Indoleacetic acid movement in the root cap

Summary When applied on the root cap of Zea mays L., indol-3yl-acetic acid (IAA) may enter the root tip and move basipetally inside the cap. From the cap to the apex (quiescent centre and meristem) the IAA transport is very slow. Polarity of IAA movement, in relation to growth, is discussed.     

Auxin biosynthesis and metabolism in isolated roots of Zea mays

The synthesis and metabolism of indole-3-acetic acid (IAA) was investigated in isolated roots of corn, Zea mays L. Roots were cultured aseptically in media supplemented with either ¹⁴C-tryptophan or ¹⁴C-IAA. Exogenously supplied IAA is rapidly and completely metabolized by root tissues. The main site in the root for the synthesis of IAA is in the apex. Removal of either the root cap or the quiescent center, or the root cap and the quiescent center from the apex has no effect on the IAA-synthesizing ability of the apex. Subdividing the terminal 2.1 cm of the root into various segments and culturing them separately stimulates IAA synthesis in these isolated root tissues. Roots in culture maintain relatively constant IAA levels, reflecting the precise controls of the level of this hormone.     

The de novo origin of the quiescent center regenerating root apices of Zea mays

Summary In roots from which the root cap and quiescent center have been removed new apical tissues regenerated in line with the main axis of the root. Regeneration of these tissues occurred from the region of the proximal meristem, which extends for no more than 350 m from the cut surface. Accompanying the regeneration of new apical tissues is a change in the architecture of the root apex and initiation and enlargement of a new quiescent center. A possible role for the quiescent center in the establishment of pattern at the apex is considered. Regeneration of the original apex failed to occur in those roots from which the root cap, quiescent center and proximal meristem were excised.Abbreviation QC quiescent center     

Hormonal Regulation of Lateral Bud (Tiller) Release in Oats (Avena sativa L.)

Stem segments containing a single node and quiescent lateral bud (tiller) were excised from the bases of oat shoots (cv. `Victory') and used to study the effects of plant hormones on release of lateral buds and development of adventitious root primordia. Kinetin (10⁻⁵ and 10⁻⁶ molar) stimulates development of tillers and inhibits development of root primordia, whereas indoleacetic acid (IAA) (10⁻⁵ and 10⁻⁶ molar) causes the reverse effects. Abscisic acid strongly inhibits kinetin-induced tiller bud release and elon-gation and IAA-induced adventitious root development. IAA, in combination with kinetin, also inhibits kinetin-induced bud prophyll (outermost leaf of the axillary bud) elongation. The IAA oxidase cofactor p-coumaric acid stimulates lateral bud release; the auxin transport inhibitor 2,3,5-triiodo-benzoic acid and the antiauxin α (p-chlorophenoxy)-isobutyric acid inhibit IAA-induced adventitious root formation. Gibberellic acid is synergistic with kinetin in the elongation of the bud prophyll. In intact oat plants, tiller release is induced by shoot decapitation, geostimulation, or the emergence of the inflorescence. Results shown support the apical dominance theory, namely, that the cytokinin to auxin ratio plays a decisive role in determining whether tillers are released or adventitious roots develop. They also indicate that abscisic acid and possibly gibberellin may act as modulator hormones in this system.     

Patterns of auxin and abscisic acid movement in the tips of gravistimulated primary roots of maize

Because both abscisic acid (ABA) and auxin (IAA) have been suggested as possible chemical mediators of differential growth during root gravitropism, we compared with redistribution of label from applied ³H-IAA and ³H-ABA during maize root gravitropism and examined the relative basipetal movement of ³H-IAA and ³H-ABA applied to the caps of vertical roots. Lateral movement of ³H-ABA across the tips of vertical roots was non-polar and about 2-fold greater than lateral movement of ³H-IAA (also non-polar). The greater movement of ABA was not due to enhanced uptake since the uptake of ³H-IAA was greater than that of ³H-ABA. Basipetal movement of label from ³H-IAA or ³H-ABA applied to the root cap was determined by measuring radioactivity in successive 1 mm sections behind the tip 90 minutes after application. ABA remained largely in the first mm (point of application) whereas IAA was concentrated in the region 2–4 mm from the tip with substantial levels found 7–8 mm from the tip. Pretreatment with inhibitors of polar auxin transport decreased both gravicurvature and the basipetal movement of IAA. When roots were placed horizontally, the movement of ³H-IAA from top to bottom across the cap was enhanced relative to movement from bottom to top whereas the pattern of movement of label from ³H-ABA was unaffected. These results are consistent with the hypothesis that IAA plays a role in root gravitropism but contrary to the idea that gravi-induced asymmetric distribution of ABA contributes to the response.     

Cytokinin biosynthesis in roots of corn

Removal of the quiescent center (QC) from the root apex of maize (Zea mays L., cv. Kelvedon 33) initiates a set of events which culiminate in the regeneration of an intact apex with a newly formed QC. Concomitant with the formation of a new QC is a marked reduction in extractable cytokinins in the tissue of the proximal meristem. Replacing the excised QC with a Dowex (acidic cation-exchange resin) bead affects both root growth and QC regeneration. Root growth is inhibited by plain Dowex beads and Dowex beads treated with zeatin; this inhibition is reversed if the beads have been treated with CaCl₂ (±zeatin). Dowex beads treated with zeatin delay the formation of a new QC; this effect is the same whether or not the beads also contain CaCl₂. The results of this investigation support the notions that cytokinin biosynthesis in roots is a result of activities of both the QC and the proximal meristem, and that cytokinins, at least if supplied exogenously, can play a role in root morphogenesis by delaying the regeneration of the QC.Abbreviations used throughout the text PM proximal meristem - QC quiescent center - RC root cap     

Evidence for Three Different Systems of Movement of Indoleacetic Acid in Intact Roots of Phaseolus coccineus

Indoleacetic acid (IAA)-5-³H (2 × 10⁻⁹M) was applied to intact roots of Phaseolus coccineus seedlings, at the apex or 2 cm above the apex, at various pHs and in the presence of Cu²⁺ and NaCl. The transport of label in the roots was then examined after 6 h by cutting the roots into 1 mm sections above and below the zone of treatment. Basipetal movement from 2 cm above the apex was unafected by pH, Cu²⁺ or NaCl. Acropetal movement from the same area decreased with increasing pH from 5.4 to 8.0, probably due to an effect of pH on the entry of IAA into the cells. pH had no effect on sucrose transport. Cu²⁺ also inhibited acropetal movement but NaCl had no effect. Basipetal movement of label from the apex was reduced by Cu²⁺ and increasing pH, but not as much as with acropetal movement, and increased by the presence of NaCl. These facts are interpreted as showing 3 different systems of IAA movement in intact roots: basipetal from 2 cm up the root in some extracellular physical system; acropetal from 2 cm up the root, and basipetal from the apex, in a metabolically dependent intracellular system, but in different tissues of the root. It is proposed that endogenous IAA not only moves into the root from the stem but is also synthesized in the root apex, and moves basipetally for a short distance to the root growing zone in a separate system from the IAA descending from the stem.     

Effects of Cations on Hormone Transport in Primary Roots of Zea mays

We examined the influence of aluminum and calcium (and certain other cations) on hormone transport in corn roots. When aluminum was applied unilaterally to the caps of 15 mm apical root sections the roots curved strongly away from the aluminum. When aluminum was applied unilaterally to the cap and ³H-indole-3-acetic acid was applied to the basal cut surface twice as much radioactivity (assumed to be IAA) accumulated on the concave side of the curved root as on the convex side. Auxin transport in the apical region of intact roots was preferentially basipetal, with a polarity (basipetal transport divided by acropetal transport) of 6.3. In decapped 5 mm apical root segments, auxin transport was acropetally polar (polarity = 0.63). Application of aluminum to the root cap strongly promoted acropetal transport of auxin reducing polarity from 6.3 to 2.1. Application of calcium to the root cap enhanced basipetal movement of auxin, increasing polarity from 6.3 to 7.6. Application of the calcium chelator, ethylene-glycol-bis-(β-aminoethylether)-N,N,N′, N′-tetraacetic acid, greatly decreased basipetal auxin movement, reducing polarity from 6.3 to 3.7. Transport of label after application of tritiated abscisic acid showed no polarity and was not affected by calcium or aluminum. The results indicate that the root cap is particularly important in maintaining basipetal polarity of auxin transport in primary roots of corn. The induction of root curvature by unilateral application of aluminum or calcium to root caps is likely to result from localized effects of these ions on auxin transport. The findings are discussed relative to the possible role of calcium redistribution in the gravitropic curvature of roots and the possibility of calmodulin involvement in the action of calcium and aluminum on auxin transport.     

Physical restraints underlying short-term inhibition by auxin of root elongation in intact maize seedlings

This report investigates physical changes associated with the short-term inhibition of root elongation in intact maize seedlings (Zea mays L. vs. Halamish) by exogenous auxin. Movement of root tips was assayed by video microscopy in control roots, roots grown for 45 min in 10^–6 M indole3-acetic acid (IAA), or roots chilled for 3 min at 11°C. IAA and chilling treatments similarly reduced root elongation rates (from 29 ± 6 m min^–1 to 6 ± 2 m min^–1). Initial rates of root tip contraction induced by 300 mOsmol mannitol were used to calculate tissue contractibility values. These allowed a comparison of effects of IAA and chilling treatments on apparent rates of water transport out of the root tip tissues. Chilling treatment reduced root tip contractibility by 66%, whereas IAA had much less effect (26% reduction). Roots were also exposed to an osmotic jump treatment; the initial osmotically induced increase in elongation rate was used to determine root tip extensibility values. Both IAA and chilling treatments reduced root tip extensibilities by 57%. Inhibition of wall-yielding properties, rather than hydraulic limitations, appeared to be primarily associated with inhibition of intact root tip elongation by exogenous IAA.     

Effects of heavy metals and strontium on division of root cap cells and meristem structural organization

In order to define relations between the behavior of quiescent center cells and the condition of root cap cells, effects of various metal salts on the root meristem structure, root growth, and division of root cap cells were investigated. Two-day-old maize (Zea mays L., cv. Diamant) seedlings were incubated on solutions containing 35 μM Ni(NO₃)₂), 10 μM Pb(NO₃)₂, or 3 mM Sr(NO₃)₂ in the absence or in the presence of 3 mM Ca(NO₃)₂. Toxic effects of metals were assessed from inhibition of the primary root length increment following 24-h and 48-h incubations as compared to the roots grown on water or on 3 mM Ca(NO₃)₂ solution. Metal localization in the root apex tissues following 24-h and 48-h incubations was determined using histochemical techniques. Cell lengths in three upper layers of root cap columella were determined, and the mitotic index in these cells was calculated. In the absence of Ca(NO₃)₂, the metals were found both in the meristem and in the root cap. Pb and Sr were revealed primarily in the cell walls, and Ni, in the cell protoplasts. In the presence of Ca(NO₃)₂, metal content in all root tissues was decreased, and their toxic effect on root growth was ameliorated. Pb and Ni inhibited cell division in the root cap. Pb caused an increase in the root cap cell length as early as following 24-h incubation, and Ni, only following 48-h incubation. Pb activated division of quiescent center cells in the direction of root cap. These effects, as well as possible involvement of dermatogen and cortex cells, resulted in a regrowth of a new root cap already after a 24-h incubation period. In this case, the meristem was transformed from a closed structure into the open one. Following 48-h incubation, Ni brought about only few divisions of quiescent center cells in the direction of root cap. It was suggested that inhibition of divisions of the root cap upper layer cells and a decrease in the sloughing off its cells can stimulate the quiescent center cell divisions. A similarity of the quiescent center and animal stem cells is discussed.     

Cell division activation in the quiescent center of excised maize root tip

The phenomenon of activating cell proliferation in the quiescent center of excised maize roots is described. The root tips were grown on wet filter paper in Petri dishes. This phenomenon was observed in 8 to 14 maize cultivars and was absent in excised Arabidopsis root tips. The distribution of mitoses in meristems greatly varied in individual seedlings roots from the same seed lot and seedlings of different cultivars. Meristem opening was observed after the removal of small root tips not longer than 3 mm and intact seminal roots. Sucrose (2%) and 10⁻⁶–10⁻⁸ M indole-3-acetic acid did not prevent meristem opening. These findings indicate that the state of quiescent center is maintained by a system of intercellular and interorgan relations, which are to be clarified.     

On ethylene and stem elongation in green pea seedlings

Maximum elongation of excised internodal stem sections of light-grown pea (Pisum sativum L.) seedlings occurred at 10⁻⁵ molar indoleacetic acid (IAA), with submaximal responses occurring at 10⁻⁴ and 10⁻³ molar. Accompanying elongation at concentrations of IAA of 10⁻⁶ to 10⁻³ molar was production of ethylene, with the amount increasing up to 10⁻⁴ molar IAA and then becoming nearly constant. Elongation of light-grown sections was not inhibited by exogenous ethylene up to 10,000 ppm in the presence of 10⁻⁵ molar IAA. Marked (up to 50%) inhibition of elongation of internodal segments in situ was observed after treating whole light-grown seedlings with exogenous ethylene for 20 hours. It is concluded that ethylene is not responsible for the submaximal elongation responses of green pea stem sections at high auxin concentrations, but that IAA per se is accountable.     

Studies on induced changes in growth patterns and polarity in roots of Vicia faba L.

Summary Roots of Vicia faba were treated with colchicine (0.025%), or IAA (4.7×10^-6 M), or both, for 3 hours and fixed at various intervals over the following 11 days. The axis of spindle orientation and the distribution of mitotic figures, lateral root primordia and xylem vessel elements was examined in the apical 10 mm of median longitudinal sections of these roots.No effect of IAA was found on the orientation of the spindle. However, evidence was obtained indicating that the systems controlling the polarity of cell division and cell expansion differ in some way.The number of lateral root primordia formed was greater in roots treated with IAA or colchicine than in control roots. These primordia were always initiated adjacent to a xylem vessel. Thus, no primordium was closer to the apex than the most apical xylem vessel, suggesting that an endogenous factor involved in primordia initiation is transported in the xylem. The primordia which develop after colchicine treatment grow out as lateral roots; this is in contrast with those which form after IAA treatment and which do not undergo elongation. These results, which it must be emphasized apply only to the apical 1 cm of treated roots, indicate that lateral root primordia become sensitive to IAA at a certain stage in their development. Exogenous IAA acts as an inhibitor.The new meristem, which forms in the primary root apex after colchicine treatment, contains both diploid and polyploid cells, i.e. it was formed from cells that were unaffected and from cells that were affected by colchicine. Following colchicine treatment the size of the meristem shrinks and this can be prevented by treatment with IAA. This and other evidence presented here, suggests that IAA is a factor involved in the control of the size of the apical meristem in normal roots.     

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

Low concentrations of auxin (e.g. 10⁻¹⁰m) do not promote the growth of intact seedling roots of maize (Zea mays L. Bear Hybrid WF 9 × 38). Higher concentrations are inhibitory. When the roots are pretreated with the ethylene biosynthesis inhibitors, cobalt and aminoethoxyvinylglycine, auxin (10⁻¹⁰ to 10⁻⁸m) 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.     

Auxin transport in roots

Summary The movement of IAA has been investigated in roots of dark-grown seedlings of Zea mays using IAA-I-¹⁴C.With 6-mm segments excised 1 mm below the apex of the root it has been shown that: (a) There is a strictly acropetal flux of IAA through the tissues, the amount of IAA found in an apical receiving block increasing almost linearly with increasing transport period up to about 6–7 hours, but thereafter declining for at least a further 18 hours. The onset of this decline appears to be dependent upon the concentration of IAA in the donor block. (b) The amount of IAA recovered in the apical receiving block increases with increasing concentration of IAA in the donor block over the range from 0.1–10 M, with transport periods of both 4 and 9 hours. (c) The radioactivity in the receiving block is confined to the IAA molecule. (d) The orientation of the segment with respect to gravity did not significantly affect the acropetal polar flux of IAA in the tissue.With non-decapitated 7-mm root apices it has been found that the presence of the apex has no effect on the strictly acropetal flux of IAA in the tissues, but that it entirely prevented the emergence of IAA into an apical receiving block.     

Three maize root-specific genes are not correctly expressed in regenerated caps in the absence of the quiescent center

The quiescent center is viewed as an architectural template in the root apical meristem of all angiosperm and gymnosperm root tips. In roots of Arabidopsis thaliana (L.) Heynh., the quiescent center inhibits differentiation of contacting initial cells and maintains the surrounding initial cells as stem cells. Here, the role of the quiescent center in the development of the maize (Zea mays L.) root cap has been further explored. Three maize root-specific genes were identified. Two of these were exclusively expressed in the root cap and one of them encoded a GDP-mannose-4,6-dehydratase. Most likely these two genes are structural, tissue-specific markers of the cap. The third gene, a putative glycine-rich cell wall protein, was expressed in the cap and in the root epidermis and, conceivably is a positional marker of the cap. Microsurgical and molecular data indicate that the quiescent center and cap initials may regulate the positional and structural expression of these genes in the cap and thereby control root cap development. Received: 22 September 1999 / Accepted: 9 November 1999     

In Vitro Transformation of Root meristem to Shoot and Plantlets in Vanilla planifolia

A study is reported of histogenesis and organogenesis duringthe processes leading up to plantlet formation in tip culturesof aerial roots of Vanilla planifolia. Young root tips excisedfrom aerial roots, less than 15 cm long, when cultured in liquidMS medium containing IAA and KN showed gravitropic responseuntil cap lysis began. With the collapse of the distal halfof the cap, the cells of the quiescent centre divided forminga hemispherical mass of cells. Further localized divisions onthe periphery of the hemisphere resulted in a number of meristemoidseach of which differentiated into a shoot meristem with leafprimordia. Procambium differentiated first beneath the apicalmeristem after two to three leaf primordia had formed and thenat the base of the leaves. After a few leaves have been formeda root meristem differentiated in close lateral proximity tothe basal end of the shoot procambium. Formation of a plateof vasculature at the nodal region of the first formed leaf,procambialization of the root and the bridging up of the shootand root vasculature with the nodal plate are described. Vanilla planifolia, root tip, in vitro, quiescent centre, meristemoid, plantlet     

Regulation of auxin transport in pea (Pisum sativum L.) by phenylacetic acid: inhibition of polar auxin transport in intact plants and stem segments

The transport of [¹⁴C]phenylacetic acid (PAA) in intact plants and stem segments of light-grown pea (Pisum sativum L. cv. Alderman) plants was investigated and compared with the transport of [¹⁴C]indiol-3yl-acetic acid (IAA). Although PAA was readily taken up by apical tissues, unlike IAA it did not undergo long-distance transport in the stem. The absence of PAA export from the apex was shown not to be the consequence of its failure to be taken up or of its metabolism. Only a weak diffusive movement of PAA was observed in isolated stem segments which readily transported IAA. When [1-¹⁴C]PAA was applied to a mature foliage leaf in light, only 5.4% of the ¹⁴C recovered in ethanol extracts (89.6% of applied ¹⁴C) had been exported from the leaf after 6.0 h. When applied to the corresponding leaf, [¹⁴C]sucrose was readily exported (46.4% of the total recovered ethanol-soluble ¹⁴C after 6.0 h). [1-¹⁴C]phenylacetic acid applied to the root system was readily taken up but, after 5.0 h, 99.3% of the recovered ¹⁴C was still in the root system.When applied to the stem of intact plants (either in lanolin at 10 mg·g^-1, or as a 10^-4 M solution), unlabelled PAA blocked the transport through the stem of [1-¹⁴C]IAA applied to the apical bud, and caused IAA to accumulate in the PAA-treated region of the stem. Applications of PAA to the stem also inhibited the basipetal polar transport of [1-¹⁴C]IAA in isolated stem segments. These results are consistent with recent observations (C.F. Johnson and D.A. Morris, 1987, Planta 172, 400–407) that no carriers for PAA occur in the plasma membrane of the light-grown pea stem, but that PAA can inhibit the carrier-mediated efflux of IAA from cells. The possible functions of endogenous PAA are discussed and its is suggested that an important role of the compound may be to modulate the polar transport and-or accumulation by cells of IAA.Abbreviations IAA indol-3yl-acetic acid - NPA N-1-naphthylphthalamic acid - PAA phenylacetic acid - IIBA 2,3,5-triiodobenzoic acid     

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm⁻¹ h⁻¹) and cell division (cells cell⁻¹ h⁻¹). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement.