Calcium Movement, Graviresponsiveness and the Structure of Columella Cells and Columella Tissues in Roots of Allium cepa L.

Roots of Allium cepa L. cv. Yellow are differentially responsiveto gravity. Long (e.g. 40 mm) roots are strongly graviresponsive,while short (e.g. 4 mm) roots are minimally responsive to gravity.Although columella cells of graviresponsive roots are largerthan those of nongraviresponsive roots, they partition theirvolumes to cellular organelles similarly. The movement of amyloplastsand nuclei in columella cells of horizontally-oriented rootscorrelates positively with the onset of gravicurvature. Furthermore,there is no significant difference in the rates of organellarredistribution when graviresponsive and nongraviresponsive rootsare oriented horizontally. The more pronounced graviresponsivenessof longer roots correlates positively with (1) their caps being9.6 times more voluminous, (2) their columella tissues being42 times more voluminous, (3) their caps having 15 times morecolumella cells, and (4) their columella tissues having relativevolumes 4·4 times larger than those of shorter, nongraviresponsiveroots. Graviresponsive roots that are oriented horizontallyare characterized by a strongly polar movement of ⁴⁵Ca²⁺ acrossthe root tip from the upper to the lower side, while similarlyoriented nongraviresponsive roots exhibit only a minimal polartransport of ⁴⁵Ca²⁺. These results indicate that the differentialgraviresponsiveness of roots of A. cepa is probably not dueto either (1) ultrastructural differences in their columellacells, or (2) differences in the rates of organellar redistributionwhen roots are oriented horizontally. Rather, these resultsindicate that graviresponsiveness may require an extensive columellatissue, which, in turn, may be necessary for polar movementof ⁴⁵Ca²⁺ across the root tip. Allium cepa, onion, root, columella tissue, columella cell, gravitropism, calcium, ultrastructure     

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     

Gravity-Induced Polar Transport of Calcium across Root Tips of Maize

Calcium movement across primary roots of maize (Zea mays, L.) was determined by application of ⁴⁵Ca²⁺ to one side of the root and collection of radioactivity in an agar receiver block on the opposite side. Ca movement across the root tip was found to be at least 20 times greater than movement across the elongation zone. The rapid movement of Ca across the tip was severely inhibited in roots from which the root cap had been removed. Ca movement across the tip was also strongly retarded in roots pretreated with 2,4-dinitrophenol or potassium cyanide. Orientation of roots horizontally had no effect on Ca movement across the elongation zone but caused a strong asymmetry in the pattern of Ca movement across the tip. In gravistimulated roots, the movement of Ca from top to bottom increased while movement from bottom to top decreased. The data indicate that gravistimulation induces polar movement of Ca toward the lower side of the root cap. An earlier report (Lee, Mulkey, Evans 1983 Science 220: 1375-1376) from this laboratory showed that artificial establishment of calcium gradients at the root tip can cause gravitropic-like curvature. Together, the two studies indicate that Ca plays a key role in linking gravistimulation to the gravitropic growth response in roots.     

Effects of Chlorpromazine on Gravitropism in Avena Coleoptiles

Chlorpromazine (CPZ), an inhibitor of the calcium-activatedform of calmodulin, is readily taken up by the roots of intactoat seedlings but poorly translocated from the roots to thecoleoptile of these plants. However, plants repeatedly rotatedthrough solutions containing low concentrations of CPZ (10^–8–10^–5M)are infiltrated, and under these conditions, CPZ significantlyinhibits the negative gravitropic response of the coleoptilewithout retarding elongation growth. This effect is observablein ‘decapitated’ (apical 1–2 mm removed) coleoptilesections and in intact whole coleoptiles. If exogenous auxinis supplied to the decapitated sections, both their growth ratesand gravitropic responsiveness are increased and, under theseconditions, CPZ can reduce the gravitropic curvature withoutreducing the overall growth rate. These results are discussedin relation to the possible role of calmodulin-dependent calcium-ionpumps in gravitropism. chlorpromazine, gravitropism, calmodulin, calcium, oat, Avena sativa     

Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.     

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.     

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.     

Effect of calmodulin antagonists on the growth and graviresponsiveness of primary roots of maize

We examined the effect of calmodulin (CaM) antagonists applied at the root tip on root growth, gravity-induced root curvature, and the movement of calcium across the root tip and auxin (IAA) across the elongation zone of gravistimulated roots. All of the CaM antagonists used in these studies delayed gravity-induced curvature at a concentration (1 M) that did not affect root growth. Calmodulin antagonists ( 1M) inhibited downward transport of label from ⁴⁵Ca²⁺ across the caps of gravistimulated roots relative to the downward transport of ⁴⁵Ca²⁺ in gravistimulated roots which were not treated with CaM antagonists. Application of CaM antagonists at the root tip ( 1M) also decreased the relative downward movement of label from ³H-IAA applied to the upper side of the elongation zone of gravistimulated roots. In general, tip application of antagonists inhibited neither the upward transport of ⁴⁵Ca²⁺ in the root tip nor the upward movement of label from ³H-IAA in the elongation zone of gravistimulated roots. Thus, roots treated with CaM antagonists ( 1 M) become less graviresponsive and exhibit reduced or even a reversal of downward polarity of calcium transport across the root tip and IAA transport across the elongation zone. The results indicate that calmodulin-regulated events play a role in root gravitropism.     

Negative gravitropic response of roots directs auxin flow to control root gravitropism

Root tip is capable of sensing and adjusting its growth direction in response to gravity, a phenomenon known as root gravitropism. Previously, we have shown that negative gravitropic response of roots (NGR) is essential for the positive gravitropic response of roots. Here, we show that NGR, a plasma membrane protein specifically expressed in root columella and lateral root cap cells, controls the positive root gravitropic response by regulating auxin efflux carrier localization in columella cells and the direction of lateral auxin flow in response to gravity. Pharmacological and genetic studies show that the negative root gravitropic response of the ngr mutants depends on polar auxin transport in the root elongation zone. Cell biology studies further demonstrate that polar localization of the auxin efflux carrier PIN3 in root columella cells and asymmetric lateral auxin flow in the root tip in response to gravistimulation is reversed in the atngr1;2;3 triple mutant. Furthermore, simultaneous mutations of three PIN genes expressed in root columella cells impaired the negative root gravitropic response of the atngr1;2;3 triple mutant. Our work revealed a critical role of NGR in root gravitropic response and provided an insight of the early events and molecular basis of the positive root gravitropism.     

Effects of MAPKK inhibitor PD98059 on the gravitropism of primary roots of maize

Gravity plays a fundamental role in plant growth and development, yet the molecular details of gravitropism is not fully understood. Here, we report the effects of PD98059, a specific inhibitor of mitogen-activated protein (MAP) kinase kinase, on the gravitropism of primary roots of maize. Unilateral application of PD98059 to horizontal roots led to different gravitropic growth. Placing PD98059-containing agar on the upper side of the root tips accelerated gravitropic curvature, whereas placing the agar on the lower side inhibited gravitropic curvature. However, no effect was detected when asymmetric application of PD98059 to vertical roots. Global application of maize primary root with PD98059 suppressed root gravitropism. Furthermore, the effects of H₂O₂ on horizontal root gravitropism and vertical root bending were compromised by pretreatment with PD98059. These results suggest an involvement of MAP kinase pathway(s) in gravitropism of maize roots.     

Organelle Sedimentation in Gravitropic Roots of Limnobium is Restricted to the Elongation Zone

Roots of the aquatic angiosperm Limnobium spongia (Bosc) Steud.were evaluated by light and electron microscopy to determinethe distribution of organelle sedimentation towards gravity.Roots of Limnobium are strongly gravitropic. The rootcap consistsof only two layers of cells. Although small amyloplasts arepresent in the central cap cells, no sedimentation of any organelle,including amyloplasts, was found. In contrast, both amyloplastsand nuclei sediment consistently and completely in cells ofthe elongation zone. Sedimentation occurs in one cell layerof the cortex just outside the endodermis. Sedimentation ofboth amyloplasts and nuclei begins in cells that are in theirinitial stages of elongation and persists at least to the levelof the root where root hairs emerge. This is the first modernreport of the presence of sedimentation away from, but not in,the rootcap. It shows that sedimentation in the rootcap is notnecessary for gravitropic sensing in at least one angiosperm.If amyloplast sedimentation is responsible for gravitropic sensing,then the site of sensing in Limnobium roots is the elongationzone and not the rootcap. These data do not necessarily conflictwith the hypothesis that sensing occurs in the cap in otherroots, since Limnobium roots are exceptional in rootcap originand structure, as well as in the distribution of organelle sedimentation.Similarly, if nuclear sedimentation is involved in gravitropicsensing, then nuclear mass would function in addition to, notinstead of, that of amyloplasts.Copyright 1994, 1999 AcademicPress Limnobium spongia, gravitropism, roots, sedimentation, cortex     

Specific Increase in Phosphatase Isozymes in Cucumber Roots Caused by Calcium Deficiency

Phosphatases in cucumber roots, whose production was inducedby Ca^2$ deficiency, were characterized chromatographically usingATP, 2'(3')-AMP and p-nitrophenyl-phosphate (PNPP) as substrates.Ca^2$ deficiency stimulated greater than 10-fold increases inthe activities with these substrates of the non-adsorbed fractionfrom a DEAE-cellulose column. Several fractions associated withthese phosphatase activities were eluted from the column withNaCl solution; their levels increased less with Ca^2$ starvation.When the non-adsorbed fraction from Ca^2$-straved roots was appliedto a Sephadex G-200 column, fractions associated with 2'(3')-AMPase(phosphatase I) and with both ATPase and PNPPase (phosphataseII) were separated. In the control roots, very weak activitiesof phosphatases I and II were observed at the same positionon the gel filtration. The phosphatase I isolated from boththe control and Ca^2$-starved roots was extremely specific tonucleoside 2'(3')-monophosphates, whereas phosphatase II fromboth types of roots had a relatively broad substrate specificity.When phosphatase I from Ca^2$-starved roots was stained with2'(3')-AMP in CaCl₂ after polyacrylamide gel electrophoresis,a single band was obtained. Phosphatase I from control rootsalso showed a single band, with the same Rf value. PhosphataseII from both types of roots contained two isozyme bands whenthe activities were stained with either ATP or PNPP. These resultsindicate that Ca^2$ starvation causes specific increases in thelevel of phosphatases I and II in cucumber roots. (Received October 28, 1981; Accepted January 19, 1982)     

Effects of Calmodulin Antagonists on the Mitochondrial and Microsomal Ca2$ Uptake in Apple Fruit

In apple fruit, active ATP-dependent microsomal Ca^2$ uptakeand respiration-dependent mitochondrial Ca^2$ uptake were observed. The mitochondrial Ca^2$ uptake was depressed by the calmodulinantagonists chlorpromazine hydrochloride (CPZ) and N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamidehydrochloride (W-7). The Ca^2$-ATPase from apple mitochondriawas also inhibited by CPZ or W-7. The apparent K_m value forCa^2$ in mitochondrial Ca^2$ uptake (K_m=0.35 mM) was similar tothat of mitochondrial Ca^2$-ATPase (K_m=0.32 mM). The inhibitoryeffect of W-7 on the activity of the mitochondrial Ca^2$ uptakewas closely correlated with the inhibition by W-7 of mitochondrialCa^2$-ATPase (r=0.996). These findings indicate that the mitochondrialuptake of Ca^2$ in apple fruit depends on the calmodulin-mediatedactivation of Ca^2$-ATPase. The microsomal Ca^2$ uptake was depressed by CPZ, suggestingthat the microsomal Ca^2$ uptake may also be modulated by calmodulin. ¹ Contribution No. C-72, Fruit Tree Research Station. (Received June 7, 1982; Accepted October 19, 1982)     

Purification of Major Isozymes of Nuclease C and Production of Active Fragments by Trypsin

Most nucleases from gametes of Chlamydomonas reinhardtii needCa^2$ for full activation. They have been named nuclease C andat least six species of isozymes have been found in the femalegamete (Ogawa and Kuroiwa 1985a). Nuclease C1&2 and C3 were purified from the vegetative cellsof this organism. Nuclease C1&2 exhibited a sharp pH optimumat 9.5, while nuclease C3 preferred a more neutral pH at 7.0–8.5.Use of the Ca^2$-EGTA [ethylene-glycol-bis-(2-aminoethyl ether)-N,N,N',N'-tetraaceticacid] buffer in the reaction mixture made it possible to determinetheir activity at the physiological Ca^2$ concentration. NucleaseC3 was not activated at low Ca^2$ concentration and exhibiteda sharp optimum at 10^–3{small tilde}10^–4 M. NucleaseC1&2 were activated at a physiological concentration of10^–6 M; increasing the Ca^2$ concentration did not affectthe activity. Nuclease C gave active fragments upon trypsin digestion. Trypticfragments of nuclease C1&2 and C3 had molecular weightsof 21,000 (referred as C6T) and 16,000 (C4T), respectively.Upon regulating the digestion, a few fragments were identifiedas intermediates of nuclease C6T by the in situ nuclease assay.These tryptic fragments were similar in molecular size to theminor components of nuclease C found in the cell lysates ofgametes and early zygotes. This finding suggests that a minorspecies of nuclease C may be produced from the major nucleaseC during gametogenesis. (Received June 26, 1985; Accepted August 28, 1985)     

Redistribution of Phosphorus, Sulfur, Potassium and Calcium in Relation to Light-Induced Gravitropic Curvature in Zea Roots

Phosphorus, sulfur, potassium and calcium were measured by particle-inducedX-ray emission (PIXE) in horizontally oriented, light-irradiatedor non-irradiated primary roots of Zea mays L., cv. Golden CrossBantam 70 which exhibit gravitropic response only after exposureto light. The content of the four elements increased in thelower half of horizontally oriented roots which had been brieflyexposed to white or red light, while there were no marked differencesin distribution between the upper and lower halves of non-irradiatedroots. The increase of each element in the lower half was observed15–30 min after irradiation in root caps and 30–60min after irradiation in the elongation zones. The effect ofred light was not reversed by far-red light given immediatelyafter the red irradiation. Ethylene glycol-bis(ß-aminoethylether)-N, N, N',N'-tetraacetic acid (EGTA) treatment of roottips inhibited the gravitropic curvature of roots, and the additionof Ca reduced this inhibition. The meaning of Ca redistributionin root caps and elongation zones during light-induced gravitropiccurvature of maize roots is discussed. (Received December 4, 1985; Accepted March 22, 1986)     

Increase in Cell Wall-Associated Phosphatase Activity in Cucumber Roots during Calcium Starvation: Binding Nature and Properties of the Phosphatase and Cell Wall Analysis

In Ca^2$-starved cucumber roots, about 23% of phosphatase assayedat pH 9.0 (ALPase) in the crude cell walls was solubilized witheither 2 M NaCl or purified endo type polygalacturonase (endo-PG)from yeast culture broth. Coexistence of NaCl and endo-PG hadlittle effect on further release of ALPase, and a small amountof the activity was solubilized from the NaCl-pretreated cellwalls by incubation with endo-PG. Ionically bound ALPase, therefore,seemed to be localized in the fraction which was hydrolyzedby endo-PG in the crude cell walls of Ca^2$-starved cucumberroots. In the control roots, however, ALPase was not effectivelysolubilized by the treatment with endoPG. Ca^2$ starvation reducedthe contents of rhamnose, uronic acids and galactose among non-cellulosicsugars in the cell walls, suggesting that the structure of pecticsubstances, possibly rhamnogalacturonan, is altered during thestarvation. Activities of both ionically and covalently bound ALPases greatlyincreased during Ca^2$ starvation. The increased ALPase in theNaCl-solubilized fraction hydrolyzed most phosphate esters tested,whereas the enzyme from control roots only cleaved nucleoside2'(3')-monophosphates and p-nitrophenylphosphate. Differencesin the properties between both types of roots were also foundwhen the effects of various inhibitors were tested. Profilesof ALPase-isozymes after polyacrylamide gel electrophoresiswere also altered by Ca^2$ starvation. (Received June 2, 1982; Accepted July 20, 1982)     

Polar Transport of Auxin across Gravistimulated Roots of Maize and Its Enhancement by Calcium

The effect of Ca on the polar movement of [³H]indoleacetic acid ([³H] IAA) in gravistimulated roots was examined using 3-day-old seedlings of maize (Zea mays L.). Transport of label was measured by placing an agar donor block containing [³H]IAA on one side of the elongation zone and measuring movement of label across the root into an agar receiver block on the opposite side. In vertically oriented roots, movement of label across the elongation zone into the receiver was slight and was not enhanced by incorporating 10 millimolar CaCl₂ into the receiver block. In horizontally oriented roots, movement of label across the root was readily detectable and movement to a receiver on the bottom was about 3-fold greater than movement in the opposite direction. This polarity was abolished in roots from which the caps were removed prior to gravistimulation. When CaCl₂ was incorporated into the receivers, movement of label across horizontally oriented intact roots was increased about 3-fold in both the downward and upward direction. The ability of Ca to enhance the movement of label from [³H]IAA increased with increasing Ca concentration in the receiver up to 5 to 10 millimolar CaCl₂. With the inclusion of CaCl₂ in the receiver blocks, gravity-induced polar movement of label into receiver blocks from applied [³H]IAA was detectable within 30 minutes, and asymmetric distribution of label within the tissue was detectable within 20 minutes. The results indicate that gravistimulation induces a physiological asymmetry in the auxin transport system of maize roots and that Ca increases the total transport of auxin across the root.     

Unidirectional Ca2+ Fluxes in Roots of Rye (Secale cereale L). A Comparison of Excised Roots with Roots of Intact Plants

The unidirectional Ca²⁺ fluxes across the plasma membrane andtonoplast were determined in both excised roots and roots ofintact seedlings of rye (Secale cereale L. cv. Rheidol). Theunidirectional Ca²⁺ fluxes across the plasma membrane and tonoplastmeasured in excised roots were of a similar order of magnitudeto those determined in roots of intact plants. Influx and effluxof Ca²⁺ across the root plasma membrane were similar (estimatedto be between 0·7 and 3·4 µmol g     

A Gradient of Endogenous Calcium Forms in Mucilage of Graviresponding Roots of Zea mays

Agar blocks that contacted the upper sides of tips of horizontally-orientedroots of Zea mays contain significantly less calcium (Ca) thanblocks that contacted the lower sides of such roots. This gravity-inducedgradient of Ca forms prior to the onset of gravicurvature, anddoes not form across tips of vertically-oriented roots or rootsof agravitropic mutants. These results indicate that (1) Cacan be collected from mucilage of graviresponding roots, (2)gravity induces a downward movement of endogenous Ca in mucilageoverlying the root tip, (3) this gravity-induced gradient ofCa does not form across tips of agravitropic roots, and (4)formation of a Ca gradient is not a consequence of gravicurvature.These results are consistent with gravity-induced movement ofCa being a trigger for subsequent redistribution of growth effectors(e.g. auxin) that induce differential growth and gravicurvature. Atomic absorption, calcium, corn, gravitropism (root), Zea mays     

Role of Calcium in Phototaxis of Physarum polycephalum

The calcium transport blockers, ruthenium red and lanthanumions, inhibit negative phototactic orientation in the acellularslime mold, Physarum polycephalum. Likewise, agents known toenhance calcium fluxes (caffeine and phosphatidic acid) affectphoto-orientation as well as trimethyl-phenyl-phosphonium^$ whichimpairs electrochemical gradients across membranes. The resultssuggest that calcium fluxes are involved in the sensory transductionof phototactic orientation in this organism. The cation transportthrough the channels occurs passively along a previously establishedgradient built by energy dependent calcium pumps. Inhibitionof these pumps by poly-L-lysine also impairs phototactic orientation.The location of the calcium transport phenomena is discussed. (Received April 17, 1985; Accepted August 12, 1985)