Gravitropism in Higher Plant Shoots : VI. Changing Sensitivity to Auxin in Gravistimulated Soybean Hypocotyls

Although the Cholodny-Went model of auxin redistribution has been used to explain the transduction phase of gravitropism for over 60 years, problems are apparent, especially with dicot stems. An alternative to an auxin gradient is a physiological gradient in which lower tissues of a horizontal stem become more sensitive than upper tissues to auxin already present. Changes in tissue sensitivity to auxin were tested by immersing marked Glycine max Merrill (soybean) hypocotyl sections in buffered auxin solutions (0, 10⁻⁸ to 10⁻² molar indoleacetic acid) and observing bending and growth of upper and lower surfaces. The two surfaces of horizontal hypocotyl sections responded differently to the same applied auxin stimulus; hypocotyls bent up (lower half grew more) in buffer alone or in low auxin levels, but bent down (upper half grew more) in high auxin. Dose-response curves were evaluated with Michaelis-Menten kinetics, with auxin-receptor binding analogous to enzyme-substrate binding. V_max for the lower half was usually greater than that for the upper half, which could indicate more binding sites in the lower half. K_m of the upper half was always greater than that of the lower half (unmeasurably low), which could indicate that upper-half binding sites had a much lower affinity for auxin than lower-half sites. Dose-response curves were also obtained for sections `scrubbed' (cuticle abraded) on top or bottom before immersion in auxin, and `gravitropic memory' experiments of L. Brauner and A. Hagar (1958 Planta 51: 115-147) were duplicated. [1-¹⁴C]Indoleacetic acid penetration was equal into the two halves, and endogenous plus exogenously supplied (not radiolabeled) free auxin in the two halves (by gas chromatography-selected ion monitoring-mass spectrometry) was also equal. Thus, differential growth occurred without free auxin redistribution, contrary to Cholodny-Went but in agreement with a sensitivity model.     

Gravitropism in Higher Plant Shoots: I. A ROLE FOR ETHYLENE

It has long been known that applied ethylene can redirect the gravitropic response, but only occasionally has it been suggested that ethylene normally plays a role in gravitropism. Two inhibitors of ethylene synthesis [Co²⁺ and aminoethoxyvinylglycine (AVG)] and two inhibitors of ethylene action (Ag⁺ and CO₂) were shown to delay the gravitropic response of cocklebur (Xanthium strumarium L.), tomato (Lycopersicon esculentum Mill.), and castor bean (Ricinus communis L.) stems. Gentle shaking on a mechanical shaker does not inhibit the gravitropic response, but vigorous hand shaking for 120 seconds delays the response somewhat. AVG and Ag⁺ further delay the response of mechanically stimulated plants. AVG delays the response of defoliated and of decapitated plants. Plants laid on their side and restricted so that they cannot bend upward store both bending energy and gravitropic stimulus; they bend immediately when released from restriction (stored energy) and continue to bend for some hours after (stored stimulus). AVG retards the storage of bending energy but not of stimulus. In gravitropism, graviperception may first stimulate ethylene evolution, which may then influence bending directly, or responses involving ethylene could be more indirect.     

Gravitropism in Higher Plant Shoots : IV. Further Studies on Participation of Ethylene

Ethylene at 1.0 and 10.0 cubic centimeters per cubic meter decreased the rate of gravitropic bending in stems of cocklebur (Xanthium strumarium L.) and tomato (Lycopersicon esculentum Mill), but 0.1 cubic centimeter per cubic meter ethylene had little effect. Treating cocklebur plants with 1.0 millimolar aminoethoxyvinylglycine (AVG) (ethylene synthesis inhibitor) delayed stem bending compared with controls, but adding 0.1 cubic centimeter per cubic meter ethylene in the surrounding atmosphere (or applying 0.1% ethephon solution) partially restored the rate of bending of AVG-treated plants. Ethylene increases in bending stems, and AVG inhibits this. Virtually all newly synthesized ethylene appeared in bottom halves of horizontal stems, where ethylene concentrations were as much as 100 times those in upright stems or in top halves of horizontal stems. This was especially true when horizontal stems were physically restrained from bending. Ethylene might promote cell elongation in bottom tissues of a horizontal stem or indicate other factors there (e.g. a large amount of `functioning' auxin). Or top and bottom tissues may become differentially sensitive to ethylene. Auxin applied to one side of a vertical stem caused extreme bending away from that side; gibberellic acid, kinetin, and abscisic acid were without effect. Acidic ethephon solutions applied to one side of young seedlings of cocklebur, tomato, sunflower (Helianthus annuus L.), and soybean (Glycine max [L.] Merr.) caused bending away from that side, but neutral ethephon solutions did not cause bending. Buffered or unbuffered acid (HCl) caused similar bending. Neutral ethephon solutions produced typical ethylene symptoms (i.e. epinasty, inhibition of stem elongation). HCl or acidic ethephon applied to the top of horizontal stems caused downward bending, but these substances applied to the bottom of such stems inhibited growth and upward bending—an unexpected result.     

Gravitropism in Higher Plant Shoots : II. Dimensional and Pressure Changes during Stem Bending

Dimensional changes during gravitropic bending of cocklebur (Xanthium strumarium L.) dicot stems were measured using techniques of stereo photogrammetry. The differential growth is from an increased growth rate on the bottom of the stem and a stopping or contraction of the top.

Contraction of the top was especially evident upon release and immediate bending of horizontal stems that had been restrained between stiff wires for 36 hours. The energy for this could have been stored in both the top and bottom, since the bottom elongated, and the top contracted.

Forces developed during bending were measured by fastening a stem tip to the end of a bar with attached strain gauges and recording electrical output from the strain gauges. Restrained mature cocklebur stems continued to accumulate potential energy for bending for about 120 hours, after which the recorded force reached a maximum.

Pressures within castor bean (Ricinus communis L.) stems were also measured with 3.5-millimeter diameter pressure transducers. As expected, the pressure on the bottom of the restrained plants increased with time; pressures decreased in vertical controls, tops of restrained stems, and bottoms of free-bending stems. Pressures increased in tops of free-bending stems. When restrained plants were released, pressure on the bottom decreased and pressure on the top increased. Results suggest a possible role for cell contraction in the top of stems bending upward in response to gravity.

     

Gravitropism in Higher Plant Shoots : III. Cell Dimensions during Gravitropic Bending; Perception of Gravity

Cross and longitudinal sections were prepared for light microscopy from vertical control plants (Xanthium strumarium L. Chicago strain), free-bending horizontal stems, plants restrained 48 hours in a horizontal position, and plants restrained 48 hours and then released, bending immediately about 130°. Top cells of free-bending stems shrink or elongate little; bottom cells continue to elongate. In restrained stems, bottom cells elongate some and increase in diameter; top cells elongate about as much but decrease in diameter. Upon release, bottom cells elongate more and decrease in diameter, while top cells shorten and increase in diameter, accounting for the bend. During restraint, bottom cells take up water while tissue pressures increase; top cells fail to take up water although tissue pressures are decreasing.

Settling of amyloplasts was observed in cells of the starch sheath.

Removal of different amounts of stem (Xanthium; Lycopersicon esculentum Miller, cv Bonny Best; Ricinus communis L. cv Yolo Wonder) showed that perception of gravity occurs in the bending (elongation) zone, although bending of fourth and fifth internodes from the top was less than in uncut controls. Uniform application of 1% indoleacetic acid in lanolin to cut stem surfaces partially restored bending. Reversing the gradient in tension/compression in horizontal stems (top under compression, bottom under tension) did not affect gravitropic bending.

     

The Geoelectric Effect in Plant Shoots: V. A DISCUSSION

In earlier papers Woodcock and Wilkins, and Hertz and Grahmreported measurements of the geoelectric effect (GEE) in Zeamays coleoptiles. While Woodcock and Wilkins attached contactelectrodes to the coleoptile directly, Grahm and Hertz employeda vibrating-electrode technique that avoided touching the plant. The results obtained by these two methods agree on all pointsexcept the magnitude of the GEE, where a difference of morethan 100 per cent is reported. In this paper this differenceis investigated and explained by certain properties characteristicof the measuring techniques used as well as by differences inexperimental procedure.     

Gravitropism: Interaction of Sensitivity Modulation and Effector Redistribution

Our increasing capabilities for quantitative hormone analysis and automated high resolution growth studies have allowed a reassessment of the classical Cholodny-Went hypothesis of gravitropism. According to this hypothesis, gravity induces redistribution of auxin toward the lower side of the organ and this causes the growth asymmetry that leads to reorientation. Arguments against the Cholodny-Went hypothesis that were based primarily on concerns over the timing and magnitude of the development of hormone asymmetry are countered by recent evidence that such asymmetry develops early and is sufficiently large to account for curvature. Thus, it appears that the Cholodny-Went hypothesis is fundamentally valid. However, recent comparative studies of the kinetics of curvature and the timing of the development of hormone asymmetry indicate that this hypothesis alone cannot account for the intricacies of the gravitropic response. It appears that time-dependent gravity-induced changes in hormone sensitivity as well as changes in sensitivity of the gravity receptor play important roles in the response.     

Evidence for a Relationship between H Excretion and Auxin in Shoot Gravitropism

The role of auxin and protons in the gravitropic response of the sunflower (Helianthus annuus L. cv Sungold) hypocotyl has been investigated. No physiological asymmetry in acid-growth capacity could be detected between the upper and lower surfaces of gravistimulated hypocotyls. These data imply that neutral buffers inhibit shoot gravitropism by preventing the establishment of a lateral proton gradient along gravitropically stimulated hypocotyls. Indirect evidence that auxin is involved in the establishment and/or maintenance of such a gradient derives from the quantitative assessment of the effects of exogenous auxin, anti-auxins, and vanadate on gravicurvature. At low concentrations, exogenous auxin accelerated curvature; at high concentrations, curvature was prevented. Vanadate, an inhibitor of auxin-enhanced H⁺ secretion, α-(p-chlorophenoxy)isobutyric acid (PCIB), an anti-auxin, and 2,3,5-triiodobenzoic acid (TIBA), an auxin-transport inhibitor, prevented observable asymmetric proton excretion using a brom cresol purple agar technique and also inhibited gravicurvature. Vanadate, PCIB, and TIBA inhibition of gravicurvature could be reversed with acid treatment to the lower surface of a gravistimulated hypocotyl. Auxin treatment to the lower surface of a gravistimulated hypocotyl did not reverse vanadate-induced inhibition, but it did partially reverse PCIB- and TIBA-induced inhibition. These results indicate a close relationship between the acid-growth theory and the differential growth responses of the sunflower hypocotyl during gravitropism.     

Ethylene Interacts with Auxin in Regulating Developmental Attenuation of Gravitropism in Flax Root

Previous research shows that gravity-sensing in flax (Linum usitatissimum) root is initiated during seed imbibition and precedes root emergence. In this study we investigated the developmental attenuation of flax root gravitropism post-germination and the involvement of ethylene. Gravity response deteriorated significantly from 3 to 11?h after root emergence, which occurred at around 19?h after imbibition (that is, from “age” 22 to 30?h). Although the root elongation rate increased from 22 to 30?h, the gravitropic curving rate declined steadily. Older roots were able to tolerate higher levels of exogenous IAA before inhibition of elongation and gravitropism occurred. The age-dependent effect of IAA on root growth and gravitropism suggests that young roots are more sensitive to auxin and respond to a smaller vertical auxin gradient than older roots upon horizontal gravistimulation. The ethylene synthesis inhibitor AVG (2-aminoethoxyvinyl glycine, 10?μM) or ethylene action inhibitor Ag⁺ (10?μM) stimulated gravitropic curvature of 30?h roots by 24 and 32%, respectively, but had no effect on 22?h roots, suggesting that as roots age, ethylene begins to play a role in root gravitropism. The auxin transport inhibitor NPA (N-naphthylphthalamic acid, 50?μM) reduced gravitropic curvature of 30?h roots by 24% but had no effect on 22?h roots. On the other hand, treating roots simultaneously with the auxin transport inhibitor and ethylene synthesis or action inhibitor stimulated gravitropic curvature of 30?h roots but not 22?h roots. Taken together, these data indicate that as roots develop, their weakened gravity response is due to decreased auxin sensitivity and possibly auxin transport regulated by ethylene.     

Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots

In 1933 Thimann and Skoog demonstrated exogenous auxin repressionof lateral bud outgrowth in decapitated shoots ofVicia faba. This evidence has given strong support for a role of auxinin apical dominance. Most, but not all, investigators have confirmedThimann and Skoog's results. In the present study, auxin treatmentswere carried out on ten different species or plant types, manyof which were treated with auxin in different forms, media andunder different light conditions. The Thimann–Skoog experimentdid work for most species (i.e. exogenous auxin did repressbud outgrowth) including thedgt tomato mutant which is knownto be insensitive to auxin in certain responses. Toxic auxinsymptoms were observed in some but not all species. The Thimann–Skoogexperiment did not work for greenhouse-grownColeus or forArabidopsis. Light was shown to reduce apical dominance inColeus andIpomoeanil . apical dominance; lateral bud outgrowth; axillary bud; auxin; IAA; decapitation; Vicia faba ; Ipomoea nil ; Pisum sativum ; Phaseolus vulgaris ; Lycopersion exculentum ; dgt ; Coleus blumei ; Arabidopsis thaliana ; Helianthus annuus ; Thimann–Skoog     

The Rate of Transport of Natural Auxin in Woody Shoots

A method is described for the estimation of the rate of movementand the quantity transported of the natural growth hormone instandard isolated segments of apple shoots. During controlledstorage diffusible auxin is collected, and later by dividingthe standard length of stem into small sections the locationof the auxin front is determined, from which the rate of transportis deduced. Temperature markedly affects both rate of transportand amount of auxin transported (cf. van der Weij, 1932), amaximum occurring at 27–30° C.; followed by a rapidfall to zero. The total diffusible auxin in a given length ofstem is not affected by storage temperatures below 30° C.but falls to zero at 42° C. The rate of transport and amounttransported are proportional to the oxygen tension over therange 0 to 5 per cent. O₂, and there is some evidence for destructionof auxin in tensions below 2 per cent.     

Auxin Relations of the Woody Shoot: The Distribution of Diffusible Auxin in Shoots of Apple and Plum Rootstock Varieties

Surveys have been made of diffusible auxin in the stem tissuesof growing shoots of apple and plum rootstock varieties. Usingagar plates as carriers auxin was collected from the lower surfaceof isolated stem sections and assayed by the Avena curvaturemethod. The stool and layer shoots studied grew for severalmonths producing many leaves and reaching considerable lengths.The data provide information on selected internodes and showthe auxin status of the shoot at various times during growth,and the auxin gradients down the stem at these various times.Free auxin content of the shoot apex was consistently less thanthat of the internodes below. In 1946 auxin content declinedthroughout growth with a steady positioning of the auxin peakin the upper shoot. In 1947, following a period of drought,when growth almost ceased, a secondary auxin peak occurred positionedin lower internodes distant from the apex. This seasonal contrastwas reflected in the auxin relations of the individual internode,and was observed both in apple and plum. The nature of the auxindecline below the peak region, and the total disappearance offree auxin from the shoot as growth subsides, is discussed.The reappearance of free auxin in mature internodes, which hasnot been transmitted from the stem apex, implies either derivationfrom a stored state or the ability of the internode to produceits own auxin.     

An Auxin Transport Inhibitor Interferes With Unicellular Gravitropism in Protonemata of the Moss Ceratodon purpureus

Abstract: Gravitropism of the protonemata of the moss Ceratodon purpureus (Hedw.) Brid. was studied after treatment with auxin transport inhibitors and auxin-related substances. The phytotropins NPA (naphthylphthalamic acid) and PBA (pyrenoylbenzoic acid), known to block auxin efflux in higher plants, strongly inhibited gravitropic curvature of the apical protonemal cell. At 3 μM NPA or PBA, approximately 60 % inhibition of curvature was observed; growth rates were less affected. Tyrphostin A47, a known antagonist of NPA effects in higher plants, released the inhibition of moss protonemal gravitropism and restored the full curvature response. Exogenous IAA, even at high concentrations (40 μM), did not interfere with protonemal gravitropism. To account for the results, modified hypotheses for auxin transport and action are discussed.     

植物生长素反应因子研究进展

生长素反应因子（ARFs）是植物生长和发育的重要调节因子,在生长素早期反应蛋白（Aux／IAAs）的参与下,通过和生长素反应基因启动子区AuxRE元件的JTGTCTC序列结合,共同调控这些基因的表达。近年来关于生长素反应因子的分子结构和ARF与Aux／IAA的相互作用及其对植物生长和发育的影响、作用的靶基因以及分子机制受到人们的重视,并在这些方面做了大量的研究。     

The Plant Oncogene rolB Alters Binding of Auxin to Plant Cell Membranes

We measured auxin-binding capacity of the membrane preparationsfrom tobacco cells transformed by rolB as compared to untransformedcontrols. In the transformed cells, the overall auxin-bindingactivity is severalfold enhanced through an increase in a bindingactivity removable from the membranes at 0.5 M salt, while thebinding activity still attached to the membranes after saltwashes remains unchanged. Antibodies against the 22 kDa maizeauxin binding protein (ABP) depress most of the membrane-attachedbinding activity in both normal and rolB-transformed cells,while they do not affect the salt-washable binding activity.In contrast, antibodies against the RolB protein prevent completelybinding of auxin to the latter activity in both normal and transformedcells, while substantially unaffecting the membrane-associatedbinding. These results point to the presence, in untransformedmembranes, of an auxin-binding activity associated with a proteinimmunologically related to RolB. This activity is much increasedin rolB cells. In contrast, the auxin-binding protein analogousto maize ABP present in tobacco membranes does not increasein the rolB-transformed cells. (Received October 1, 1993; Accepted April 22, 1993)     

PINOID Kinase Regulates Root Gravitropism through Modulation of PIN2-Dependent Basipetal Auxin Transport in Arabidopsis

Reversible protein phosphorylation is a key regulatory mechanism governing polar auxin transport. We characterized the auxin transport and gravitropic phenotypes of the pinoid-9 (pid-9) mutant of Arabidopsis (Arabidopsis thaliana) and tested the hypothesis that phosphorylation mediated by PID kinase and dephosphorylation regulated by the ROOTS CURL IN NAPHTHYLPHTHALAMIC ACID1 (RCN1) protein might antagonistically regulate root auxin transport and gravity response. Basipetal indole-3-acetic acid transport and gravitropism are reduced in pid-9 seedlings, while acropetal transport and lateral root development are unchanged. Treatment of wild-type seedlings with the protein kinase inhibitor staurosporine phenocopies the reduced auxin transport and gravity response of pid-9, while pid-9 is resistant to inhibition by staurosporine. Staurosporine and the phosphatase inhibitor, cantharidin, delay the asymmetric expression of DR5∷revGFP (green fluorescent protein) at the root tip after gravistimulation. Gravity response defects of rcn1 and pid-9 are partially rescued by treatment with staurosporine and cantharidin, respectively. The pid-9 rcn1 double mutant has a more rapid gravitropic response than rcn1. These data are consistent with a reciprocal regulation of gravitropism by RCN1 and PID. Furthermore, the effect of staurosporine is lost in pinformed2 (pin2). Our data suggest that reduced PID kinase function inhibits gravitropism and basipetal indole-3-acetic acid transport. However, in contrast to PID overexpression studies, we observed wild-type asymmetric membrane distribution of the PIN2 protein in both pid-9 and wild-type root tips, although PIN2 accumulates in endomembrane structures in pid-9 roots. Similarly, staurosporine-treated plants expressing a PIN2∷GFP fusion exhibit endomembrane accumulation of PIN2∷GFP, but no changes in membrane asymmetries were detected. Our data suggest that PID plays a limited role in root development; loss of PID activity alters auxin transport and gravitropism without causing an obvious change in cellular polarity.A variety of important growth and developmental processes, including gravity response, embryo and vascular development, and the branching of roots and shoots, are controlled by the directional and regulated transport of auxin in higher plants. Reversible protein phosphorylation is an important regulatory strategy that may modulate auxin transport and dependent processes such as root gravitropism, perhaps through action of the PINOID (PID) kinase (for review, see DeLong et al., 2002; Galvan-Ampudia and Offringa, 2007). PID is an AGC family Ser/Thr kinase (Christensen et al., 2000) and belongs to an AGC kinase clade containing WAG1, WAG2, AGC3-4, and D6PK/AGC1-1 (Santner and Watson, 2006; Galvan-Ampudia and Offringa, 2007; Zourelidou et al., 2009). PID activity has been demonstrated in vitro and in vivo (Christensen et al., 2000; Michniewicz et al., 2007), and several pid mutant alleles exhibit altered auxin transport in the inflorescence and a floral development defect resembling that of auxin transport mutants (Bennett et al., 1995). Overexpression of the PID gene results in profound alterations in root development and responses to auxin transport inhibitors, reduced gravitropism and auxin accumulation at the root tip (Christensen et al., 2000; Benjamins et al., 2001; Michniewicz et al., 2007), as well as enhanced indole-3-acetic acid (IAA) efflux in tobacco (Nicotiana tabacum) cell cultures (Lee and Cho, 2006) and altered PINFORMED1 (PIN1), PIN2, and PIN4 localization patterns (Friml et al., 2004; Michniewicz et al., 2007), consistent with PID being a positive regulator of IAA efflux. However, the effects of pid loss-of-function mutations on auxin transport activities and gravitropic responses in roots have not yet been reported (Robert and Offringa, 2008).In contrast, auxin transport and gravitropism defects of a mutant with reduced protein phosphatase activity have been characterized in detail. The roots curl in naphthylphthalamic acid1 (rcn1) mutation, which ablates the function of a protein phosphatase 2A regulatory subunit, causes reduced PP2A activity in vivo and in vitro (Deruère et al., 1999). Roots and hypocotyls of rcn1 seedlings have elevated basipetal auxin transport (Deruère et al., 1999; Rashotte et al., 2001; Muday et al., 2006), and rcn1 roots exhibit a significant delay in gravitropism, consistent with altered auxin transport (Rashotte et al., 2001; Shin et al., 2005). These data indicate that PP2A is a negative regulator of basipetal transport and suggest that if PID-dependent phosphorylation regulates root auxin transport and gravitropism, then it may act in opposition to PP2A-dependent dephosphorylation.In roots, auxin transport is complex, with distinct sets of influx and efflux carriers that define tissue-specific and opposing directional polarities (for review, see Leyser, 2006). IAA moves acropetally, from the shoot toward the root apex, through the central cylinder (Tsurumi and Ohwaki, 1978), and basipetally, from the root apex toward the base, through the outer layer of cells (for review, see Muday and DeLong, 2001). When plants are reoriented relative to the gravity vector, auxin becomes asymmetrically distributed across the root tip, as a result of a process termed lateral auxin transport (for review, see Muday and Rahman, 2008). Several carriers that mediate root basipetal IAA transport have been clearly defined and include the influx carrier AUXIN-INSENSITIVE1 (AUX1; Marchant et al., 1999; Swarup et al., 2004; Yang et al., 2006) and efflux carriers of two classes, PIN2 (Chen et al., 1998; Müller et al., 1998; Rashotte et al., 2000) and ATP-BINDING CASSETTE TYPE B TRANSPORTER4/MULTIDRUG-RESISTANT4/P-GLYCOPROTEIN4 (ABCB4/MDR4/PGP4; Geisler et al., 2005; Terasaka et al., 2005; Lewis et al., 2007). Lateral transport at the root tip may be mediated by PIN3, an efflux carrier with a gravity-dependent localization pattern (Friml et al., 2002; Harrison and Masson, 2007).Gravitropic curvature of Arabidopsis (Arabidopsis thaliana) roots requires changes in IAA transport at the root tip (for review, see Muday and Rahman, 2008). Auxin transport inhibitors (Rashotte et al., 2000) and mutations in genes encoding basipetal transporters, including aux1 (Bennett et al., 1996), pin2/agr1 (Chen et al., 1998; Müller et al., 1998), and abcb4/mdr4/pgp4 (Lin and Wang, 2005; Lewis et al., 2007), alter gravitropism. Auxin-inducible reporters exhibit asymmetric expression across the root tip prior to differential growth, and this asymmetry is abolished by treatment with auxin transport inhibitors that prevent gravitropic curvature (Rashotte et al., 2001; Ottenschläger et al., 2003). Additionally, the pin3 mutant exhibits slightly reduced rates of gravitropic curvature (Harrison and Masson, 2007), and PIN3 is expressed in the columella cells, which are the site of gravity perception (Blancaflor et al., 1998; Friml et al., 2002). The PIN3 protein relocates to membranes on the lower side of columella cells after gravitropic reorientation, consistent with a role in facilitating asymmetric IAA transport at the root tip (Friml et al., 2002; Harrison and Masson, 2007).The available data suggest a model in which PID and RCN1 antagonistically regulate basipetal transport and gravitropic response in root tips (Fig. 1). In this model, the regions with the highest IAA concentrations in the epidermal and cortical cell layers are indicated by shading, and the arrows indicate the direction and relative amounts of basipetal auxin transport. Our previous work suggests that elevated basipetal IAA transport in rcn1 roots impairs gravitropic response, presumably due to the inability of roots either to form or to perceive a lateral auxin gradient in the context of a stronger polar IAA transport stream (Rashotte et al., 2001). Enhanced basipetal transport may increase the initial auxin concentration along the upper side of the root, impeding the establishment or perception of a gradient in rcn1 and cantharidin-treated wild-type roots (Fig. 1, right). Based on the published pid inflorescence transport data (Bennett et al., 1995), we hypothesize that pid seedling roots and staurosporine-treated wild-type roots have reduced basipetal auxin transport (Fig. 1, left). Upon reorientation of roots relative to the gravity vector, the reduced basipetal IAA transport in pid may lead to slower establishment of an auxin gradient across the root. This model then predicts that cantharidin treatment of pid-9 or staurosporine treatment of rcn1 seedlings would enhance or restore gravitropism in these mutants. Similarly, a double mutant might be expected to exhibit a corrected gravitropic response relative to the single mutants.Open in a separate windowFigure 1.Auxin transport defects in pid-9 and rcn1 mutants alter auxin redistribution after reorientation relative to the gravity vector. This model predicts that differences in basipetal auxin transport activities of wild-type, pid-9, and rcn1 roots will affect the formation of lateral auxin gradients. The shaded area in each root represents the region of highest IAA concentration in epidermal and cortical cells, with darker shading in the central columella cells, believed to be the auxin maxima. The direction and amount of basipetal IAA transport are indicated by arrows. The region of differential growth during gravitropic bending is indicated by the shaded rectangle. If auxin transport is reduced (as shown in the pid-9 mutant or in staurosporine-treated seedlings), this would lead to a slower formation of an auxin gradient in root tips. The rcn1 mutation (or treatment with cantharidin) has already been shown to lead to increased basipetal transport and a reduced rate of gravitropic bending, consistent with altered formation or perception of an auxin gradient. The antagonistic effects of kinase and phosphatase inhibition are predicted to lead to normal gravity responses in the pid-9 rcn1 double mutant as well as in pid-9 and rcn1 single mutants treated with the “reciprocal” inhibitor.The experiments described here were designed to test this model by examining gravitropism and root basipetal IAA transport in pid and staurosporine-treated seedlings. We investigated the regulation of gravity response by PID kinase and RCN1-dependent PP2A activities and observed antagonistic interactions between the rcn1 and pid-9 loss-of-function phenotypes that are consistent with reciprocal kinase/phosphatase regulation. We found that loss of kinase activity in the pid mutant and in staurosporine-treated wild-type plants inhibits basipetal auxin transport and the dependent physiological process of root gravitropism. Our results suggest that staurosporine acts to regulate these processes through inhibition of PID kinase and that PID effects are PIN2 dependent. In both wild-type and pid-9 roots, we observed polar membrane distribution of the PIN2 protein; unlike wild-type roots, though, pid-9 roots exhibited modest accumulation of PIN2 in endomembrane structures. Similarly, we detected asymmetric distribution and endomembrane accumulation of PIN2∷GFP in staurosporine-treated roots. Our data suggest that PID plays a limited role in root development; loss of PID activity alters PIN2 trafficking, auxin transport, and gravitropism without causing an obvious loss of cellular polarity. Together, these experiments provide insight into phosphorylation-mediated control of the gravity response and auxin transport in Arabidopsis roots.     

The Geoelectric Effect in Plant Shoots: I. THE CHARACTERISTICS OF THE EFFECT

A comparative study has been made of the geoelectric effectin Helianthus hypocotyls and Zea coleoptiles using two electrodesystems. With the static-drop electrode system a potential differencedeveloped between the upper and lower surfaces of the shootsimmediately after they were turned into the horizontal position.The lower surface of the shoot became positively charged withrespect to the upper surface. In non-decapitated shoots thispotential difference continued to increase for at least 20 min,whereas in decapitated shoots no further increase occurred afterabout 10 min from the moment of reorientation. In contrast,with a flowing-solution electrode system no potential differencedeveloped in non-decapitated shoots until about 12 min afterthey were placed in the horizontal position. Thereafter thelower surface became increasingly positively charged with respectto the upper surface for at least a further 12 min. In decapitatedshoots there was no tendency whatsoever for the lower surfaceof the horizontal shoot to become positively charged with respectto the upper surface, even after 25 min in the horizontal position.The static-drop electrode system has an inherent sensitivityto reorientation in a gravitational field; a potential differencedevelops immediately after reorientation, and increases to amaximum value within the first 10 min of reorientation, regardlessof whether or not the electrodes are in contact with plant tissue.The continued increase in the potential difference measuredacross the shoots with the static-drop electrode system, andthe entire development of the potential difference measuredwith the flowing-solution electrode system, are both dependentupon the shoot apex being intact. These facts have enabled usto show that the electro-potential difference measured acrosshorizontally placed non-decapitated tissues with the static-dropelectrode system is the resultant of two distinct processes:(a) an immediate, purely physical electrical effect generatedin the electrodes themselves, and (b) a delayed geoelectriceffect which arises solely in the living tissues of the shootand which is dependent upon the apex of the shoot being intact.