The role of auxin in hairy root induction

Summary We have investigated the relative role of auxin and of Agrobacterium rhizogenes T-DNA in the induction of hairy roots. By infecting carrot discs with suitably constructed bacterial strains containing different T-DNA complements, we have shown that both auxin and the presence of T-DNA in the carrot cells are required for root growth on the discs. Auxin added alone or in combination with cytokinin is not sufficient to induce rooting on uninfected discs. Also cells transformed by T-DNA containing only auxin synthetic genes very rarely differentiate into roots. On the other hand auxin is necessary for hairy root induction since A. rhizogenes devoid of T-DNA-borne auxin genes is not capable of eliciting symptoms in the absence of hormone. Auxin is not required for either T-DNA transfer or T-DNA expression in the transformed host. Cells infected in the absence of auxin, which do not respond by rooting, do contain T-DNA whose expression is shown by the synthesis of hairy root opines; subsequent addition of auxin to these quiescent transformed cells results in root development. A model for hairy root induction where the action of T-DNA is envisaged as conferring auxin responsiveness to the transformed cells is discussed.     

The role of endogenous auxin in root initiation

This paper describes the process of the formation of adventitious roots. There appears to be good agreement that this consists of four stages, defifferentiation coupled with the formation of a meristematic locus, cell division to form a radially symmetrical cluster of cells, further divisions coupled with organisation into a bilaterally symmetrical meristem and finally growth of cells in the basal part of the meristem which causes its protursion through the epidermis. Evidence for the involvement of auxins in these various stages is reviewed and the extent to which rooting of easy- and hard-to-root species can be accounted for in terms of auxin content discussed. Peaks of IAA occur soon after excision of cuttings in some species and there is some evidence suggesting that this is correlated with changes in peroxidase activity. The possible involvement of cytokinins with auxins is briefly considered.     

The role of endogenous auxin in root initiation

This paper is the second part of a review which considers evidence for the involvement of auxin in root initiation. Part II examines the research being carried out with transformed plant tissues. Agrobacterium rhizogenes causes abundant root initiation at the site of inoculation. Ri plasmid T-DNA contains several genes which encode enzymes involved in the biosynthesis and metabolism of indole-3-acetic acid. Transfer of various fragments of the Ri plasmid has also been reported to confer increased sensitivity to auxin upon plant cells. Controlled expression of these genes in the plant genome potentially offer an insight for developmental plant physiologists into the role of plant growth substances in the process of root initiation. The importance of absolute levels of IAA in the stimulation of root initiation is discussed.     

Localization of intracellular calcium in root meristematic cells and root cap cells of maize

The localization of Ca²⁺ in cells of the periblem and dermatogen in the root meristem and the columella and peripheral cells of the root cap of maize was examined by the precipitation method of potassium pyroantimonate and EGTA-treatment. In periblem and dermatogen cells, Ca²⁺ was found to be localized in the nucleoplasm and granular zone of the nucleolus. Ca²⁺ was also found in most cell organelles: in the matrix in mitochondria, on the thylakoid membrane in proplastids, in the vacuoles and on the plasma membranes. Ca²⁺ was also distributed throughout the cytoplasmic ground matrix. Much Ca²⁺ was present in the cell wall soon after its formation during the cell division. Ca²⁺ was also conspicuous in the vesicles of Golgi in the dermatogen cells. In columella and peripheral cells, there was less Ca²⁺ in the organelles and cytoplasmic ground matrix, but Ca²⁺ was present in Golgi vesicles in the peripheral cells. Electron microscopic and X-ray microanalysis showed that Ca²⁺ was also present in the mucilaginous layer, the outermost cell wall of the peripheral cells.     

Cd-induced system of defence in the garlic root meristematic cells

Studies on cadmium effects in the root meristematic cells of Allium sativum L. were carried out using electron microscopy in order to explain the possible mechanisms of garlic seedlings’ tolerance to Cd stress. Seedlings were treated with 0.01, 0.10 and 1.00 mM CdCl₂ solutions for 0.5, 1, 2, 4, 8, 10, 12, 24 and 48 h, respectively. The results indicated that cell walls, plasma membrane and main organelles actively participated in Cd detoxification and tolerance at low Cd concentrations. Once excessive Cd ions entered the cytosol, a defence mechanism becomes activated, protecting the cells against cadmium toxicity. However, under high Cd content in cells, the cell structure was damaged, even leading to cells death.     

Endogenous and exogenous auxin in the control of root growth

The endogenous indol-3yl-acetic acid (IAA) of detipped apical segments from roots of maize (cv ORLA) was greatly reduced by an exodiffusion technique which depended upon the preferential acropetal transport of the phytohormone into buffered agar. When IAA was applied to the basal cut ends of freshly prepared root segments only growth inhibitions were demonstrable but after the endogenous auxin concentration had been reduced by the exodiffusion technique it became possible to stimulate growth by IAA application. The implications of the interaction between exogenous and endogenous IAA in the control of root segment growth are discussed with special reference to the role of endogenous IAA in the regulation of root growth and geotropism.Abbreviations IAA indol-3yl-acetic acid - GC-MS gas chromatography-mass spectrometry     

The role of reactive oxygen species in cell growth: lessons from root hairs

Reactive oxygen species (ROS) play a diversity of roles in plants. In recent years, a role for NADPH oxidase-derived ROS during cell growth and development has been discovered in a number of plant model systems. These studies indicate that ROS are required for cell expansion during the morphogenesis of organs such as roots and leaves. Furthermore, there is evidence that ROS are required for root hair growth where they control the activity of calcium channels required for polar growth. The role of ROS in the control of root hair growth is reviewed here and results are highlighted that may provide insight into the mechanism of plant cell growth in general.     

Zinc-induced vacuolation in root meristematic cells of Festuca rubra L.

Abstract. The effect of Zn on vacuole development in root meristematic cells was examined in three cultivars of Festuca rubra: a Zn-tolerant cultivar (Merlin), a salt-tolerant cultivar (Hawk) and a non-tolerant cultivar (S.59) in order to determine whether or not vacuole development was related to Zn tolerance. Treatment with Zn greatly increased the percentage of cells in the apical meristem which were vacuolated in all three cultivars (7.01-fold increase in Merlin, 3.61-fold increase in Hawk and 5.39-fold increase in S.59 over the range 0–0.5 μg Zn cm^?3). Morphometric analyses on electromicrographs indicated that the percentage total vacuolar volume fraction of meristematic tissue was also increased by Zn treatment. Most of this increase was due to an increase in a particular component of the vacuole which was 0.2–0.5 μm in diameter, spherical or ovoid in outline and possessed a distinct amorphous electron dense matrix (Type A intravacuolar body). X-ray microanalysis revealed that this matrix was rich in calcium and phosphorus in control plants. In Zn-treated roots, elevated levels of Zn were found in the matrix in Merlin and Hawk, but not in S.59. In addition, intravacuolar membranous whorls or myelin bodies were more highly organized in Zn-treated Merlin and Hawk but not S.59. Elevated Zn levels were not found in any other vacuolar component nor in nuclei, nucleoli, cell walls or ground cytoplasm. Control roots of Merlin possessed a greater type A vacuolar volume fraction than either Hawk or S.59 which may confer a greater capacity to compartmentalize Zn at the onset of exposure to toxic metals than the other cultivars. Predictably, the EM procedures resulted in loss of Zn from the roots to the fixative and dehydrating solutions. However, the rate of loss was the same (approximately 75–80%) regardless of cultivar. These findings are discussed in relation to their possible role in the mechanisms of Zn tolerance in these cultivars.     

Short-term boron deprivation inhibits endocytosis of cell wall pectins in meristematic cells of maize and wheat root apices

By using immunofluorescence microscopy, we observed rapidly altered distribution patterns of cell wall pectins in meristematic cells of maize (Zea mays) and wheat (Triticum aestivum) root apices. This response was shown for homogalacturonan pectins characterized by a low level (up to 40%) of methylesterification and for rhamnogalacturonan II pectins cross-linked by a borate diol diester. Under boron deprivation, abundance of these pectins rapidly increased in cell walls, whereas their internalization was inhibited, as evidenced by a reduced and even blocked accumulation of these cell wall pectins within brefeldin A-induced compartments. In contrast, root cells of species sensitive to the boron deprivation, like zucchini (Cucurbita pepo) and alfalfa (Medicago sativa), do not internalize cell wall pectins into brefeldin A compartments and do not show accumulation of pectins in their cell walls under boron deprivation. For maize and wheat root apices, we favor an apoplastic target for the primary action of boron deprivation, which signals deeper into the cell via endocytosis-mediated pectin signaling along putative cell wall-plasma membrane-cytoskeleton continuum.     

F-actin-dependent endocytosis of cell wall pectins in meristematic root cells. Insights from brefeldin A-induced compartments

Brefeldin A (BFA) inhibits exocytosis but allows endocytosis, making it a valuable agent to identify molecules that recycle at cell peripheries. In plants, formation of large intracellular compartments in response to BFA treatment is a unique feature of some, but not all, cells. Here, we have analyzed assembly and distribution of BFA compartments in development- and tissue-specific contexts of growing maize (Zea mays) root apices. Surprisingly, these unique compartments formed only in meristematic cells of the root body. On the other hand, BFA compartments were absent from secretory cells of root cap periphery, metaxylem cells, and most elongating cells, all of which are active in exocytosis. We report that cell wall pectin epitopes counting rhamnogalacturonan II dimers cross-linked by borate diol diester, partially esterified (up to 40%) homogalacturonan pectins, and (1-->4)-beta-D-galactan side chains of rhamnogalacturonan I were internalized into BFA compartments. In contrast, Golgi-derived secretory (esterified up to 80%) homogalacturonan pectins localized to the cytoplasm in control cells and did not accumulate within characteristic BFA compartments. Latrunculin B-mediated depolymerization of F-actin inhibited internalization and accumulation of cell wall pectins within intracellular BFA compartments. Importantly, cold treatment and protoplasting prevented internalization of wall pectins into root cells upon BFA treatment. These observations suggest that cell wall pectins of meristematic maize root cells undergo rapid endocytosis in an F-actin-dependent manner.     

Mitotic index of meristematic cells and root growth of Pisum sativum is affected by inositol cycle modulators

Relationships between cell division and inositol cycle modulation caused by different effectors in roots of Pisum sativum were studied. Stimulation of the inositol cycle by myoinositol increased the mitotic index of meristematic cells and root length, while the inhibition of the cycle with Li+ and a heavy metal Gd3+ considerably decreased mitotic activity and growth. Exposure of roots to 10 mM CaCl2 and 15 mM myoinositol resulted in the accumulation of chromosome aberrations. Changes in the activity of inositol cycle are assumed to be involved in the root growth control.     

The reaction of Lupinus angustifolius L. root meristematic cell nucleoli to lead

The effect of 2–48 h treatment of Lupinus angustifolius L. roots with lead nitrate at the concentration of 10⁻⁴ M on the nucleoli in meristematic cells was investigated. In the lead presence the number of ring-shaped as well as segregated nucleoli increased especially after 12–48 h of treatment, while spindle-shaped nucleoli appeared after 24 h and 48 h. Lead presence also increased the frequency of cells with silver-stained particles in the nucleus and the number of these particles especially from the 12th hour of treatment. It was accompanied by significant decline of nucleolar area. Analysis of these cells in transmission electron microscope confirmed the presence of ring-shaped and segregated nucleoli. Moreover, electron microscopy revealed compact structure nucleoli without granular component. Additionally, one to three oval-shaped fibrillar structures attached to nucleolus or lying free in the nucleoplasm were visible. The possible mechanism of lead toxicity to the nucleolus is briefly discussed.     

An auxin transport independent pathway is involved in phosphate stress-induced root architectural alterations in Arabidopsis. Identification of BIG as a mediator of auxin in pericycle cell activation

Arabidopsis (Arabidopsis thaliana) plants display a number of root developmental responses to low phosphate availability, including primary root growth inhibition, greater formation of lateral roots, and increased root hair elongation. To gain insight into the regulatory mechanisms by which phosphorus (P) availability alters postembryonic root development, we performed a mutant screen to identify genetic determinants involved in the response to P deprivation. Three low phosphate-resistant root lines (lpr1-1 to lpr1-3) were isolated because of their reduced lateral root formation in low P conditions. Genetic and molecular analyses revealed that all lpr1 mutants were allelic to BIG, which is required for normal auxin transport in Arabidopsis. Detailed characterization of lateral root primordia (LRP) development in wild-type and lpr1 mutants revealed that BIG is required for pericycle cell activation to form LRP in both high (1 mm) and low (1 microm) P conditions, but not for the low P-induced alterations in primary root growth, lateral root emergence, and root hair elongation. Exogenously supplied auxin restored normal lateral root formation in lpr1 mutants in the two P treatments. Treatment of wild-type Arabidopsis seedlings with brefeldin A, a fungal metabolite that blocks auxin transport, phenocopies the root developmental alterations observed in lpr1 mutants in both high and low P conditions, suggesting that BIG participates in vesicular targeting of auxin transporters. Taken together, our results show that auxin transport and BIG function have fundamental roles in pericycle cell activation to form LRP and promote root hair elongation. The mechanism that activates root system architectural alterations in response to P deprivation, however, seems to be independent of auxin transport and BIG.     

Fractionation and characterization of polyadenylated RNA from broad bean meristematic root cells

Several populations of polyadenylated RNA from Vicia faba méristematic root cells were fractionated by stepwise thermal elution from poly(U)-Sepharose following sequential phenol extraction. Analysis of these fractions showed that the size of the poly(A) segment could influence this fractionation, but in some cases other characteristics of the molecule are involved. Evidence was obtained that 45–60% of the nucleotides of plant polyadenylated RNA are in base paired regions, as was previously demonstrated for mammalian mRNA.

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Resume Après extraction séquentielle au phénol des ARN totaux de cellules méristématiques de racine de Fêve, les ARN polyadénylés, fixés sur colonne de poly(U)-Sépharose, sont séparés en plusieurs populations par une élution thermique à 40° et 50° C. L'analyse structurale de ces fractions montre que si la taille du segment des poly(A) joue le principal rôle dans ce partage, d'autres caractéristiques de molécules d'ARN peuvent interférer. Nous montrons de plus pour la première fois que 45 à 60% des nucléotides des ARN polyadénylés végétaux sont engagés dans des structures à double brin, comme c'est le cas dans les ARN polyadénylés animaux.

     

Plant growth regulators and adventitious root development in relation to auxin

Adventitious root formation in stem cuttings of mung bean was enhanced by ethrel, which had an additive effect when employed simultaneously with indolebutyric acid (IBA). Abscisic acid (ABA) did not influence the number of roots per cutting whereas gibberellic acid (GA₃) and kinetin were without effect on rooting at lower concentrations but were inhibitory at higher concentrations. Nevertheless, all three of these chemicals showed synergistic interactions with IBA and/or indol-3-ylacetic acid (IAA) and thereby significantly promoted root formation. A localised application of morphactin to the epicotyl of cuttings totally inhibited root production irrespective of which of the foregoing growth regulators were suppliedvia the hypocotyl. Morphactin application also prevented root formation in cuttings treated with vitamin D₂. The various growth regulators employed had differing effects on growth of roots but there was no simple relationship between their effects on root formation and subsequent root growth.