Photosynthetic sucrose drives the lateral root clock in Arabidopsis seedlings.

The development of plant roots is subject to control by light. Here, we show that, similar to monotonous root elongation, the periodic induction of lateral roots (LRs) depends on the activation by light of photomorphogenic and photosynthetic photoreceptors in the shoot in a hierarchical order. The prevailing belief is that the plant hormone auxin serves as a mobile signal transmitter, responsible for interorgan communication, including light-controlled shoot-to-root connections. Alternatively, it has been proposed that the transcription factor HY5 assumes the role as a mobile shoot-to-root signal transmitter. Here, we provide evidence that photosynthetic sucrose produced in the shoot acts as the long-distance signal carrier regulating the local, tryptophan-based biosynthesis of auxin in the LR generation zone of the primary root tip, where the LR clock controls the pace of LR initiation in an auxin-tunable manner. Synchronization of LR formation with primary root elongation allows the adjustment of overall root growth to the photosynthetic performance of the shoot and the maintenance of a constant LR density during light-dark changes in a variable light environment.

The plant hormone auxin beats the time for oscillating light-regulated lateral root induction.

The molecular mechanism underlying the periodic induction of lateral roots, a paradigmatic example of clock-driven organ formation in plant development, is a matter of ongoing, controversial debate. Here, we provide experimental evidence that this clock is frequency modulated by light and that auxin serves as a mediator for translating continuous light signals into discontinuous gene activation signals preceding the initiation of lateral roots in Arabidopsis seedlings. Based on this evidence, we propose a molecular model of an ultradian biological clock involving auxin-dependent degradation of an AUX/IAA-type transcription repressor as a flexible, frequency-controlling delay element. This model widens the bandwidth of biological clocks by adding a new type that allows the pace of organ formation to adapt to the changing environmental demands of the growing plant.

Priming and positioning of lateral roots in Arabidopsis. An approach for an integrating concept.

Branching by de novo formation of lateral roots along the primary root of Arabidopsis seedlings follows a complex longitudinal and transverse pattern. How this pattern is generated is presently under debate. The 'bending hypothesis' proposes that lateral root primordia are initiated by a local accumulation of auxin at the convex side of bends resulting from deflections through obstacles, gravitropic bending, or other means. In contrast, the 'oscillation hypothesis' proposes the existence of an endogenous clock-type oscillator mechanism producing periodic pulses of gene expression in the root tip that determine the future sites of primordium initiation. Here we report physiological experiments dissecting periodic priming signals, pre-disposing the root to rhythmic lateral root formation, from bending-mediated signals responsible for the subsequent positioning of their initiation along the growing root. While the frequency of lateral roots can be promoted by auxin in the mature root, their positioning follows a pre-formed pattern determined by previous bending. Both types of signals turn out to be necessary, complementary components in an integrating concept of lateral root patterning.

Photosynthetic sucrose acts as cotyledon-derived long-distance signal to control root growth during early seedling development in Arabidopsis.

The most hazardous span in the life of green plants is the period after germination when the developing seedling must reach the state of autotrophy before the nutrients stored in the seed are exhausted. The need for an economically optimized utilization of limited resources in this critical period is particularly obvious in species adopting the dispersal strategy of producing a large amount of tiny seeds. The model plant Arabidopsis thaliana belongs to this category. Arabidopsis seedlings promote root development only in the light. This response to light has long been recognized and recently discussed in terms of an organ-autonomous feature of photomorphogenesis directed by the red/blue light absorbing photoreceptors phytochrome and cryptochrome and mediated by hormones such as auxin and/or gibberellin. Here we show that the primary root of young Arabidopsis seedlings responds to an interorgan signal from the cotyledons and that phloem transport of photosynthesis-derived sugar into the root tip is necessary and sufficient for the regulation of root elongation growth by light.

Oxygen activation at the plasma membrane: relation between superoxide and hydroxyl radical production by isolated membranes.

Production of reactive oxygen species (hydroxyl radicals, superoxide radicals and hydrogen peroxide) was studied using EPR spin-trapping techniques and specific dyes in isolated plasma membranes from the growing and the non-growing zones of hypocotyls and roots of etiolated soybean seedlings as well as coleoptiles and roots of etiolated maize seedlings. NAD(P)H mediated the production of superoxide in all plasma membrane samples. Hydroxyl radicals were only produced by the membranes of the hypocotyl growing zone when a Fenton catalyst (FeEDTA) was present. By contrast, in membranes from other parts of the seedlings a low rate of spontaneous hydroxyl radical formation was observed due to the presence of small amounts of tightly bound peroxidase. It is concluded that apoplastic hydroxyl radical generation depends fully, or for the most part, on peroxidase localized in the cell wall. In soybean plasma membranes from the growing zone of the hypocotyl pharmacological tests showed that the superoxide production could potentially be attributed to the action of at least two enzymes, an NADPH oxidase and, in the presence of menadione, a quinone reductase.

Naphthoquinone-dependent generation of superoxide radicals by quinone reductase isolated from the plasma membrane of soybean.

Using a tetrazolium-based assay, a NAD(P)H oxidoreductase was purified from plasma membranes prepared from soybean (Glycine max) hypocotyls. The enzyme, a tetramer of 85 kD, produces O2(.-) by a reaction that depended on menadione or several other 1,4-naphthoquinones, in apparent agreement with a classification as a one-electron-transferring flavoenzyme producing semiquinone radicals. However, the enzyme displayed catalytic and molecular properties of obligatory two-electron-transferring quinone reductases of the DT-diaphorase type, including insensitivity to inhibition by diphenyleneiodonium. This apparent discrepancy was clarified by investigating the pH-dependent reactivity of menadionehydroquinone toward O2 and identifying the protein by mass spectrometry and immunological techniques. The enzyme turned out to be a classical NAD(P)H:quinone-acceptor oxidoreductase (EC 1.6.5.2, formerly 1.6.99.2) that reduces menadione to menadionehydroquinone and subsequently undergoes autoxidation at pH > or = 6.5. Autoxidation involves the production of the semiquinone as an intermediate, creating the conditions for one-electron reduction of O2. The possible function of this enzyme in the generation of O2(.-) and H2O2 at the plasma membrane of plants in vivo is discussed.

Biomechanics of plant growth.

Growth of turgid cells, defined as an irreversible increase in cell volume and surface area, can be regarded as a physical process governed by the mechanical properties of the cell wall and the osmotic properties of the protoplast. Irreversible cell expansion is produced by creating a driving force for water uptake by decreasing the turgor through stress relaxation in the cell wall. This mechano-hydraulic process thus depends on and can be controlled by the mechanical properties of the wall, which in turn are subject to modification by wall loosening and wall stiffening reactions. The biochemical mechanisms of these changes in mechanical wall properties and their regulation by internal signals (e.g., hormones) or external signals (e.g., light, drought stress) are at present incompletely understood and subject to intensive research. These signals act on walls that have the properties of composite materials in which the molecular structure and spatial organization of polymers rather than the distribution of mechanical stresses dictate the allometry of cell and organ growth and thus cell and organ shape. The significance of cell wall architecture for allometric growth can be demonstrated by disturbing the oriented deposition of wall polymers with microtubule-interfering drugs such as colchicine. Elongating organs (e.g., cylindrical stems or coleoptiles) composed of different tissues with different mechanical properties exhibit longitudinal tissue tensions resulting in the transfer of wall stress from inner to peripheral cell layers that adopt control over organ growth. For physically analyzing the growth process leading to seed germination, the same mechanical and hydraulic parameters as in normal growth are principally appropriate. However, for covering the influences of the tissues that restrain embryo expansion (seed coat, endosperm), an additional force and a water permeability term must be considered.

Sensitive detection and localization of hydroxyl radical production in cucumber roots and Arabidopsis seedlings by spin trapping electron paramagnetic resonance spectroscopy.

As reactive oxygen species are important for many fundamental biological processes in plants, specific and sensitive techniques for their detection in vivo are essential. In particular, the analysis of hydroxyl radical (OH*) formation in biological reactions has rarely been attempted. Here, it is shown that spin trapping electron paramagnetic resonance (EPR) spectroscopy allows the detection and quantitative estimation of OH* production in vivo in one single cucumber seedling root. It is possible to localize the OH* production site to the growth zone of the root by varying the position of the intact seedling inside the resonator cavity of the EPR spectrometer. Moreover, the demonstration of impaired OH* formation in the root of the Arabidopsis mutant rhd2 impaired in a superoxide-producing Nicotimamide adenine dinucleotide phosphate (NADPH) oxidase has been accomplished. Spin trapping EPR provides a valuable tool for analyzing the production of OH*in vivo with high resolution in small tissue samples.

Production of reactive oxygen intermediates (O(2)(.-), H(2)O(2), and (.)OH) by maize roots and their role in wall loosening and elongation growth.

Cell extension in the growing zone of plant roots typically takes place with a maximum local growth rate of 50% length increase per hour. The biochemical mechanism of this dramatic growth process is still poorly understood. Here we test the hypothesis that the wall-loosening reaction controlling root elongation is effected by the production of reactive oxygen intermediates, initiated by a NAD(P)H oxidase-catalyzed formation of superoxide radicals (O(2)(.-)) at the plasma membrane and culminating in the generation of polysaccharide-cleaving hydroxyl radicals ((.)OH) by cell wall peroxidase. The following results were obtained using primary roots of maize (Zea mays) seedlings as experimental material. (1) Production of O(2)(.-), H(2)O(2), and (.)OH can be demonstrated in the growing zone using specific histochemical assays and electron paramagnetic resonance spectroscopy. (2) Auxin-induced inhibition of growth is accompanied by a reduction of O(2)(.-) production. (3) Experimental generation of (.)OH in the cell walls with the Fenton reaction causes wall loosening (cell wall creep), specifically in the growing zone. Alternatively, wall loosening can be induced by (.)OH produced by endogenous cell wall peroxidase in the presence of NADH and H(2)O(2). (4) Inhibition of endogenous (.)OH formation by O(2)(.-) or (.)OH scavengers, or inhibitors of NAD(P)H oxidase or peroxidase activity, suppress elongation growth. These results show that juvenile root cells transiently express the ability to generate (.)OH, and to respond to (.)OH by wall loosening, in passing through the growing zone. Moreover, inhibitor studies indicate that (.)OH formation is essential for normal root growth.

Evidence for the involvement of cell wall peroxidase in the generation of hydroxyl radicals mediating extension growth.

Hydroxyl radicals (*OH), produced in the cell wall, are capable of cleaving wall polymers and can thus mediate cell wall loosening and extension growth. It has recently been proposed that the biochemical mechanism responsible for *OH generation in the cell walls of growing plant organs represents an enzymatic reaction catalyzed by apoplastic peroxidase (POD). This hypothesis was investigated by supplying cell walls of maize ( Zea mays L.) coleoptiles and sunflower ( Helianthus annuus L.) hypocotyls with external NADH, an artificial substrate known to cause *OH generation by POD in vitro. The effects of NADH on wall loosening, growth, and *OH production in vivo were determined. NADH mediates cell wall extension in vitro and in vivo in an H2O2-dependent reaction that shows the characteristic features of POD. NADH-mediated production of *OH in vivo was demonstrated in maize coleoptiles using electron paramagnetic resonance spectroscopy in combination with a specific spin-trapping reaction. Kinetic properties and inhibitor/activator sensitivities of the *OH-producing reaction in the cell walls of coleoptiles resembled the properties of horseradish POD. Apoplastic consumption of external NADH by living coleoptiles can be traced back to the superimposed action of two enzymatic reactions, a KCN-sensitive reaction mediated by POD operating in the *OH-forming mode, and a KCN-insensitive reaction with the kinetic properties of a superoxide-producing plasma-membrane NADH oxidase the activity of which can be promoted by auxin. Under natural conditions, i.e. in the absence of external NADH, this enzyme may provide superoxide (O2*-) (and H2O2 utilized by POD for) *OH production in the cell wall.

Polysaccharide degradation by Fenton reaction--or peroxidase-generated hydroxyl radicals in isolated plant cell walls.

The formation of hydroxyl radicals (OH*) by peroxidase was confirmed by EPR spectroscopy using ethanol/alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone as a spin-trapping system specific of OH*. The effect of OH*, generated either non-enzymatically with the Fenton reaction (H(2)O(2) + Fe(2+)) or with horseradish peroxidase in the presence of O(2) and NADH, on cell walls isolated from maize (Zea mays) coleoptiles or soybean (Glycine max) hypocotyls was investigated. OH* produced by these reactions attack polysaccharides in the wall, demonstrated by the release of a heterogeneous mixture of polymeric breakdown products into the incubation medium. The peroxidase-catalyzed degradation of cell-wall polysaccharides can be inhibited by KCN and superoxide radical (O(2)*) or OH* scavengers. These data support the hypothesis that OH*, produced by cell-wall peroxidases in vivo, act as wall-loosening agents in plant extension growth.

Evidence that hydroxyl radicals mediate auxin-induced extension growth.

Reactive oxygen intermediates, i.e. the superoxide radical (O*-)(2), hydrogen peroxide (H2O2) and the hydroxyl radical (*OH), are generally regarded as harmful products of oxygenic metabolism causing cell damage in plants, animals and microorganisms. However, oxygen radical chemistry may also play a useful role in polymer breakdown leading to wall loosening during extension growth of plant cells controlled by the phytohormone auxin. Backbone cleavage of cell wall polysaccharides can be accomplished in vitro by (*OH) produced from H2O2 in a Fenton reaction or in a reaction catalyzed by peroxidase supplied with O2 and NADH. Here, we show that coleoptile growth of maize seedlings is accompanied by the release of reactive oxygen intermediates in the cell wall. Auxin promotes release of (O*-)(2) and subsequent generation of (*OH)when inducing elongation growth. Experimental generation of (*OH) in the wall causes an increase in wall extensibility in vitro and replaces auxin in inducing growth. Auxin-induced growth can be inhibited by scavengers of (O*-)(2), H2O2 or (*OH), or inhibitors interfering with the formation of these molecules in the cell wall. These results provide the experimental background for a novel hypothesis on the mechanism of plant cell growth in which (*OH), produced from (O*-)(2) and H2O2 by cell wall peroxidase, acts as a wall-loosening agent.