Hydrotropism and its interaction with gravitropism in roots

We have studied hydrotropism and its interaction with gravitropism in agravitropic roots of a pea mutant and normal roots of peas (Pisum sativum L.) and maize (Zea mays L.). The interaction between hydrotropism and gravitropism in normal roots of peas or maize were also examined by nullifying the gravitropic response on a clinostat and by changing the stimulus-angle for gravistimulation. Depending on the intensity of both hydrostimulation and gravistimulation, hydrotropism and gravitropism of seedling roots strongly interact with one another. When the gravitropic response was reduced, either genetically or physiologically, the hydrotropic response of roots became more unequivocal. Also, roots more sensitive to gravity appear to require a greater moisture gradient for the induction of hydrotropism. Positive hydrotropism of roots occurred due to a differential growth in the elongation zone; the elongation was much more inhibited on the moistened side than on the dry side of the roots. It was suggested that the site of sensory perception for hydrotropism resides in the root cap, as does the sensory site for gravitropism. Furthermore, an auxin inhibitor, 2,3,5-triiodobenzoic acid (TIBA), and a calcium chelator, ethyleneglycol-bis-(-aminoethylether)-N,N,N,N- tetraacetic acid (EGTA), inhibited both hydrotropism and gravitropism in roots. These results suggest that the two tropisms share a common mechanism in the signal transduction step.     

Phototropism and gravitropism in lateral roots of Arabidopsis

Gravitropism and, to a lesser extent, phototropism have been characterized in primary roots, but little is known about structural/functional aspects of these tropisms in lateral roots. Therefore, in this study, we report on tropistic responses in lateral roots of Arabidopsis thaliana. Lateral roots initially are plagiogravitropic, but when they reach a length of approximately 10 mm, these roots grow downward and exhibit positive orthogravitropism. Light and electron microscopic studies demonstrate a correlation between positive gravitropism and development of columella cells with large, sedimented amyloplasts in wild-type plants. Lateral roots display negative phototropism in response to white and blue light and positive phototropism in response to red light. As is the case with primary roots, the photoresponse is weak relative to the graviresponse, but phototropism is readily apparent in starchless mutant plants, which are impaired in gravitropism. To our knowledge, this is the first report of phototropism of lateral roots in any plant species.     

Basipetal auxin transport is required for gravitropism in roots of Arabidopsis

Auxin transport has been reported to occur in two distinct polarities, acropetally and basipetally, in two different root tissues. The goals of this study were to determine whether both polarities of indole-3-acetic acid (IAA) transport occur in roots of Arabidopsis and to determine which polarity controls the gravity response. Global application of the auxin transport inhibitor naphthylphthalamic acid (NPA) to roots blocked the gravity response, root waving, and root elongation. Immediately after the application of NPA, the root gravity response was completely blocked, as measured by an automated video digitizer. Basipetal [(3)H]IAA transport in Arabidopsis roots was inhibited by NPA, whereas the movement of [(14)C]benzoic acid was not affected. Inhibition of basipetal IAA transport by local application of NPA blocked the gravity response. Inhibition of acropetal IAA transport by application of NPA at the root-shoot junction only partially reduced the gravity response at high NPA concentrations. Excised root tips, which do not receive auxin from the shoot, exhibited a normal response to gravity. The Arabidopsis mutant eir1, which has agravitropic roots, exhibited reduced basipetal IAA transport but wild-type levels of acropetal IAA transport. These results support the hypothesis that basipetally transported IAA controls root gravitropism in Arabidopsis.     

Behavior of amyloplasts in photo- and gravitropism of the moss protonema

The protonema of mosses Ceratodon purpureus and Pottia intermedia is negatively gravitropic in darkness and grows on the substrate surface under illumination. However, the putative mechanisms of these growth responses are not well understood so far. For gravitropism, sedimentation of amyloplasts has been widely assumed to be the first step of the signal transduction chain. This model was supported by numerous observations where amyloplasts' number or size in a protonema apical cell correlated with its gravisensitivity. Unlike multicellular graviperceptive organs, a protonema apical cell is the same site for both of gravity and light perception and realization of growth movements. In addition, red light is known to change the cell responses to gravity. Therefore, we analysed the influence of red light on the events associated with graviperception and growth movements of protonema apical cells, namely: plastid behavior, size, and number, starch content, chlorophyll fluorescence intensity and alpha-amylase activity, under gravistimulation of dark-grown protonema.     

Cell dynamics studies on the pericycle of radish seedling roots

Pericycle cells of the radish seedling root were found arrested in G2 of the cell cycle, and could be stimulated by auxin into rapid cell cycling, leading to the formation of lateral root primordia.     

Colonisation of seedling roots by 2,4-D degrading bacteria: A plant-microbial model

The development of model plant-microbial associations between Gram negative soil microbes capable of degrading phenoxyacetate herbicides, such as 2,4-D and 2,4-D methyl ester, and the crops canola and wheat was described. Both an Acinetobacter baumannii pJP4 transconjugant and Alcaligenes eutrophus JMP 134 colonised non-parasitically on the roots of sterilised seedlings in a hydroponic system. Laser scanning confocal microscopy has shown that colonisation occurred both on the root surface and deeper inside the mucilage layer or inside some surface root cells. When 2,4-D was added to the hydroponic medium supporting the growth of those seedlings colonised by 2,4-D degrading bacteria, the gas chromatographic analysis showed a rapid decrease in the concentration of this herbicide. These bacteria colonising the root system were shown to be responsible for the degradation of 2,4-D. Plants inoculated with the 2,4-D degrading microbes were subsequently found to be less susceptible to damage by the herbicide in such hydroponic systems.     

Micromanipulation of amyloplasts with optical tweezers in Arabidopsis stems

Intracellular sedimentation of highly dense, starch-filled amyloplasts toward the gravity vector is likely a key initial step for gravity sensing in plants. However, recent live-cell imaging technology revealed that most amyloplasts continuously exhibit dynamic, saltatory movements in the endodermal cells of Arabidopsis stems. These complicated movements led to questions about what type of amyloplast movement triggers gravity sensing. Here we show that a confocal microscope equipped with optical tweezers can be a powerful tool to trap and manipulate amyloplasts noninvasively, while simultaneously observing cellular responses such as vacuolar dynamics in living cells. A near-infrared (λ=1064 nm) laser that was focused into the endodermal cells at 1 mW of laser power attracted and captured amyloplasts at the laser focus. The optical force exerted on the amyloplasts was theoretically estimated to be up to 1 pN. Interestingly, endosomes and trans-Golgi network were trapped at 30 mW but not at 1 mW, which is probably due to lower refractive indices of these organelles than that of the amyloplasts. Because amyloplasts are in close proximity to vacuolar membranes in endodermal cells, their physical interaction could be visualized in real time. The vacuolar membranes drastically stretched and deformed in response to the manipulated movements of amyloplasts by optical tweezers. Our new method provides deep insights into the biophysical properties of plant organelles in vivo and opens a new avenue for studying gravity-sensing mechanisms in plants.     

Reduced gravitropism in inflorescence stems and hypocotyls, but not roots, of Arabidopsis mutants with large plastids

The sites of gravity perception are columella cells in roots and endodermal cells in hypocotyls and inflorescence stems. Since plastids are likely to play a role in graviperception, we investigated gravitropism in plastid mutants of Arabidopsis . Previous studies have shown that the arc 6 and arc 12 ( a ccumulation and r eplication of c hloroplasts) mutants have an average of two large plastids per leaf mesophyll cell. In this study, we found that these arc mutants have altered plastid morphology throughout the entire plant body, including the cells involved in gravity perception. There were no major differences in total starch content per cell in endodermal and columella cells of the wild-type (WT) compared to arc 6 and arc 12 as assayed by iodine staining. Thus, the total mass of plastids per cell in arc 6 and arc 12 is similar to their respective WT strains. Results from time course of curvature studies demonstrated that the plastid mutation affected gravitropism only of inflorescence stems and hypocotyls, but not roots. Thus, roots appear to have different mechanisms of gravitropism compared to stems and hypocotyls. Time course of curvature studies with light-grown seedlings were performed in the presence of latrunculin B (Lat-B), an actin-depolymerizing drug. Lat-B promoted gravitropic curvature in hypocotyls of both the WT and arc 6 but had little or no effect on gravitropism in roots of both strains. These results suggest that F-actin is not required for hypocotyl gravitropism.     

Growth and gravitropism of corn roots in solution

Growth and early gravitropic responses of corn roots in solution have been studied using time-lapse photography. Aeration was required for both root growth and gravitropism. The optimum pH for gravitropism was in the range 5 to 6. The bending response seemed to be greater for roots in non-buffered solution than in buffered solution. Fastest growth and maximum curvature occurred with about 0.2 mol m⁻³ Ca²⁺. Under some conditions, the gravitropic response started with apparently negligible time delay after the start of the gravitropic stimulus. This may denote graviperception in or near the elongation zone itself. This mechanism for early but relatively weak gravitropism may help to explain a variety of gravitropic responses such as the ‘early wrong way’ curvature, and the behaviour of roots whose columella cells lack amyloplasts. More rapid bending appears to start at about 20 min, which is consistent with observations on roots in humid air and with the accepted statolith model of perception in the root cap.     

Inhibition of gravitropism in primary roots of Zea mays by chloramphenicol

Primary roots of Zea mays seedlings germinated and grown in 0.1 mM chloramphenicol (CMP) were significantly less graviresponsive than primary roots of seedlings germinated and grown in distilled water. Elongation rates of roots treated with CMP were significantly greater than those grown in distilled water. Caps of control and CMP-treated roots possessed extensive columella tissues comprised of cells containing numerous sedimented amyloplasts. These results indicate that the reduced graviresponsiveness of CMP-treated roots is not due to reduced rates of elongation, the absence of the presumed gravireceptors (i.e., amyloplasts in columella cells), or reduced amounts of columella tissue. These results are consistent with CMP altering the production and/or transport of effectors that mediate gravitropism.     

Enhanced gravitropism of roots with a disrupted cap actin cytoskeleton

The actin cytoskeleton has been proposed to be a major player in plant gravitropism. However, understanding the role of actin in this process is far from complete. To address this problem, we conducted an analysis of the effect of Latrunculin B (Lat B), a potent actin-disrupting drug, on root gravitropism using various parameters that included detailed curvature kinetics, estimation of gravitropic sensitivity, and monitoring of curvature development after extended clinorotation. Lat B treatment resulted in a promotion of root curvature after a 90 degrees reorientation in three plant species tested. More significantly, the sensitivity of maize (Zea mays) roots to gravity was enhanced after actin disruption, as determined from a comparison of presentation time of Lat B-treated versus untreated roots. A short 10-min gravistimulus followed by extended rotation on a 1-rpm clinostat resulted in extensive gravitropic responses, manifested as curvature that often exceeded 90 degrees. Application of Lat B to the cap or elongation zone of maize roots resulted in the disruption of the actin cytoskeleton, which was confined to the area of localized Lat B application. Only roots with Lat B applied to the cap displayed the strong curvature responses after extended clinorotation. Our study demonstrates that disrupting the actin cytoskeleton in the cap leads to the persistence of a signal established by a previous gravistimulus. Therefore, actin could function in root gravitropism by providing a mechanism to regulate the proliferation of a gravitropic signal originating from the cap to allow the root to attain its correct orientation or set point angle.     

The roles of phytochromes in elongation and gravitropism of roots

Gravitropic orientation and the elongation of etiolated hypocotyls are both regulated by red light through the phytochrome family of photoreceptors. The importance of phytochromes A and B (phyA and phyB) in these red light responses has been established through studies using phy mutants. To identify the roles that phytochromes play in gravitropism and elongation of roots, we studied the effects of red light on root elongation and then compared the gravitropic curvature from roots of phytochrome mutants of Arabidopsis (phyA, phyB, phyD and phyAB) with wild type. We found that red light inhibits root elongation approximately 35% in etiolated seedlings and that this response is controlled by phytochromes. Roots from dark- and light-grown double mutants (phyAB) and light-grown phyB seedlings have reduced elongation rates compared with wild type. In addition, roots from these seedlings (dark/light-grown phyAB and light-grown phyB) have reduced rates of gravitropic curvature compared with wild type. These results demonstrate roles for phytochromes in regulating both the elongation and gravitropic curvature of roots.     

Chromosaponin I specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots

We have found that chromosaponin I (CSI), a gamma-pyronyl-triterpenoid saponin isolated from pea (Pisum sativum L. cv Alaska), specifically interacts with AUX1 protein in regulating the gravitropic response of Arabidopsis roots. Application of 60 microM CSI disrupts the vertically oriented elongation of wild-type roots grown on agar plates but orients the elongation of agravitropic mutant aux1-7 roots toward the gravity. The CSI-induced restoration of gravitropic response in aux1-7 roots was not observed in other agravitropic mutants, axr2 and eir1-1. Because the aux1-7 mutant is reduced in sensitivity to auxin and ethylene, we examined the effects of CSI on another auxin-resistant mutant, axr1-3, and ethylene-insensitive mutant ein2-1. In aux1-7 roots, CSI stimulated the uptake of [(3)H]indole-3-acetic acid (IAA) and induced gravitropic bending. In contrast, in wild-type, axr1-3, and ein2-1 roots, CSI slowed down the rates of gravitropic bending and inhibited IAA uptake. In the null allele of aux1, aux1-22, the agravitropic nature of the roots and IAA uptake were not affected by CSI. This close correlation between auxin uptake and gravitropic bending suggests that CSI may regulate gravitropic response by inhibiting or stimulating the uptake of endogenous auxin in root cells. CSI exhibits selective influence toward IAA versus 1-naphthaleneacetic acid as to auxin-induced inhibition in root growth and auxin uptake. The selective action of CSI toward IAA along with the complete insensitivity of the null mutant aux1-22 toward CSI strongly suggest that CSI specifically interacts with AUX1 protein.     

Circumnutation and gravitropism cause root waving in Arabidopsis thaliana

Arabidopsis thaliana roots grow in a wavy pattern on inclinedagar plates. This waving behaviour has been interpreted as representinga gravitropism-dependent thigmotropic response. We argue insteadthat this root waving represents primarily a flattened spiralgrowth pattern resulting from circumnutation and gravitropism. Key words: Arabidopsis, circumnutation, gravitropism, roots, thigmotropism     

Hydrotropism in roots: sensing of a gradient in water potential by the root cap

Roots of the agravitropic pea (Pisum sativum L.) mutant, ageotropum, responded to a gradient in water potential as small as 0.5 MPa by growing toward the higher water potential. This positive response occurred when a sorbitol-containing agar block was unilaterally applied to the root cap but not when applied to the elongation region. Unilateral application of higher concentrations of sorbitol to the elongation region caused root curvature toward the sorbitol source, presumably because of growth reduction on the water-stressed side. The control blocks of plain agar applied to either the root cap or the elongation region did not cause significant curvature of the roots. These results demonstrate that hydrotropism in roots occurs following perception of a gradient in water potential by the root cap.     

The Arabidopsis heavy metal P-type ATPase HMA5 interacts with metallochaperones and functions in copper detoxification of roots

Since copper (Cu) is essential in key physiological oxidation reactions, organisms have developed strategies for handling Cu while avoiding its potentially toxic effects. Among the tools that have evolved to cope with Cu is a network of Cu homeostasis factors such as Cu-transporting P-type ATPases that play a key role in transmembrane Cu transport. In this work we present the functional characterization of an Arabidopsis Cu-transporting P-type ATPase, denoted heavy metal ATPase 5 (HMA5), and its interaction with Arabidopsis metallochaperones. HMA5 is primarily expressed in roots, and is strongly and specifically induced by Cu in whole plants. We have identified and characterized plants carrying two independent T-DNA insertion alleles, hma5-1 and hma5-2. Both mutants are hypersensitive to Cu but not to other metals such as iron, zinc or cadmium. Interestingly, root tips from Cu-treated hma5 mutants exhibit a wave-like phenotype at early stages and later on main root growth completely arrests whereas lateral roots emerge near the crown. Accordingly, these lines accumulate Cu in roots to a greater extent than wild-type plants under Cu excess. Finally, yeast two-hybrid experiments demonstrate that the metal-binding domains of HMA5 interact with Arabidopsis ATX1-like Cu chaperones, and suggest a regulatory role for the plant-specific domain of the CCH Cu chaperone. Based on these findings, we propose a role for HMA5 in Cu compartmentalization and detoxification.     

Inhibition of polar calcium movement and gravitropism in roots treated with auxin-transport inhibitors

Primary roots of maize (Zea mays L.) and pea (Pisum sativum L.) exhibit strong positive gravitropism. In both species, gravistimulation induces polar movement of calcium across the root tip from the upper side to the lower side. Roots of onion (Allium cepa L.) are not responsive to gravity and gravistimulation induces little or no polar movement of calcium across the root tip. Treatment of maize or pea roots with inhibitors of auxin transport (morphactin, naphthylphthalamic acid, 2,3,5-triiodobenzoic acid) prevents both gravitropism and gravity-induced polar movement of calcium across the root tip. The results indicate that calcium movement and auxin movement are closely linked in roots and that gravity-induced redistribution of calcium across the root cap may play an important role in the development of gravitropic curvature.Abbreviations 9-HFCA 9-hydroxyfluorenecarboxylic acid - NPA naphthylphthalamic acid - TIBA 2,3,5-triiodobenzoic acid - IAA indole-3-acetic acid     

TSA1 interacts with CSN1/CSN and may be functionally involved in Arabidopsis seedling development in darkness

The COP9 signalosome (CSN) is a multiprotein complex which participates in diverse cellular and developmental processes.CSN1,one of the subunits of CSN,is essential for assembly of the multiprotein complex via PCI (proteasome,COP9 signalosome and initiation factor 3) domain in the C-terminal half of CSN 1.However,the role of the N-terminal domain (NTD) of CSN 1,which is critical for the function of CSN,is not completely understood.Using a yeast two-hybrid (Y2H) screen,we found that the NTD of CSN1 interacts with TSK-associating protein 1 (TSA1),a reported Ca2+-binding protein.The interaction between CSN1 and TSA1 was confirmed by co-immunoprecipitation in Arabidopsis.tsal mutants exhibited a short hypocotyl phenotype in darkness but were similar to wild-type Arabidopsis under white light,which suggested that TSA1 might regulate Arabidopsis hypocotyl development in the dark.Furthermore,the expression of TSA1 was significantly lower in a csnl null mutant (fus6),while CSN1 expression did not change in a tsal mutant with weak TSA1 expression.Together,these findings suggest a functional relationship between TSA1 and CSN1 in seedling development.