was inhibited by glucose but the type of competition could not be clearly distinguished. Glucosamine, 50 millimolar, inhibited the respiration rate by 30%. The glucosamine uptake was pH-dependent, with maximum uptake at around pH 7. Lack of enhancement of uptake by low pH as well as the permanent membrane depolarization suggest a uniport mechanism for the charged species of the substrate and an electroneutral diffusion of the uncharged species.