Modulation of the root epidermal phenotype by hormones,inhibitors and iron regime

Patterning of epidermal cells is subject to genetic regulation but also influenced by environmental stimuli. To adapt to unfavorable environmental conditions plants have developed various mechanisms to increase the plasma membrane's surface area of epidermal root cells, for example through the formation of root hairs and differentiation of rhizodermal transfer cells. Mechanisms controlling cell fate speciation in the rhizodermis were investigated by application of hormones and hormone antagonists. In addition, the effect of Fe deficiency on root epidermal patterning and Fe(III)-reduction activity was examined. In the iron-hyperaccumulating pea mutants dgl and brz and in the Arabidopsis mutant man1 Fe(III)-reduction activity was found to be up-regulated under both high and low iron supply. In contrast, morphological responses such as the development of transfer cells and extranumerary root hairs was repressed by a high iron concentration in the external medium. All morphological responses can be mimicked by exogenous application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) or the auxin analog 2,4-dichlorophenoxyacetic acid (2,4-D). Conversely, Fe(III)-reduction rates were not influenced or only slightly affected by the hormone treatment. Application of inhibitors of ethylene synthesis, ethylene action or auxin transport was effective only in inhibiting the formation of extra root hairs, indicating that these hormones are not required for transfer cell formation or expression of Fe(III) reduction. These data suggest that the Fe reductase induced by iron stress does not depend on the formation of transfer cells and further imply separate regulatory pathways for the two responses. The data are compatible with a model in which root reduction activity is modulated by a shoot-borne signal coordinating iron uptake with the shoot demand, while the epidermal phenotype is primarily dependent on the intracellular iron concentration of root cells.     

Acclimative changes in root epidermal cell fate in response to Fe and P deficiency: a specific role for auxin?

Summary Root hair formation and the development of transfer cells in the rhizodermis was investigated in various existing auxinrelated mutants ofArabidopsis thaliana and in the tomato mutantdiageotropica. Wild-type Arabidopsis plants showed increased formation of root hairs when the seedlings were cultivated in Fe- or P-free medium. These extranumerary hairs were located in normal positions and in positions normally occupied by nonhair cells, e.g., over periclinal walls of underlying cortical cells. Defects in auxin transport or reduced auxin sensitivity inhibited the formation of root hairs in response to Fe deficiency completely but did only partly affect initiation and elongation of hairs in P-deficient roots. Application of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid or the auxin analog 2,4-dichlorophenoxyacetic acid did not rescue the phenotype of the auxin-resistantaxr2 mutant under control and Fe-deficient conditions, indicating that functionalAXR2 product is required for translating the Fe deficiency signal into the formation of extra hairs. The development of extra hairs inaxr2 roots under P-replete conditions was not affected by auxin antagonists, suggesting that this process is independent of auxin signaling. In roots of tomato, growth under Fe-deficient conditions induced the formation of transfer cells in the root epidermis. Transfer cell frequency was enhanced by application of 2,4-dichlorophenoxyacetic acid but was not inhibited by the auxin transport inhibitor N-1-naphthylphthalamic acid. In thediageotropica mutant, which displays reduced sensitivity to auxin, transfer cells appeared to develop in both Fe-sufficient and Fe-deficient roots. Similar to the wild type, no reduction in transfer cell frequency was observed after application of the above auxin transport inhibitor. These data suggest that auxin has no primary function in inducing transfer cell development; the formation of transfer cells, however, appears to be affected by the hormonal balance of the plants.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - TIBA triiodobenzoic acid - NPA N-1-naphthylphthalamic acid - STS silver thiosulfate     

Genetic analysis of two tomato mutants affected in the regulation of iron metabolism

Iron is one of the most important micronutrients for plants. Like other organisms, plants have developed active mechanisms for the acquisition of sufficient iron from the soil. Nevertheless, very little is known about the genetic mechanisms that control the active uptake. In tomato, two spontaneously derived mutants are available, which are defective in key steps that control this process. The recessive mutationchloronerva (chln) affects a gene which controls the synthesis of the non-protein amino acid nicotianamine (NA), a key component in the iron physiology of plants. The root system of the recessive mutantfer is unable to induce any of the characteristic responses to iron deficiency and iron uptake is thus completely blocked. We present a characterization of the double mutant, showing that thefer gene is epistatic over thechln gene and thus very likely to be one of the major genetic elements controlling iron physiology in tomato. In order to gain access to these two genes at the molecular level, both mutants were precisely mapped onto the high density RFLP map of tomato. Thechln gene is located on chromosome 1 and thefer gene is on chromosome 6 of tomato. Using this high-resolution map, a chromosome walk has been started to isolate thefer gene by map-based cloning. The isolation of thefer gene will provide new insights into the molecular mechanisms of iron uptake control in plants.     

Interaction of an immature seed-specific trans-acting factor with the 5′ upstream region of a rice glutelin gene

Summary Mutants of Arabidopsis thaliana (L) Heyhn were induced by soaking seed in the mutagen, ethyl methanesulfonate (EMS). The progeny of the mutagen-treated generation were screened for abnormal root gravitropism and three mutants were isolated: agr-1, agr-2 and agr-3. Genetic analysis showed these to be alleles of one locus distinct from that of two previously described allelic mutations, aux-1 and aux-2. The agr phenotypes are recessive and analysis of agr-1 suggests monofactorial inheritance. The roots of agr-1 appear to be totally agravitropic while those of agr-2 and agr-3 show reduced responses to gravity. agr-3 also shows negative root gravitropism as a variable trait. The roots of agr-1 and agr-2 were found to have normal responses to the inhibitory effects of 2,4-dichlorophenoxyacetic acid (2,4-D) but those of agr-3 were significantly more sensitive. No other phenotypic abnormalities were detected.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - EMS ethyl methanesulfonate     

The effect of plant growth regulators on growth, morphology and condensed tannin accumulation in transformed root cultures of Lotus corniculatus

This study concerns the effects of four different classes of plant growth regulators on root morphology, patterns of growth and condensed tannin accumulation in transgenic root cultures of Lotus corniculatus L. (Bird's-foot trefoil). Growth of transformed roots in 2,4-dichlorophenoxyacetic acid (2,4-D) resulted in decreased tannin levels relative to controls at concentrations of 10^-6 M and above, while gibberellic acid (GA₃) inhibited tannin accumulation at concentrations of 10^-7 M and above. Benzyladenine (BA) had little effect at low concentrations (10^-7 M and below) but resulted in an increase in tannin levels at 10^-6 M. Abscisic acid had little effect on levels of condensed tannins at any of the concentrations used. Experiments involving growth regulator addition and medium transfer demonstrated that 2,4-D inhibition of tannin accumulation could be reversed by GA₃ and BA, while GA₃ downregulation could only be reversed by the addition of 2,4-D. Although 2,4-D inhibited tannin accumulation, addition of 2,4-D to root cultures grown for 14 or 28 days in the absence of plant growth regulators stimulated both growth and tannin biosynthesis. Characteristic alterations in root morphologies accompanied growth regulator-mediated modulation of tannin biosynthesis. Growth in 2,4-D resulted in partially de-differentiated root cultures while growth in GA₃ produced roots with an elongated phenotype. Restoration of tannin biosynthesis in 2,4-D-treated roots was accompanied by root re-differentiation and the production of new lateral roots.Abbreviations ABA abscisic acid - BA benzyladenine - 2,4-d 2,4-dichlorophenoxyacetic acid - GA₃ gibberellic acid 3 - FW fresh weight     

Auxin binding in roots: A comparison between maize roots and coleoptiles

Auxin binding onto membrane fractions of primary roots of maize seedlings has been demonstrated using naphth-1yl-acetic acid (NAA) and indol-3yl-acetic acid (IAA) as ligands. This binding is compared with the already well characterized interaction between auxins and coleoptile membranes. The results indicate that while kinetic parameters are of the same order for root and coleoptile binding, a number of differences occur with respect to location in cells and relative affinity. The possible significance of the existence of such binding sites in root cells is discussed in relation to auxin action.Abbreviations 4-Cl-PA 4-chlorophenoxyacetic acid - EDTA ethylene diamine tetracetic acid - IAA indol-3yl-acetic acid - MCPA 2-methyl-4-chlorophenoxyacetic acid - NAA naphth-1yl-acetic acid - 2-NAA naphth-2yl-acetic acid - Tris 2-amino-2-(hydroxymethyl) propane-1,3 diol - TIBA 2,3,5 triiodobenzoic acid - NPA naphthylphthalamic acid - PCIB 4-chlorophenoxyisobutyric acid - PCPP 4-chlorophenoxyisopropionic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

High-frequency induction of adventitious shoots from hypocotyl segments ofLiquidambar styracifiua L. by thidiazuron

The effects of thidiazuron (TDZ) on adventitious bud and shoot formation from hypocotyl segments of sweetgum (Liquidambar styracifiua) were tested alone and in combination with 2,4-dichlorophenoxyacetic acid (2,4-D). The combination of 1 mg/1 TDZ with 0.01 mg/l 2,4-D resulted in the highest frequency of bud production. Lower concentrations of TDZ stimulated shoot production, generating the most shoots at 0.1 mg/1 TDZ with 0.01 mg/1 of 2,4-D. Inhibition of shoot elongation by TDZ was overcome by transferring shoot cultures to a shoot proliferation medium lacking TDZ or containing naphthaleneacetic acid and benzyladenine in addition to TDZ. Shoot production in liquid culture was significantly greater than that in solid culture. Comparisons of in vitro and ex vitro rooting of the adventitious shoots demonstrated that ex vitro rooting produced plants with faster growth rates and more extensive root systems.Abbreviations BA Benzyladenine - IBA indole-3-butyric acid - NAA naphthaleneacetic acid - PGR plant growth regulator - TDZ thidiazuron - 2,4-D 2,4-dichlorophenoxyacetic acid     

Expression of an auxin-inducible promoter of tobacco in Arabidopsis thaliana

The expression of the auxin-inducible Nt103-1 gene of tobacco was studied in Arabidopsis thaliana. For this purpose we introduced a gene fusion between the promoter of the gene and the -glucuronidase reporter gene (GUS) into Arabidopsis thaliana. The expression and location of GUS activity were studied histochemically in time and after incubation of seedlings on medium containing auxins or other compounds. The auxins 2,4-dichlorophenoxyacetic acid (2,4-D), indole-3-acetic acid (IAA), and 1-naphthylacetic acid (1-NAA) were able to induce GUS activity in the root tips of transgenic seedlings. The auxin transport inhibitor 2,3,5-triiodobenzoic acid was able to induce GUS activity not only in the root tip, but also in other parts of the root. Induction by the inactive auxin analog 3,5-dichlorophenoxyacetic acid was much weaker. Compounds like glutathione and the heavy metal CuSO₄ were weak inducers. GUS activity observed after induction by glutathione was located in the transition zone. Salicylic acid and compounds increasing the concentration of hydrogen peroxide in the cell were also very well able to induce GUS activity in the roots. The possible involvement of hydrogen peroxide as a second messenger in the pathway leading to the induction of the Nt103-1 promoter is discussed.     

Hormonal regulation of ripening in the strawberry,a non-climacteric fruit

Anthocyanin accumulation is one measure of ripening in the strawberry (Fragaria ananassa Duch.), a non-climacteric fruit. Neither aminoethoxyvinylglycine, an inhibitor of 1-aminocyclopropane carboxylic acid synthase, nor inhibitors of ethylene action (silver, norbornadiene) affected anthocyanin accumulation in ripening fruit. When the achenes were removed from one half of an unripe fruit there was an accelerated accumulation of anthocyanin and induction of phenylalanine ammonia lyase on the de-achened portion of the ripening fruit. These effects of achene removal could be prevented by the application of the synthetic auxins 1-naphthaleneacetic acid or 2,4-dichlorophenoxyacetic acid to the de-achened surface. The introduction of 1-naphthalene acetic acid into intact unripe strawberry fruit through the peduncle delayed their subsequent ripening, as measured by the accumulation of anthocyanin, loss of chlorophyll and decrease in firmness. These findings suggest that the decline in the concentration of auxin in the achenes as strawberry fruit mature modulates the rate of fruit ripening.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - AVG aminoethoxyvinylglycine - NAA 1-naphthaleneacetic acid - PA1 phenylalanine ammonia-lyase - POA phenoxyacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Tissue culture of Hypericum brasiliense Choisy: Shoot multiplication and callus induction

Hypericum brasiliense, a non-domesticated plant has been shown to have useful medicinal properties. This plant has not been cultivated so a protocol for mass propagation based on selection of superior clones was developed and a protocol established for the culture of callus cells that could be used for in vitro metabolite production. A micropropagation method based on amplification of nodal buds was developed, by selection, from ten seedling clones that were examined for growth rate, multiplication rate and rooting. The effect of various basal media, growth regulator types and concentrations were examined for optimal callus induction. Optimal callus induction occured on either Murashige and Skoog's or Gamborg's media supplemented with 1 to 2 mg l^–1 of 2,4-dichlorophenoxyacetic acid.Abbreviations B5 Gamborg's medium - 2,4-Dscd 2,4-dichlorophenoxyacetic acid - IAA indolacetic acid - MS Murashige & Skoog's medium - NAA naphtaleneacetic acid     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

Plant regeneration from shoot apex explants of foxtail millet

Green and etiolated shoot apices of foxtail millet (Setaria italica L.) cv. Nese 2A were cultured on Murashige and Skoog medium with four concentrations of 2,4-dichlorophenoxyacetic acid or 2,4,5-trichlorophenoxyacetic acid. In all treatments, embryogenic calli capable of plant regeneration were induced after ten weeks in culture. Calli induced on 2 mg l^-1 of 2,4-d from green apices gave a higher rate of plant regeneration in comparison with etiolated apices on the other treatments. Plant regeneration was obtained from one year-old cultures. Regenerated plants were successfully established in soil, reached maturity and produced seeds.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - EC embryogenic calli - NE nonembryogenic calli - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid     

Hernandulcin in hairy root cultures of Lippia dulcis

The hairy root culture of Lippia dulcis Trev., Verbenaceae, was established by transformation with Agrobacterium rhizogenes A4. The transformed roots grew well in Murashige and Skoog medium containing 2% sucrose. The roots turned light green when they were cultured under 16 h/day light. The green hairy roots produced the sweet sesquiterpene hernandulcin (ca. 0.25 mg/g dry wt) together with 20 other mono- and sesquiterpenes, while no terpenes were detected in the nontransformed root cultures. The growth and hernandulcin production in the hairy root cultures were influenced by the addition of auxins to the medium. The addition of a low concentration of chitosan (0.2 – 10.0 mg / l) enhanced the production of hernandulcin 5-fold.Abbreviations Cht chitosan - IAA indole-3-acetic acid - NAA 1-naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog(1962)     

Establishment of synchrony by starvation and readdition of auxin in suspension cultures of Catharanthus roseus cells

A system of synchronous cell division was established by starvation of auxin and its readdition to suspension cultures of cells of Catharanthus roseus L. cv. Little-Pinky. When cells in the stationary phase were transferred to fresh medium free of 2,4-dichlorophenoxyacetic acid (2,4-D), cells were arrested preferentially at the G₁ phase. After cells had been cultured for 2 days in medium without 2,4-D, readdition of 2,4-D induced the synchronous division of cells. In this system, 70–80% of cells divided synchronously within 3 to 4h, and the mitotic index increased sharply in parallel with the increase in cell number. Active synthesis of DNA was demonstrated by measurements of incorporation of [³H]-thymidine into the DNA fraction. The induction of cell division by the addition of 2,4-D was inhibited by treating cells with analogues of auxin, such as 2,4,6-trichlorophenoxyacetic acid and p-chlorophenoxyisobutyric acid.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - DAPI 4,6-diamidino-2-phenylindole - IAA indole-3-acetic acid - MS Murashige & Skoog - NAA -naphthalenacetic acid - PCIB p-chlorophenoxyisobutyric acid - 2,4,6-T 2,4,6-trichlorophenoxyacetic acid     

(+)-4-Carboxymethyl-2,4-dimethylbut-2-en-4-olide as dead-end metabolite of 2,4-dimethylphenoxyacetic acid or 2,4-dimethylphenol by Alcaligenes eutrophus JMP 134

2,4-Dimethylphenoxyacetic acid and 2,4-dimethylphenol are not growth substrates for Alcaligenes eutrophus JMP 134 although being cooxidized by 2,4-dichlorophenoxyacetate grown cells. None of the relevant catabolic pathways were induced by the dimethylphenoxyacetate. 3,5-Dimethylcatechol is not subject to metacleavage. The alternative ortho-eleavage is also unproductive and gives rise to (+)-4-carboxymethyl-2,4-dimethylbut-2-en-4-olide as a dead-end metabolite. High yields of this metabolite were obtained with the mutant Alcaligenes eutrophys JMP 134-1 which constitutively expresses the genes of 2,4-dichlorophenoxyacetic acid metabolism.     

In vitro culture ofBellevalia romana (L.) Rchb. I. Plant regeneration through adventitious shoots and somatic embryos

Summary Callus induction, adventitious shoot and root formation, and somatic embryogenesis were investigated in root, cotyledon and mesocotyl cultures ofBellevalia romana (L.) Rchb. grown on a synthetic nutrient medium containing different plant hormones. The combination of naphtaleneacetic acid plus benzylaminopurine was very effective in causing callus growth and plant regeneration from mesocotyl explants. On the contrary 2,4-dichlorophenoxyacetic acid caused suppression of shoot bud development in the same type of callus. Both cotyledon and root derived calli showed a low growth rate and did not regenerate shoots but only roots. Differentiation of somatic embryos which eventually developed into plantlets was promoted by 2,4-dichlorophenoxyacetic acid in suspension cultures. The results are discussed in relation to studies on nuclear behaviour during different morphogenetic pathways.     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

Somatic embryogenesis and organogenesis in Encephalartos dyerianus and E. natalensis

Callus cultures were initiated from zygotic embryos of Encephalartos dyerianus and E. natalensis. Callus of both species were transferred onto a modified B5 medium containing different combinations of 2,4-dichlorophenoxyacetic acid and kinetin. Somatic embryogenesis and shoot organogenesis occurred in both species. The embryos were dicotyledonary. To date none of the embryos have matured.Abbreviations ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid     

Influence of auxin type and concentration on peanut somatic embryogenesis

Somatic embryogenesis in peanut (Arachis hypogaea L.) using immature cotyledonary explants was induced on a wide range of 2,4-dichlorophenoxyacetic acid (2,4-D) (5 to 60mg l^–1) and naphthaleneacetic acid (NAA) (20 to 50 mg l^–1) levels. Percent embryogenesis ranged from 31 to 94%. As auxin level increased in induction medium, percent embryogenesis decreased and was associated with browning of explants. However, with higher 2,4-D induction levels (40 mg l^–1 and over), embryogenic explants had dense masses of embryogenic areas and repetitive embryogenesis was enhanced. Higher auxin concentrations during induction decreased precocious germination of embryos, but had no marked effect on somatic embryo morphology. The use of 2,4-D compared to NAA in the induction medium resulted in greater per cent embryogenesis and mean number of embryos. Embryos induced on NAA were harder, less pliant, and less succulent; cultures exhibited more extensive root development and nonembryogenic callus proliferation.Abbreviations B5 Gamborg et al. (1968) - BA benzyladenine - 2,4-D dichlorophenoxyacetic acid - IAA indole-3-acetic acid - MS Murashige & Skoog (1962) - NAA naphthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid     

Metabolism of 2,4-dichlorophenoxyacetic acid in cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.)

Cell suspension cultures of soybean (Glycine max L.) and wheat (Triticum aestivum L.) incorporated 2,4-dichlorophenoxyacetic acid (2,4-D) into a metabolite fraction which was insoluble in ethanol, water, and hot sodium dodecylsulphate. Further treatment with hot dimethylformamide solubilized a material which by the following criteria appeared to consist of 2,4-D derivatives covalently bound to lignin: i) co-chromatography of radioactivity and of UV-absorbing material upon gel permeation chromatography; ii) spectral similarity with authentic lignins (IR- and UV-spectra, phloroglucinol reaction), 2,4-D appeared to be incorporated as the intact molecule, as shown by comparison of ring- and sidechain-labeled 2,4-D and by detection of monohydroxylated and intact 2,4-D as the major radioactive products of acid hydrolysis. The same compounds were released from the metabolite material which could not be solubilized in dimethylformamide. The incorporation of xenobiotics or their metabolites into lignin, followed by deposition in the cell wall, is suggested as a general pathway for local excretion and detoxification by plant cells.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4-OH-2,5-D 4-hydroxy-2,5-dichlorophenoxyacetic acid - SDS sodium dodecylsulphate - DMF dimethylformamide