Auxin transport: a new synthetic inhibitor

The new synthetic plant growth regulator DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo [5,1-a] isoindol-8-one) was examined for its effects on auxin transport. At a concentration of 0.5 mm in the receiver agar cylinders DPX1840 significantly inhibited the basipetal transport of naphthaleneacetic acid-1-¹⁴C in stem sections of Vigna sinensis Endl., Pisum sativum L., Phaseolus vulgaris L., Glycine max L., Helianthus annuus L., Gossypium hirsutum L., and Zea mays L. without significantly reducing total auxin uptake or recovery. The time sequence of the effect varied with the plant species. A similar inhibition of the basipetal movement of indoleacetic acid-1-¹⁴C was observed in intact seedlings of Phaseolus vulgaris L. In contrast to basipetal auxin transport DPX1840 had no significant effect on the acropetal movement of indoleacetic acid-1-¹⁴C in stem sections of Gossypium hirsutum L. Qualitatively the effect of DPX1840 on basipetal auxin transport was similar to that of other known auxin transport inhibitors. Quantitative differences, however, suggested the following order of activity: Naptalam>morphactin[unk]DPX1840>2,3,5-triiodobenzoic acid.     

Failure of Ethylene to Change the Distribution of Indoleacetic Acid in the Petiole of Coleus blumei X frederici during Epinasty

The effect of ethylene on the distribution of applied indoleacetic acid in the petiole of Coleus blumei Benth. X C. frederici G. Taylor has been investigated during the development of epinastic curvature. Using intact plants, ¹⁴C-IAA was applied to the distal region of the leaf lamina and the accumulation of label in the abaxial and adaxial halves of 5 mm petiole sections was determined after 1.5, 3, and 6 hours. Over this period the label was transported out of the lamina into the petiole at a rate of at least 66 mm hr⁻¹. Of the total amount of label in the petiole sections, 24 to 30% was located in the adaxial half and this distribution was not altered significantly by exposing plants to an atmosphere containing 50 μl/l ethylene. Thus when epinastic curvature is induced by ethylene there is no associated increase in the IAA content of the expanding adaxial half. The role of endogenous IAA in petiole epinasty was studied by restricting its movement with DPX 1840 (3,3a-dihydro-2-[p-methoxyphenyl]-8H-pyrozolo{5,1-a}isoindol-8-one). The leaf petioles still showed an initial epinastic response to ethylene. It is concluded that ethylene-induced epinasty is not dependent upon either any change in the transport of IAA or its redistribution within the petiole.     

The Control of Lateral Root Development in Cultured Pea Seedlings. II. Root Fasciation Induced by Auxin Inhibitors

Lateral root development in cultured seedlings of Pisum sativum (cv. Alaska) was modified by the application of auxin transport inhibitors or antagonists. When applied either to replace the root tip or beneath the cotyledonary node, two auxin transport inhibitors, 2,3,5-triiodobenzoic acid (TIBA) and 3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-α]isoindol-8-one (DPX-1840), increased cell division activity opposite the protoxylem poles. This resulted in the formation of masses of cells, which we are calling root primordial masses (RPMs), 2 to 3 days after treatment. RPMs differed from lateral root primordia in that they lacked apical organization. Some roots however developed both RPMs and lateral roots indicating that both structures were similar in terms of the timing and location of cell division in the pericycle and endodermis leading to their initiation. Removal of the auxin transport inhibitors allowed many of the RPMs to organize later into lateral root primordia and to emerge in clusters. When the auxin, indoleacetic acid (IAA) was added to the growth medium along with DPX-1840, 3 ranks of RPMs now in the form of fasciated lateral roots emerged from the primary root. The auxin antagonist, p-chlorophenoxy-isobutyric acid (PCIB), also induced RPM formation. In contrast to DPX-1840 treatment, the addition of IAA during PCIB treatment caused normal lateral root development.     

Sites of auxin action: regulation of geotropism, growth, and ethylene production by inhibitors of auxin transport

The inhibitors of auxin transport-NPA (N-1-naphthylphthalamic acid), DPX1840 (3,3a-dihydro-2-(p-methoxyphenyl)-8H-pyrazolo[5,1-a] isoindol-8-one), and TIBA (2,3,5-triiodobenzoic acid)-inhibited geotropism in roots of intact Pisum sativum L. seedlings. NPA and DPX1840 also caused cellular swelling in the roots. The swelling was due to a greater inhibition of elongation than increase in weight and looked identical to the one caused by ethylene. However, ethylene did not act as an intermediate in the action of auxin transport inhibitors because all three failed to stimulate ethylene production and some of their growth-inhibiting effect was retained in the presence of saturating levels of ethylene. In the presence of 10 mum indoleacetic acid the growth-inhibiting effect of auxin transport inhibitors was lost after 18 hours. On the other hand, auxin transport inhibitors did not interfere with the ability of auxin to promote ethylene production. Growth inhibition caused by auxin transport inhibitors was reversible. Pea root sections resumed normal growth following flushing of treated sections with inhibitor-free solutions. Experiments with (14)C-2, 4-dichlorophenoxyacetic acid revealed that the herbicide and auxin transport inhibitors may have the same binding site. It was concluded that a class of structurally dissimilar compounds may share a similar physiological role since they all appear to compete with endogenous auxin for certain binding sites and they all have similar growth-regulating activities.     

Branch development in Lupinus angustifolius L.#I. Not all branches have the same potential growth rate

Although the basal and uppermost lateral branches of Lupinus angustifolius L. frequently grow and contribute to yield, buds formed in the axils of leaves 6-12 (referred to as middle buds) rarely grow. This may be due to an inherent limitation of these buds, or some form of apical dominance or competition imposed by the plant. The hypothesis that middle buds have the full capacity to grow, but remain suppressed on intact plants was tested. The main stem apex and buds from the axils of leaves 1 and 8 (bud 1 and bud 8) were excised and cultured on sterile agar. The buds were removed from culture and weighed every 2-3 d for 21 d. The growth rate of apices from the main stem was approximately 5.8 mg d^-1, compared to 2.4 mg d^-1 for bud 1 and 0.9 mg d^-1 for bud 8. Buds in the axils of leaves 6-10 on intact plants were painted six times with a synthetic cytokinin, benzylaminopurine, from 40 d after sowing. This promoted rapid elongation and thickening of these buds, visible as early as 5 d after painting began. The rapid growth of these branches was associated with a reduction in the length of the remaining branches on the plant. However, excision of lower branches did not increase the growth of the middle buds. It is concluded that buds 6-12 of Lupinus angustifolius L. have a partial potential to grow. This potential appears to be limited by innate factors in the bud, and may be structural and/or hormonal. The limitation appears to develop very early in the plant, and potential growth is not modified by subsequent nutrition of the plant.     

Changes in the content of endogenous auxins in apical buds ofchenopodium rubrum L. Induced with respect to the endogenous rhythm in capacity to flower

The content of endogenous auxins was examined in apical buds ofChenopodium rubrum plants induced by a photoperiodic cycle of 16h darkness and 8h light followed by a dark period of various duration so as to correspond with either maximal or minimal flowering response in the endogenous rhythm in capacity to flower initiated by the photoperiodic treatment. Apical buds of potentially generative plants contained less auxins than apical buds of plants which remained in the vegetative state. Apical buds from plants treated with kinetin (1. 10^-3 M) and therefore remaining in the vegetative state showed an auxin level comparable to that of untreated plants exhibiting minimal flowering response irrespective of the duration of the second dark period. Plants cultivated on a sucrose solution (0.6 M) during the second dark period became generative even at the normal minimum of flowering. The auxin content of the apical buds was low, similarly as in untreated plants induced for a period leading to maximal flowering response. On the other hand, apical buds from plants grown on sucrose solution during a dark period leading to the manifestation of maximal flowering response showed a relatively high auxin content comparable to that found in untreated plants which had obtained a more extended induction by three photoperiodic cycles. The results are discussed with respect to the possible role of endogenous auxins in the regulation of the changes in growth correlations occurring in the shoot apex during photoperiodic induction and in the expression of the competence to flower.     

Release of lateral buds from apical dominance by glyphosate in soybean and pea seedlings

Application of a sublethal dose of glyphosate (N-[phosphonomethyl]glycine) to the seedlings of soybean (Glycine max L. Merr. cv. Evans) and pea (Pisum sativum L. cv. Alaska) promoted growth of the cotyledonary and other lateral buds. The pattern of the glyphosate-induced lateral bud growth was different from that induced by decapitation. Under the experimental condition, glyphosate did not kill the apical buds. Feeding stem sections of the seedlings with radiolabeled indole-3-acetic acid ([2¹⁴C]IAA) and subsequent analysis of free [2-¹⁴C]IAA and metabolite fractions revealed that the glyphosate-treated plants had higher rates of IAA metabolism than the control plants. The treated pea plants metabolized 75% of [2-¹⁴C]IAA taken up in the 4-h incubation period compared to 46.5% for the control, an increase of 61%. The increase was small but consistent in soybean seedlings. As a result, the glyphosate-treated plants had less free IAA and ethylene than the control plants. The increase of IAA metabolism induced by glyphosate is likely to change the auxin-cytokinin balance and contribute to the release of lateral buds from apical dominance in these plants.     

Plant regeneration via somatic embryogenesis and shoot organogenesis from immature cotyledons of Camellia nitidissima Chi

Camellia nitidissima Chi (Theaceae) is a world-famous economic and ornamental plant with golden-yellow flowers. It has been classified as one of the rarest and most endangered plants in China. Our objective was to induce somatic embryogenesis, shoot organogenesis and plant regeneration for C. nitidissima. Three types of callus (whitish, reddish and yellowish) were induced from immature cotyledons on improved woody plant medium (WPM) with different plant growth regulators (PGRs). Among the callus, whitish callus was induced by 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and reddish and yellowish callus were induced by strongly active cytokinins, thidiazuron (TDZ) or 6-benzylaminopurine (BAP), singly or combined with weakly active auxin, α-naphthaleneacetic acid (NAA). The embryogenic callus could differentiate into somatic embryos, nodular embryogenic structures (large embryo-like structures) or adventitious shoots depending on the PGR used in WPM. BAP was best for adventitious buds and zeatin was best for somatic embryogenesis while kinetin (Kt) was best for the formation of nodular embryogenic structures. The three regeneration pathways often occurred in the same embryogenic callus clumps. Most shoots (80.0%) developed roots in WPM supplemented with 24.6 μM IBA and 0.3 μM NAA while 47.5% of somatic embryos could germinate directly and develop into plantlets on induction medium supplemented with 0.9 μM BAP and 0.1 μM NAA. The nodular embryogenic structures could be sub-cultured and cyclically developed in one of two differentiation pathways: shoot organogenesis or somatic embryogenesis. Plantlets derived from shoot buds rooted and somatic embryos germinated when transplanted into soil in a greenhouse; 66.7% of plantlets from shoot culture and 78.6% of plantlets from somatic embryos survived after 8 weeks’ acclimatization.     

Subclasses of Adenosine Receptors in Brain Membranes from Adult Tissue and from Primary Cultures of Chick Embryo

Abstract: Membranes from adult chicken brain have high-affinity binding sites for N⁶-cyclohexyl[³H]adenosine (CHA) (K_D= 4 nM, Bmax = 0.6 pmol/mg protein). This CHA binding could be attributed to adenosine receptors of the A₁ type, since substituted adenosine analogs, e.g. N⁶-(l -2-phenylisopropyl)adeno sine (IC50 = 60 nM), were very potent displacers. Binding sites for 1,3-diethyl- 8-[³H]phenylxanthine (DPX) in adult brain membranes have a moderate affinity (K_D= 50 nM, Bmax = 1.5 pmol/mg). The association of DPX with these sites could be completely displaced by 8-phenyltheophylline (IC₅₀= 300 nM) and other xanthines, but only 45% of specific DPX binding could be displaced by phenylisopropyladenosine. This suggests that about half of DPX sites are putative A₁ receptors and the other half are of the A₂ type. Primary cultures of pure glial and neuronal cells from chick embryo brain were also examined for adenosine receptors. Specific binding of CHA could not be detected in these preparations, but both glial and neuronal membranes have specific sites for DPX. At a [³H]DPX concentration of 20 nM, specific binding was 50% higher (per mg protein) in glial than in neuronal membranes. The maximum binding of DPX to glial membranes (B_max= 1.6 pmol/mg) was comparable to values for adult brain, but the glial affinity (K_D= 90 nM) was somewhat less. Phenylisopropyladenosine was able to displace less than 20% of the total glial sites for DPX. This finding was in accord with the lack of CHA sites and demonstrates that A₁ receptors make little contribution to DPX binding in glial membranes. In decreasing order of potency, 8-phenyltheophylline, CHA, theophylline, caffeine, and 3-isobutyl-I-methylxanthine completely displace DPX association with glia. DPX binding to glial membranes thus appears due to a single class of receptors, which may prove to be of the A₂ type.     

Carbon Dioxide and Flowering in Pharbitis nil Choisy

The effects of photoperiod on floral and vegetative development of Pharbitis nil were modified by atmospheric CO₂ concentrations maintained during plant growth. Short day (SD) photoperiods caused rapid flowering in Pharbitis plants growing in 0.03 or 0.1% CO₂, while plants in long day (LD) conditions remained vegetative. At 1 or 5% CO₂, however, flower buds were developed under both the SD and LD photoperiods. Flowering was earliest in the plants exposed to SD at low CO₂ concentrations which formed floral buds at stem node 3 or 4. At high CO₂ concentrations, floral buds did not form until stem node 6 or 7. Both high CO₂ concentrations and LD photoperiods tended to enhance stem elongation and leaf formation.     

Insulin-Like Growth Factors Are Expressed in the Taste System,but Do Not Maintain Adult Taste Buds

Growth factors regulate cell growth and differentiation in many tissues. In the taste system, as yet unknown growth factors are produced by neurons to maintain taste buds. A number of growth factor receptors are expressed at greater levels in taste buds than in the surrounding epithelium and may be receptors for candidate factors involved in taste bud maintenance. We determined that the ligands of eight of these receptors were expressed in the E14.5 geniculate ganglion and that four of these ligands were expressed in the adult geniculate ganglion. Of these, the insulin-like growth factors (IGF1, IGF2) were expressed in the ganglion and their receptor, insulin-like growth factor receptor 1 (IGF1R), were expressed at the highest levels in taste buds. To determine whether IGF1R regulates taste bud number or structure, we conditionally eliminated IGF1R from the lingual epithelium of mice using the keratin 14 (K14) promoter (K14-Cre::Igf1r^lox/lox). While K14-Cre::Igf1r^lox/lox mice had significantly fewer taste buds at P30 compared with control mice (Igf1r^lox/lox), this difference was not observed by P80. IGF1R removal did not affect taste bud size or cell number, and the number of phospholipase C β2- (PLCβ2) and carbonic anhydrase 4- (Car4) positive taste receptor cells did not differ between genotypes. Taste buds at the back of the tongue fungiform taste field were larger and contained more cells than those at the tongue tip, and these differences were diminished in K14-Cre::Igf1r^lox/lox mice. The epithelium was thicker at the back versus the tip of the tongue, and this difference was also attenuated in K14-Cre::Igf1r^lox/lox mice. We conclude that, although IGFs are expressed at high levels in the taste system, they likely play little or no role in maintaining adult taste bud structure. IGFs have a potential role in establishing the initial number of taste buds, and there may be limits on epithelial thickness in the absence of IGF1R signaling.     

Functional and Structural Analysis of a β-Glucosidase Involved in β-1,2-Glucan Metabolism in Listeria innocua

Despite the presence of β-1,2-glucan in nature, few β-1,2-glucan degrading enzymes have been reported to date. Recently, the Lin1839 protein from Listeria innocua was identified as a 1,2-β-oligoglucan phosphorylase. Since the adjacent lin1840 gene in the gene cluster encodes a putative glycoside hydrolase family 3 β-glucosidase, we hypothesized that Lin1840 is also involved in β-1,2-glucan dissimilation. Here we report the functional and structural analysis of Lin1840. A recombinant Lin1840 protein (Lin1840r) showed the highest hydrolytic activity toward sophorose (Glc-β-1,2-Glc) among β-1,2-glucooligosaccharides, suggesting that Lin1840 is a β-glucosidase involved in sophorose degradation. The enzyme also rapidly hydrolyzed laminaribiose (β-1,3), but not cellobiose (β-1,4) or gentiobiose (β-1,6) among β-linked gluco-disaccharides. We determined the crystal structures of Lin1840r in complexes with sophorose and laminaribiose as productive binding forms. In these structures, Arg572 forms many hydrogen bonds with sophorose and laminaribiose at subsite +1, which seems to be a key factor for substrate selectivity. The opposite side of subsite +1 from Arg572 is connected to a large empty space appearing to be subsite +2 for the binding of sophorotriose (Glc-β-1,2-Glc-β-1,2-Glc) in spite of the higher K_m value for sophorotriose than that for sophorose. The conformations of sophorose and laminaribiose are almost the same on the Arg572 side but differ on the subsite +2 side that provides no interaction with a substrate. Therefore, Lin1840r is unable to distinguish between sophorose and laminaribiose as substrates. These results provide the first mechanistic insights into β-1,2-glucooligosaccharide recognition by β-glucosidase.     

Transformation of Brassica napus canola cultivars with Arabidopsis thaliana acetohydroxyacid synthase genes and analysis of herbicide resistance

Summary A survey of selected crop species and weeds was conducted to evaluate the inhibition of the enzyme acetohydroxyacid synthase (AHAS) and seedling growth in vitro by the sulfonylurea herbicides chlorsulfuron, DPX A7881, DPX L5300, DPX M6316 and the imidazolinone herbicides AC243,997, AC263,499, AC252,214. Particular attention was given to the Brassica species including canola cultivars and cruciferous weeds such as B. kaber (wild mustard) and Thlaspi arvense (stinkweed). Transgenic lines of B. napus cultivars Westar and Profit, which express the Arabidopsis thaliana wild-type AHAS gene or the mutant gene csr1-1 at levels similar to the resident AHAS genes, were generated and compared. The mutant gene was essential for resistance to the sulfonylurea chlorsulfuron but not to DPX A7881, which appeared to be tolerated by certain Brassica species. Cross-resistance to the imidazolinones did not occur. The level of resistance to chlorsulfuron in transgenic canola greatly exceeded the levels that were toxic to the Brassica species or cruciferous weeds. Direct selection of transgenic lines with chlorsulfuron sprayed at field levels under greenhouse conditions was achieved.     

Regeneration of calabrian pine from juvenile needles

Cytokinins applied in an agar medium induced adventitious buds on cultured needles from seedling of Pinus brutia Ten. Cytokinins applied as pulses to the explants prior to culture were less effective. Irrespective of the mode of cytokinin application, 8 weeks was the time required to bring about bud formation. Organogenetic potential of the cultured needles decreased with chronological age of the explanted seedlings. The induced buds grew into elongated shoots on culture medium without cytokinins, but the inclusion of activated charcoal (1%) doubled the elongation rate. There was an indication that mixtures of cytokinins were more effective than separate cytokinins in producing buds on explants, but the difference between treatments did not achieve statistical significance. Parenchyma cells in mesophyll layers were evidently the target cells responding to the culture conditions. After a period of activity and division in these cells, meristematic zones developed which later led to formation of bud primordia, and subsequently these primordia developed into well-formed adventitious buds. Subsequent rooting (64%) of shoots was achieved using a combination of two auxins and a low level of cytokinin.     

Changes after Decapitation in Concentrations of Indole-3-Acetic Acid and Abscisic Acid in the Larger Axillary Bud of Phaseolus vulgaris L. cv Tender Green

Early changes in the concentrations of indole-3-acetic acid (IAA) and abscisic acid (ABA) were investigated in the larger axillary bud of 2-week-old Phaseolus vulgaris L. cv Tender Green seedlings after removal of the dominant apical bud. Concentrations of these two hormones were measured at 4, 6, 8, 12 and 24 hours following decapitation of the apical bud and its subtending shoot. Quantitations were accomplished using either gas chromatography-mass spectrometry-selected ion monitoring (GS-MS-SIM) with [¹³C₆]-IAA or [²H₆]-ABA as quantitative internal standards, or by an indirect enzyme-linked immunosorbent assay, validated by GC-MS-SIM. Within 4 hours after decapitation the IAA concentration in the axillary bud had increased fivefold, remaining relatively constant thereafter. The concentration of ABA in axillary buds of decapitated plants was 30 to 70% lower than for buds of intact plants from 4 to 24 hours following decapitation. Fresh weight of buds on decapitated plants had increased by 8 hours after decapitation and this increase was even more prominent by 24 hours. Anatomical assessment of the larger axillary buds at 0, 8, and 24 hours following decapitation showed that most of the growth was due to cell expansion, especially in the intermodal region. Thus, IAA concentration in the axillary bud increases appreciably within a very few hours of decapitation. Coincidental with the rise in IAA concentration is a modest, but significant reduction in ABA concentration in these axillary buds after decapitation.     

Plant regeneration,developmental pattern and genetic fidelity of somatic embryogenesis derived Musa spp.

Multiplication of banana cvs. Grand Naine (Musa AAA, Cavendish-sub group) and Rasthali (Musa AAB, Silk-sub group) were attempted through somatic embryogenesis. The influence of position of male flower buds, amino acid supplements in the induction of somatic embryogenesis and field performance of embryogenic cell suspension (ECS) derived banana plants were studied. Differentiated immature male flower buds positioned at 6–8?th bract whorl as explants showed better callus induction and somatic embryogenesis. Supplementation with glutamine at 400?mg?L^?1 along with 20:20?g?L^?1sucrose: maltose in maturation media induced a 10-fold increase in somatic embryo formation compared to control. Cotyledonary stage somatic embryos desiccated for 2?h showed higher germination compared to non-desiccated embryos. The plantlets generated were hardened, and the genetic fidelity of the plantlets was confirmed using ISSR marker. To check the field performance of ECS derived plants, plantlets were hardened and planted in the field along with meristem and sucker. During the field growth, these ECS derived plants were morphologically similar to those of control plants. In this experiment, it was observed that ECS derived banana plants displayed normal phenotype as that of plants grown from meristem and sucker. The protocol developed could be useful highly for large-scale micropropagation or genetic manipulation studies in these commercially important banana cultivars.     

A new class of synthetic auxin transport inhibitors

Auxin transport inhibition by a new class of synthetic plant growth regulants, the 2-(3-aryl-5-pyrazolyl)benzoic acids, was examined in bean (Phaseolus vulgaris L.) using the donor-receiver agar cylinder technique. These compounds can be prepared by the dehydrogenation and ring cleavage of compounds like DPX-1840 (2-(4-methoxyphenyl)-3,3adihydro-8H-pyrazolo[5,1-a] isoindol-8-one) which was previously reported (Plant Physiol. 1972. 50: 322-327) to be a potent inhibitor of auxin transport. These new growth regulators inhibit auxin transport more than DPX-1840 does as evidenced by their consistently greater reduction of basipetal auxin transport capacity in bean when incorporated into the receiver agar cylinder or applied foliarly to intact plants. Direct comparisons of the effect of DPX-1840, its dehydrogenation product (2-(4-methoxyphenyl)-8H-pyrazolo [5,1-a]isoindol-8-one), and its open-ring form (2-(3-(4-methoxyphenyl)-5-pyrazolyl) benzoic acid) on auxin transport indicated the following order of activity: ring-open > dehydrogenated form > DPX-1840. DPX-1840-(14)C, applied at 0.5 mg/l to etiolated bean hypocotyl hooks followed by extraction and thin layer chromatography, indicated the biological conversion of DPX-1840 to its open-ring form. Collectively, these results suggest that the biologically active forms of DPX-1840-type compounds are the open-ring (2-(3-aryl-5-pyrazolyl) benzoic acids.