Effects of abscisic acid and xanthoxin on elongation and gravitropism in primary roots of Zea mays.

We examined the involvement of abscisic acid (ABA) and xanthoxin (Xan) in maize root gravitropism by (1) testing the ability of ABA to allow positive gravitropism in dark-grown seedlings of the maize cultivar LG11, a cultivar known to require light for positive gravitropism of the primary root, (2) comparing curvature in roots in which half of the cap had been excised and replaced with agar containing either ABA or indole-3-acetic acid (IAA), (3) measuring gravitropism in roots of seedlings submerged in oxygenated solutions of ABA or IAA and (4) testing the effect of Xan on root elongation. Using a variety of methods of applying ABA to the root, we found that ABA did not cause horizontally-oriented primary roots of dark-grown seedlings to become positively gravitropic. Replacing half of the root cap of vertically oriented roots with an agar block containing ABA had little or no effect on curvature relative to that of controls in which the half cap was replaced by a plain agar block. Replacement of the removed half cap with IAA either canceled or reversed the curvature displayed by controls. When light-grown seedlings were submerged in ABA they responded strongly to gravistimulation while those in IAA did not. Xan (up to 0.1 mM) did not affect root elongation. The results indicate that ABA is not a likely mediator of root gravitropism and that the putative ABA precursor, Xan, lacks the appropriate growth-inhibiting properties to serve as a mediator of root gravitropism.

Localization of calcium in roots and microsomal membranes of corn by direct pyroantimonate precipitation.

Many cell membrane systems, including microsomal vesicles of corn, are able to regulate calcium levels both in vivo and in vitro, often in an ATP-dependent, calmodulin-stimulated fashion. The purpose of this study was to determine calcium distribution in meristematic cells of intact tissue and microsomal vesicles from corn roots using direct pyroantimonate-osmium fixation. In root cells, precipitates were localized in mitochondria, plastids, the nucleus, endoplasmic reticulum, Golgi apparatus, and along the plasma membrane. Plasma membrane-enriched microsomal vesicles isolated from corn roots incubated in media to permit calcium transport before pyroantimonate-osmium fixation show internal precipitates associated with the membrane and in the lumen of the vesicles. De-staining of the sections with 1 mM EDTA or EGTA removed precipitate from the sections, confirming the presence of calcium in the antimonate precipitates. These data support biochemical data that this same membrane preparation exhibited ATP-dependent calcium sequestration that was stimulated by calmodulin, as measured by retention of 45Ca. This provides evidence that these membranes are responsible for ATP-requiring, calmodulin-stimulated calcium transport in the intact cell.

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.

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.

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 (45)Ca(2+) 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.

Reversible loss of gravitropic sensitivity in maize roots after tip application of calcium chelators.

The application of calcium chelating agents (EDTA or EGTA) to the tips of maize roots caused a loss of gravitropic sensitivity. When the chelator was replaced with calcium chloride, gravitropic sensitivity was restored. Asymmetric application of calcium chloride near the tip of a vertical root caused curvature toward the calcium source. When the calcium was applied to the upper surface of the tip of a root oriented horizontally, the root curved upward even though control roots exhibited strong downward curvature. Application of calcium chloride to the tips of decapped roots, which are known to be gravitropically insensitive, did not restore gravitropic sensitivity. However, asymmetric application of calcium chloride near the tips of decapped roots caused curvature toward the calcium source. Calcium may play a key role in linking gravity detection to gravitropic curvature in roots.

The kinetics of abscisic acid action on root growth and gravitropism.

Using an auxanometer and time-lapse cinematography we have studied the timing of abscisic acid (ABA) effects on elongation, gravitropic curvature, and hydrogen-ion efflux in several cultivars of maize (Zea mays L.). The effect of high concentrations (e.g. 0.1 mM) of ABA on root elongation is triphasic, including 1) a period of promotion lasting approximately 12 h, 2) a subsequent period of increasing inhibition lasting approximately 12h, and 3) gradual recovery to a rate within approximately 80% of the control rate. With lower concentrations of ABA (e.g. 0.1 µM) only the transient promotive phase is seen. Abscisic acid enhances ethylene biosynthesis in roots of maize but suppression of ethylene biosynthesis does not prevent the long-term inhibitory action of ABA on growth. Application of ABA (0.1 mM) to the upper surface of horizontally placed roots accelerates positive gravitropism. Application of ABA to the lower surface retards gravitropism and in some cases causes the roots to curve upward against the direction of gravity. These observations are consistent with our finding that the initial effect of ABA on root elongation is stimulatory. Since root gravitropism is rapid enough to be completed within the stimulatory phase of ABA action, the data argue against hypotheses of gravitropism based upon accumulation of ABA to inhibitory levels on the lower side of a hirizontal root.

Promotion of growth and hydrogen ion efflux by auxin in roots of maize pretreated with ethylene biosynthesis inhibitors.

Low concentrations of auxin (e.g. 10(-10)m) do not promote the growth of intact seedling roots of maize (Zea mays L. Bear Hybrid WF 9 x 38). Higher concentrations are inhibitory. When the roots are pretreated with the ethylene biosynthesis inhibitors, cobalt and aminoethoxyvinylglycine, auxin (10(-10) to 10(-8)m) strongly promotes their growth. The promotion of growth by auxin in pretreated roots is preceded by enhanced hydrogen ion secretion from the roots. The data indicate that hormone-enhanced hydrogen ion secretion may play a role in the rapid promotion of root growth by auxin. The ability of auxin to promote the growth of intact roots is discussed in relation to the Cholodny/Went hypothesis of hormonal control of root geotropism.

Geotropism in corn roots: evidence for its mediation by differential Acid efflux.

The elongation zone in intact growing corn roots secretes acid leading to a reduced pH along the surface of the root and in the adjacent medium. This can be detected by placing the root on an agar medium containing the pH indicator dye bromocresol purple. When the root is treated with a growth inhibitory concentration of the hormone indole-3-acetic acid, the acid efflux is reversed and growth is greatly retarded. When the root is mounted vertically, acid secretion is uniform along the elongation zone, and the root grows straight downward. When the root is placed horizontally, there is enhanced acid efflux along the upper surface of the elongation zone and reduced acid efflux along the lower surface. An increased rate of elongation of the upper cells relative to the lower cells then results in downward curvature of the root. The correlation between acid efflux patterns and growth patterns indicates that proton efflux is important in the control of root growth.

Correlations between proton-efflux patterns and growth patterns during geotropism and phototropism in maize and sunflower.

By placing seedlings of sunflower (Helianthus annuus L.) or maize (Zea mays L.) on agar plates containing a pH indicator dye it is possible to observe surface pH patterns along the growing seedling by observing color changes of the indicator dye. Using this method we find that in geotropically stimulated sunflower hypocotyls or maize coleoptiles there is enhanced proton efflux on the lower surface of the organ prior to the initiation of curvature. As curvature develops the pattern of differential acid efflux becomes more intense. A similar phenomenon is observed when these organs are exposed to unilateral illumination, i.e. enhanced acid efflux occurs on the dark side of the organ prior to the initiation of phototropic curvature and the pattern of differential acid efflux intensifies as phototropic curvature develops. These observations indicate that differential acid efflux occurs in response to tropistic stimuli and that the acid efflux pattern may mediate the development of tropistic curvatures.

Auxin action on proton influx in corn roots and its correlation with growth.

At concentrations inhibitory to the elongation of corn (Zea mays L.) roots, the auxins, indole-3-acetic acid (IAA) and α-naphthaleneacetic acid (α-NAA), cause an increase in the pH of the bathing medium; this increase occurs with an average latent period shorter than the latent period for the inhibitory effect of these auxins on elongation. Indole-2-carboxylic acid, an inactive structural analogue of IAA, and ß-naphthaleneacetic acid, an inactive analogue of α-NAA, affect neither growth nor the pH of the medium. Since acid pH is known to promote and basic pH to inhibit root elongation, the data are consistent with the hypothesis that hormone-induced modification of cell-wall pH plays a role in the control of elongation of roots, as has been proposed for elongation of stems and coleoptiles.