Dynamics in PIN2 auxin carrier ubiquitylation in gravity-responding Arabidopsis roots

Plant tropisms are decisively influenced by dynamic adjustments in spatiotemporal distribution of the growth regulators auxin. Polar auxin transport requires activity of PIN-type auxin carrier proteins, with their distribution at the plasma membrane significantly contributing to the directionality of auxin flow. Control of PIN protein distribution involves regulation of their endocytosis and further sorting into the lytic vacuole for degradation and recently, protein ubiquitylation has been demonstrated to control degradative sorting of plasma membrane proteins in plants.1-6 Here we show dynamic adjustments in PIN2 ubiquitylation in gravity-stimulated roots, a response that coincides with establishment of a lateral PIN2 expression gradient. Our results imply that perception and transduction of gravity signals triggers differential ubiquitylation of PIN2, which might feed back on the coordination of auxin distribution in root meristems.     

Phosphatidylinositol turnover in isolated soybean membranes stimulated by the synthetic growth hormone 2,4-dichlorophenoxyacetic acid

Phospholipids of plant membranes isolated from homogenates of dark-grown hypocotyls of soybean (Glycine max L.) undergo rapid and specific degradative changes. The degradation of phosphatidylinositol (PI) in such membranes is enhanced in the presence of the synthetic auxin, 2,4-dichlorophenoxyacetic acid (2,4-D), measured as the hydrolysis of PI or by an enhancement of [3H]inositol incorporation into membrane-associated PI stimulated by Mn2+, but not dependent upon added CTP, Mg2+, or diglyceride. The response is rapid and enhanced by auxin throughout the physiological range of growth-promoting concentrations (optimum at about 7 X 10(-7) M). The growth-inactive 2,4-D analogue, 2,3-dichlorophenoxyacetic acid (2,3-D), is without effect. These findings suggest a cell-free response of isolated membranes to the hormone mediated by a definable enzymatic reaction.     

Cytokinins in cut carnation flower III. The influence of indoleacetic acid on growth,cytokinin content and metabolism of 14C-benzyladenine in cultured ovaries

Exogenous applications of auxin to in vitro grown carnation ovaries resulted in an increase in dry mass and a decrease in the levels of endogenous cytokinins within the ovaries. Untreated ovaries showed no significant increase in dry mass. There was however, an increase in endogenous cytokinins over the same period. When ¹⁴C-BA was applied to ovaries both with and without exogenous auxin the pattern of growth and cytokinin changes followed a similar trend. Although the BA-metabolites were similar in both treatments, degradative metabolism of the cytokinin was faster and the increase in ovary dry mass greater when auxin was included in the treatment.     

Self-organization of the vascular system in plant leaves: inter-dependent dynamics of auxin flux and carrier proteins

The vegetative hormone Auxin is involved in vascular tissues formation throughout the plant. Trans-membrane carrier proteins transporting auxin from cell to cell and distributed asymmetrically around each cell give to auxin a polarized movement in tissues, creating streams of auxin that presume future vascular bundles. According to the canalization hypothesis, auxin transport ability of cells is thought to increase with auxin flux, resulting in the self-enhancement of this flux along auxin paths. In this study we evaluate a series of models based on canalization hypothesis using carrier proteins, under different assumptions concerning auxin flux formation and carrier protein dynamics. Simulations are run on a hexagonal lattice with uniform auxin production. A single cell located in the margin of the lattice indicates the petiole, and acts as an auxin sink. The main results are: (1) We obtain branching auxin distribution patterns. (2) The type of self-enhancement described by the functional form of the carrier proteins regulation responding to the auxin flux intensity in different parts of a cell, has a strong effect on the possibility of generating the branching patterns. For response functions with acceleration in the increase of carrier protein numbers compared to the auxin flux, branching patterns are likely to be generated. For linear or decelerating response functions, no branching patterns are formed. (3) When branching patterns are formed, auxin distribution greatly differs between the case in which the number of carrier proteins in different parts of a cell are regulated independently, and the case in which different parts of a cell compete for a limited number of carrier proteins. In the former case, the auxin level is lower in veins than in the surrounding tissue, while in the latter, the auxin is present in greater abundance in veins. These results suggest that canalization is a good candidate for describing plant vein pattern formation.     

The auxin-binding protein 1 is essential for the control of cell cycle

The phytohormone auxin has been known for >50 years to be required for entry into the cell cycle. Despite the critical effects exerted by auxin on the control of cell division, the molecular mechanism by which auxin controls this pathway is poorly understood, and how auxin is perceived upstream of any change in the cell cycle is unknown. Auxin Binding Protein 1 (ABP1) is considered to be a candidate auxin receptor, triggering early modification of ion fluxes across the plasma membrane in response to auxin. ABP1 has also been proposed to mediate auxin-dependent cell expansion, and is essential for early embryonic development. We investigated whether ABP1 has a role in the cell cycle. Functional inactivation of ABP1 in the model plant cell system BY2 was achieved through cellular immunization via the conditional expression of a single-chain fragment variable (scFv). This scFv was derived from a well characterized anti-ABP1 monoclonal antibody previously shown to block the activity of the protein. We demonstrate that functional inactivation of ABP1 results in cell-cycle arrest, and provide evidence that ABP1 plays a critical role in regulation of the cell cycle by acting at both the G1/S and G2/M checkpoints. We conclude that ABP1 is essential for the auxin control of cell division and is likely to constitute the first step of the auxin-signalling pathway mediating auxin effects on the cell cycle.     

Probing the actin-auxin oscillator

The directional transport of the plant hormone auxin depends on transcellular gradients of auxin-efflux carriers that continuously cycle between plasma membrane and intracellular compartments. This cycling has been proposed to depend on actin filaments. However, the role of actin for the polarity of auxin transport has been disputed. To get insight into this question, actin bundling was induced by overexpression of the actin-binding domain of talin in tobacco BY-2 cells and in rice plants. This bundling can be reverted by addition of auxins, which allows to address the role of actin organization on the flux of auxin. In both systems, the reversion of a normal actin configuration can be restored by addition of exogenous auxins and this fully restores the respective auxin-dependent functions. These findings lead to a model of a self-referring regulatory circuit between polar auxin transport and actin organization. To further dissect the actin-auxin oscillator, we used photoactivated release of caged auxin in tobacco cells to demonstrate that auxin gradients can be manipulated at a subcellular level.Key words: actin, auxin, BY-2, caged compounds, cell division, coleoptile, rice, tobacco     

Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane

Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.     

The involvement of auxin in the ripening of climacteric fruits comes of age: the hormone plays a role of its own and has an intense interplay with ethylene in ripening peaches

Ethylene has long been regarded as the main regulator of ripening in climacteric fruits. The characterization of a few tomato mutants, unable to produce climacteric ethylene and to ripen their fruits even following treatments with exogenous ethylene, has shown that other factors also play an important role in the control of climacteric fruit ripening. In climacteric peach and tomato fruits it has been shown that, concomitant with ethylene production, increases in the amount of auxin can also be measured. In this work a genomic approach has been used in order to understand if such an auxin increase is functional to an independent role played by the hormone during ripening of the climacteric peach fruits. Besides the already known indirect activity on ripening due to its up-regulation of climacteric ethylene synthesis, it has been possible to show that auxin plays a role of its own during ripening of peaches. In fact, the hormone has shown the ability to regulate the expression of a number of different genes. Moreover, many genes involved in biosynthesis and transport and, in particular, the signalling (receptors, Auxin Response Factors and Aux/IAA) of auxin had increased expression in the mesocarp during ripening, thus strengthening the idea that this hormone is actively involved in the ripening of peaches. This study has also demonstrated the existence of an important cross-talk between auxin and ethylene, with genes in the auxin domain regulated by ethylene and genes in the ethylene domain regulated by auxin.     

Function of the ubiquitin-proteasome pathway in auxin response

The plant hormone auxin regulates many aspects of growth and development. Despite the importance of this hormone, the molecular basis for auxin action has remained elusive. Recent advances using molecular genetics in Arabidopsis have begun to elucidate the mechanisms involved in auxin signaling. These results suggest that protein degradation by the ubiquitin pathway has a central role in auxin response.     

Do Trees Grow on Money? Auxin as the Currency of the Cellular Economy

Auxin plays a role in nearly every aspect of a plant''s life. Signals from the developmental program, physiological status, and encounters with other organisms all converge on the auxin pathway. The molecular mechanisms facilitating these interactions are diverse; yet, common themes emerge. Auxin can be regulated by modulating rates of biosynthesis, conjugation, and transport, as well as sensitivity of a cell to the auxin signal. In this article, we describe some well-studied examples of auxin''s interactions with other pathways.It has often been said—sometimes in joyful wonderment, sometimes in teeth-grinding frustration—auxin does everything. Although the major molecular details of the auxin pathway are largely known, many questions remain about how this one simple signaling molecule is responsible for directing so many diverse responses. The focus of this article is to try to illustrate several themes of how auxin acts in concert with other pathways to trigger specific cellular events in time and space.One potentially useful analogy in trying to understand auxin''s complicated roles is to think of it as money. Auxin does not have much intrinsic value—it stores very little energy or raw materials. However, like paper currency, it has great symbolic value, as an easily circulated means of facilitating transactions in the dynamic economy of plant life. As with currency, the amount, form, and location of auxin affects which transactions are possible. Other factors, such as what commodities are available in a given time and place, constrain which transactions auxin can facilitate. For instance, you cannot usually buy a goldfish at a shoe store, and it is quite challenging to purchase an ice cream sundae at 8 am.Here, we describe some of the ways in which the developmental program, the abiotic environment, the circadian clock, other hormones, and other organisms modify the auxin response. Auxin can be regulated by modulating rates of biosynthesis, conjugation, and transport, as well as sensitivity of a cell to the auxin signal. In addition, the dominant pathway of biosynthesis, the composition and levels of IAA conjugates, the direction of auxin transport, and the downstream consequences of sensing auxin can all be radically altered by the cellular milieu. These context-specific networks help shape the outcome (transactions) of cellular exposure to auxin. Each of the networks impinging on the cellular response to auxin could easily fill a long article (or entire collection) on their own. For brevity''s sake, only a handful of examples will be described highlighting some of the molecular mechanisms by which other signaling networks intersect with the auxin pathway.     

The effect of auxin concentration on cytokinin stability and metabolism

The stability of [³H]zeatin riboside supplied to freshly excised tobacco pith explants was found to be inversely related to -naphthaleneacetic acid concentration in the incubation medium. At higher concentrations of -naphthaleneacetic acid greater breakdown of [³H]zeatin riboside was indicated by higher levels of degradative metabolites (adenine, adenosine and adenosine nucleotides) formed. This auxin effect on cytokinin metabolism appears to be mediated, at least in part, through cytokinin oxidase. The results of in-vitro assays carried out with partially purified enzyme from corn kernels substantiale this conclusion. These findings are discussed in relation to recent observations of auxin and cytokinin levels in crown-gall tumours with altered morphology.Abbreviations FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - IP isopentenyladenine - NAA naphthaleneacetic acid - ZR zeatin riboside     

Effect of Protein Synthesis Inhibitors, Auxin, and Gibberellic Acid on Abscission

Chloramphenicol, actinomycin D, and other inhibitors of protein synthesis promote abscission in several plant genera. Abscission is accelerated in species where an abscission layer is present, as well as in tissue where no abscission layer develops prior to abscission. The inhibitors promote abscission in species where cell division is reported to precede the separation processes as well as in tissues where no cell division is associated with the initiation of abscission. Indoleacetic acid (IAA) or auxin precursors, when applied with chloramphenicol and aclinomycin D, overcome the promotive effects of the inhibitors on abscission. These inhibitors apparently do not promote abscission through their effects on auxin precursor conversion, IAA transport, and IAA destruction in the petiole. IAA increases the incorporation of leucine-1-¹⁴C into a trichloroacetic acid precipitable fraction of the abscission zone under conditions where abscission is retarded. A low concentration of IAA which accelerates abscission, decreases incorporation of leucine into protein. Other promoters of abscission — chloramphenicol, d-aspartic acid, and gibberellic acid —also decrease the incorporation of leucine into the protein of the abscission zone. The data indicate that enzymes required for the degradative processes associated with abscission are already present in the abscission zone whereas a continuous synthesis of protein is required for the retention of the leaf.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

生长素与乙烯信号途径及其相互关系研究进展

长期的研究表明, 生长素在调节植物生长发育的各种生理活动中起关键作用, 但对它如何调控这些生理活动却缺乏系统和深入的了解。最近, 细胞核内生长素信号途径的发现为揭示其作用机制带来了曙光。乙烯参与果实成熟及植物对逆境的反应等生理活动, 其信号途径也已得到部分阐明。越来越多的证据表明, 乙烯的作用与生长素对植物生长发育的调控之间有密切的联系。该文概述了生长素与乙烯信号途径的研究进展及其相互关系, 讨论了生长素在植物三重反应中的作用; 并对生长素与乙烯相互关系研究中存在的问题及研究前景进行了探讨。     

Auxin and the developing root of Arabidopsis thaliana

The plant hormone auxin has long been known to play a crucial role in plant growth and development, but how it affects so many different processes has remained a mystery. Recent evidence from genetic and molecular studies has begun to reveal a possible mechanism for auxin action. In this article we will present an overview with specific emphasis on auxin's role in roots of Arabidopsis thaliana , focusing on cell division, elongation and differentiation.     

Effetti del trattamento In Vivo con auxlna di internodi di pisello sulla fosforilazione ossidativa dei mitocondri In Vitro

Abstract

EFFECT OF TREATMENT WITH IAA OF PEA INTERNODE SECTIONS ON THE OXIDATIVE PHOSPHORYLATION OF THEIR MITOCHONDRIA. — It was previously shown that auxin treatment raises the level of ATP in pea stem sections, under the conditions where the hormone stimulates growth (MARRé AND FORTI). It is also known that under the same conditions auxin stimulates oxygen uptake (MARRé, FORTI e ARRIGONI; MARRÉ and FORTI), and that the auxin induced respiration is most probably mediated by cytochrome oxidase (MARRÉ, FORTI and GAUR).

However, auxin has no effect when added to isolated mitochondria. In this paper, the effect of auxin treatment of the tissue on the activity of mitochondria isolated after the hormone treatment has been studied. It has been found that oxidative phosphorylation of mitochondria from the auxin treated pea stem sections is 13% higher than that of controi sections. The auxin effect is significant at 96% probabilities. There is no effect of the hormone on the P/O ratio.