Effect of auxin on acropetal auxin transport in roots of corn

Acropetal [¹⁴C]indoleacetic acid (IAA) transport was investigated in roots of corn. At least 40 to 50% of this movement is dependent on activities in the root apex. Selective excision of various populations of cells comprising the root apex, e.g. the root cap, quiescent center, or proximal meristem show that the proximal meristem is the critical region in the apex with regard to influencing IAA movement. The quiescent center has no influence and the root cap has only a minor effect. Excision and replacement of the proximal meristem with an exogenous supply of 10⁻⁸ to 10⁻⁹ molar IAA prevents the reduction in acropetal IAA transport which would normally occur in the absence of this meristem. Substituting 10⁻⁹ molar IAA for the excised root cap brings about a significant increase in the amount of IAA moved acropetally, as compared to intact roots with the root cap still in place. From this and previous work, it is concluded that IAA synthesis occurring in the proximal meristem stimulates the movement of IAA from the basal to apical end of the root.     

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.     

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     

THE QUIESCENT CENTER IN CULTURED ROOTS OF CONVOLVULUS ARVENSIS L.

Cultured roots of Convolvulus arvensis were incubated in 0.2–0.3 μc/ml methyl-³H-thymidine for different intervals of time. In roots supplied with tritiated thymidine for 12 hr, 14 hr, 48 hr, or 14 hr followed by transfer to fresh medium for 24 hr, autoradiographs prepared of serial, longitudinal sections of the root tips showed the presence of a subterminal quiescent center in the root proper at the distal poles of the central cylinder and cortex. In addition, a zone of unlabelled cells in the columella, distal to the root cap initials, was present. In roots supplied continuously with tritiated thymidine for 64 hr, 96 hr, and 120 hr, the quiescent center was either reduced in size or was not present.     

THE ISOLATION AND CULTURE IN VITRO OF THE QUIESCENT CENTER OF ZEA MAYS

Using 3-day-old seedling roots of Zea mays L., cv. Kelvedon 33, it was possible to remove the root cap by a simple surgical manipulation without damage to the root proper. By a further small cut, the quiescent center (QC) itself was isolated. This double-convex lens-shaped tissue piece 100 X 250 μm is composed of 1000–1500 cells representing only 0.25 mm³ in volume. The explant was demonstrated unequivocally by ³H-thymidine incorporation before excision and then by autoradiography to be composed of the specific cells usually designated the quiescent center. Using sterile techniques, the QC's were placed on nutrient agar slants and allowed to grow in culture. Of a number of nutrient media tested, only a medium supplemented with organic nitrogen components, indoleacetic acid, kinetin and inorganic nutrients plus sucrose (S2M + K -2,4-D) was effective in eliciting development. Thirty to 40 percent of the 150 isolated QC's grown on this medium formed elongated roots, up to 2 cm in length in 3–4 weeks. Roots developing on agar medium showed in their proximal portion a vascular pattern with 5–6 metaxylem elements or variations of this pattern, but as the root elongated, the vascular pattern was progressively reduced in complexity at the more distal end to a small central group of metaxylem elements. When agar-grown roots were transferred after one week in culture to a liquid nutrient medium of the same composition, the initially reduced vascular pattern evident in the proximal tissues became progressively more complex in the distal portion of the root and after 2 cm of elongation, showed an essentially normal primary vascular tissue pattern characteristic of the seedling root.     

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.     

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.     

Localization and identification of auxin in roots of Zea mays

Summary Roots of 3.5-day-old seedlings of Zea mays cv. Giant White Horsetooth contain an extractable auxin which has chromatographic properties and reactions to chromogenic sprays identical with those of indole-3-acetic acid (IAA). By separating stele from cortex (and root tips) before extraction it was shown that the auxin is localized predominantly in the stele, with little being found in the cortex. Whole roots, isolated cortices and isolated steles accumulate and metabolize exogenously applied IAA-1-¹⁴C. The stelar tissue is distinguished from whole roots and cortical tissue in having a different pattern of IAA metabolism.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

Transport of [3H]IAA label in gravistimulated primary roots of maize

The curvature of roots in response to gravity is attributed to the development of a differential concentration gradient of IAA in the top and bottom of the elongation region of roots. The development of the IAA gradient has been attributed to the redistribution of IAA from the stele to cortical tissues in the elongation region. The gravistimulated redistribution of IAA was investigated by applying [³H]IAA to the cut surface of 5 mm apical primary root segments. The movement of label from the stele-associated [³H]IAA into the root, tip, root cap, and cortical tissues on the top and bottom of the elongation region was determined in vertically growing roots and gravistimulated roots. Label from the stele moved into the region of cell differentiation (root tip) prior to accumulating in the elongation region. Little label was observed in the root cap. Gravistimulation did not increase the amount of label moving from the stele; but gravistimulation did increase the amount of label accumulating in cortical tissues on the lower side of the elongation region, and decreased the amount of label accumulating in cortical tissues on the upper side of the elongation region. Removal of the cap prior to or immediately following gravity stimulation rendered the roots partially insensitive to gravity and also prevented gravity-induced asymmetric redistribution of label. However, removal of the root cap following 30 min of gravistimulation did not alter root curvature or the establishment of an IAA asymmetry across the region of root elongation. These results suggest that a signal originating in the root cap directs auxin redistribution in tissues behind the root cap, leading to the development of an asymmetry of IAA concentration in the elongation region that in turn causes the differential growth rate in the elongation region of a graviresponding root.     

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     

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     

An Auxin Gradient and Maximum in the Arabidopsis Root Apex Shown by High-Resolution Cell-Specific Analysis of IAA Distribution and Synthesis

Local concentration gradients of the plant growth regulator auxin (indole-3-acetic acid [IAA]) are thought to instruct the positioning of organ primordia and stem cell niches and to direct cell division, expansion, and differentiation. High-resolution measurements of endogenous IAA concentrations in support of the gradient hypothesis are required to substantiate this hypothesis. Here, we introduce fluorescence-activated cell sorting of green fluorescent protein–marked cell types combined with highly sensitive mass spectrometry methods as a novel means for analyses of IAA distribution and metabolism at cellular resolution. Our results reveal the presence of IAA concentration gradients within the Arabidopsis thaliana root tip with a distinct maximum in the organizing quiescent center of the root apex. We also demonstrate that the root apex provides an important source of IAA and that cells of all types display a high synthesis capacity, suggesting a substantial contribution of local biosynthesis to auxin homeostasis in the root tip. Our results indicate that local biosynthesis and polar transport combine to produce auxin gradients and maxima in the root tip.     

The effect of zeatin and iso-pentenyladenine on IAA transport from the shoot to the root of Pinus pinea seedlings

The tips of the tap roots of Pinus pinea seedlings were dipped in zeatin or iso-pentenyladenine solutions. Immediately after cytokinin application to the root tip or after a 24 h lag phase, [2-¹⁴C]IAA was applied to the shoot apex. Treating with zeatin resulted in an increase in [2-¹⁴C]IAA transport from the shoot to the root. Iso-pentenyladenine also caused a slight increase in transport of radioactivity to the root but this was less pronounced compared to the results obtained with zeatin. With zeatin treatment increasing amounts of radioactivity accumulated in the lateral root emerging zone of the tap root (Section III). This was in sharp contrast to the treatment with iso-pentenyladenine where little radioactivity accumulated in this section of the root. Recovery of radioactivity 48 h after applying [2-¹⁴C]IAA showed that 33% of the recovered radioactivity co-chromatographed with authentic IAA. The implications of the effect of different cytokinins on the distribution of radioactivity along the tap root of Pinus pinea following [2-¹⁴C]IAA application to the shoot are discussed.Abbreviations Z zeatin - iP iso-pentenyladenine - TCL thin-layer chromatography     

ROOT APEX STRUCTURE IN EPHEDRA MONOSPERMA AND EPHEDRA CHILENSIS (EPHEDRACEAE)

The root meristem of E. monosperma and E. chilensis possesses a central group of distinctive, large cells. These cells have large nuclei with scattered heterochromatin, proplastids with no starch, small vacuoles, mitochondria, few dictyosomes and endoplasmic reticulum cisternae, and lipid deposits. Over a 24 hr labelling period, the large cells fail to incorporate ³H-thymidine, whereas cells both distal and proximal to this region do. A quiescent center which includes these large cells is present therefore. Both species have an extensive root cap, the length being contributed by mitoses in many tiers of cells distal to the quiescent center. The root cap consists of a columella and peripheral regions. Distinctive amyloplasts, an increase in the number of endoplasmic reticulum cisternae and dictyosomes, large vacuoles, and lipid deposits are characteristic of differentiated columella cells. Peripheral cells elongate, lose most of their starch, and are eventually sloughed from the root.     

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.     

The transport and metabolism of 14C-labelled indoleacetic acid in intact pea seedlings

Summary Part of the IAA-I- or IAA-2-¹⁴C applied at low concentrations to the apices of intact, light-grown dwarf pea seedling was transported unchanged to the root system The calculated velocity of transport in the stem was 11 mm per hour. In the root the label accumulated in the developing lateral root primordia.A large proportion of the applied IAA was converted by tissues of the apical bud, stem and root to indole-3-acetyl-aspartic acid (IAAsp). This compound was not transported. In addition evidence was obtained for the formation of IAA-protein complexes in the apex and roots, but not in the fully-expanded internodes.Large quantities of a decarboxylation product of IAA, tentatively indentified as indole-3-aldehyde (IAld), and several minor metabolites of IAA, were detected in extracts of the roots and first internodes, but not in the above-ground organs exposed to light. These compounds were readily transported through stem and root tissues. Together, the decarboxylation of IAA and the formation of IAAsp operated to maintain a relatively constant level of free IAA-¹⁴C in the root system.     

Localization of a phytohormone using immunocytochemistry

The localization of cytokinins in corn root tips was investigated using antibodies or antibody fragments directed against dihydrozeatin riboside and labeled with rhodamine or colloidal gold. Roots were sectioned at -30 degrees to -40 degrees for immunofluorescence or freeze-substituted in ethanol or acetone and embedded in plastic for electron microscopy. Meristematic cells surrounding the quiescent center as well as root cap cells were specifically labeled using direct immunofluorescence techniques, whereas cells of the quiescent center did not bind label. Tissue sections treated with colloidal gold-labeled antibody fragments had gold particles widely distributed in the cytoplasm. The results show that the quiescent center is not the major site of cytokinin localization in root tips.     

DURATION OF CELL CYCLES IN CULTURED ROOTS OF CONVOLVULUS

The durations of the cell cycle in physiologically different regions of the meristem of cultured roots of Convolvulus arvensis were determined by the metaphase-accumulation technique involving colchicine. The cell cycle in the root cap increases from 13 hr in the actively dividing initials of the first tier to 155 hr in the slowly dividing initials of tiers 2–4 to an indeterminate value for derivatives of the initials in the root cap columella. The cycle times for the cells of the central cylinder and cortex are 21 and 27 hr, respectively. The cells of the quiescent center have a cycle of an estimated 420 hr. The duration of the cell cycle in these different regions is discussed in relation to the increased duration of G₁ in slowly or non-dividing cells. The possible regulation of cell division by the synthesis of a cell-division factor in the quiescent center is also discussed.     

The Role of Boron in Plant Growth: IV. INTERRELATIONSHIPS BETWEEN BORON AND INDOL-3YL-ACETIC ACID IN THE METABOLISM OF BEAN RADICLES

The hypothesis that boron deficiency is equivalent to a stateof IAA toxicity was explored. Bioassays showed that extractsof substances similar to IAA taken from boron-deficient rootswere significantly more inhibitory to the growth of bean-rootsegments than those from normal roots. Supplied IAA and borondeficiency together restricted root growth to a greater extentthan either deficiency or IAA treatment separately. Roots werefound to recover more quickly from the inhibitory effects ofsupplied IAA if boron was present at high (0.5 ppm) rather thanlow (0.01 ppm) concentrations. Experiments with ¹⁴C-labelled IAA showed that deficient rootsabsorbed ¹⁴C more slowly than boron-fed roots and there wasalso a lower rate of decarboxylation in the deficient tissue.Consideration of the published evidence showed that many ofthe effects of boron deficiency could follow from an upset inIAA metabolism. It is suggested that boron-deficient tissuesuffers from excess auxin either because the element is necessaryfor some growth process, such as cell wall formation or nucleicacid synthesis, which, when impaired, results in the accumulationof auxin, or because the IAA-oxidation system is affected byphenolic inhibitors which boron normally inactivates by complexformation.