Developmental and genotypic differences in the response of pea stem segments to auxin

The objective of this investigation was to examine the response to exogenous auxin (indole-3-acetic acid; IAA)of stem segments at two developmental stages. The standard auxin response of excised stem segments and intact plants consists of an initial growth response and a prolonged growth response. We found that this biphasic response does not occur in internodes at very early stages. Stem segments of light grown pea of various genotypes were cut when the fourth internode was at 6–13% of full expansion (early-expansion) or at 18–25% of full expansion (mid-expansion). Length measurements of excised segments were made after 48 hours of incubation on buffer with or without auxin. An angular position transducer linked to a computerized data collection system provided high-resolution measurement of growth of stacks of segments incubated in buffer over 20 hours. Early-expansion segments of all genotypes deviated from the standard auxin response, while mid-expansion segments responded in a manner consistent with previous reports. Early-expansion segments of tall, light-grown plants were unique in showing an auxin-induced inhibition of growth. The auxin-induced inhibition correlated with high endogenous auxin content, as determined by HPLC and GC/MS, across genotypes and between early-expansion and mid-expansion segments of tall plants. Measurement of ethylene evolved from stem segments in response to auxin, and treatment of segments with the ethylene action inhibitor, norbornadiene, showed the inhibition to be mediated in part by heightened ethylene sensitivity. Growth of early-expansion segments of dwarf and severe dwarf plants was stimulated by exogenous auxin, but the growth rate increase was delayed compared to that in mid-expansion segments. This is the first time that such a growth response, termed the delayed growth response has been emonstrated. It is concluded that developmental stage and endogenous hormone content affect tissue response to exogenous auxin.     

Complementary action of Gibberellic acid and auxins in Pea internode extension

Gibberellic acid (GA) induced extension of green pea-stem sectionsin light only if an auxin was also present. Of the auxins tested3-indolylacetic acid, 2-methyl-4-chloro-phenoxyacetic acid,2: 4-dichloro-phenoxyacetic acid and I-naphthylacetic acid wereeffective in increasing extension of sections and in elicit-inga response to GA. Excised internodes from plants pre-treatedwith GA extended appreciably faster in vitro than those fromuntreated plants only if an auxin was supplied in the incubationmedium. This and other evidence suggests that in the intactplant GA elicits a growth-response only in the presence of auxin.By comparing growth-rates of excised internodes in vitro andof intact internodes in vivo under comparable conditions, usinguntreated plants and plants pre-treated with GA, evidence hasbeen obtained that in untreated plants growth-rate is somehowlimited to a level below that made potentially possible by theendogenous auxin supply; treatment with GA appears to releasethe plant from this state of inhibition. Growth of intact peainternodes is considered to be regulated by a three-factor system,consisting of auxin, an inhibitory system, and a hormone withphysiological properties similar to those of GA.     

Relationships between the development of adventitious roots and the biosynthesis of anthocyanins in first internodes of sorghum

The initiation and subsequent growth of adventitious roots in excised first internodes of Sorghum vulgare var. Wheatland milo were studied to determine the effect of these processes on anthocyanin biosyntheses. Segmentation of the internodes inhibited both adventitious root growth and accumulation of cyanidin equally in all segments; these results can be interpreted as a common requirement for bidirectional longitudinal transport. The presence of the coleoptile, especially in the absence of the base of the internode, inhibited the growth of the roots, but increased the number of root initials. High intensities of white and blue light which induced cyanidin synthesis slightly decreased adventitious root growth. Anaerobic conditions produced by solution infiltration strongly inhibited the growth of adventitious roots and greatly increased the accumulation of apigeninidin and luteolinidin. Addition of indoleacetic acid, kinetin and cofactors such as pyridoxine produced effects on the initiation and subsequent growth of these roots similar to those effects reported in the literature. But unlike root formation in hypocotyls, the initiation of adventitious roots in Sorghum internodes was not always directly correlated with the accumulation of anthocyanins, and the subsequent growth of these roots was frequently inversely correlated with some of the anthocyanin biosyntheses. The possible nature of these correlations is discussed. Comparisons are made with related Sorghum lines and mutants.     

Spatial-specific regulation of root development by phytochromes in Arabidopsis thaliana

Distinct tissues and organs of plants exhibit dissimilar responses to light exposure – cotyledon growth is promoted by light, whereas hypocotyl growth is inhibited by light. Light can have different impacts on root development, including impacting root elongation, morphology, lateral root proliferation and root tropisms. In many cases, light inhibits root elongation. There has been much attention given to whether roots themselves are the sites of photoperception for light that impacts light-dependent growth and development of roots. A number of approaches including photoreceptor localization in planta, localized irradiation and exposure of dissected roots to light have been used to explore the site(s) of light perception for the photoregulation of root development. Such approaches have led to the observation that photoreceptors are localized to roots in many plant species, and that roots are capable of light absorption that can alter morphology and/or gene expression. Our recent results show that localized depletion of phytochrome photoreceptors in Arabidopsis thaliana disrupts root development and root responsiveness to the plant hormone jasmonic acid. Thus, root-localized light perception appears central to organ-specific, photoregulation of growth and development in roots.     

PRIMARY PHLOEM REGENERATION WITHOUT CONCOMITANT XYLEM REGENERATION: ITS HORMONE CONTROL IN COLEUS

Phloem regeneration in the absence of xylem regeneration was evoked in number 5 internodes of Coleus blumei Benth. by severing xylemless phloem bundles. Its quantitative extent was estimated. To determine whether phloem regeneration is directly affected by auxin, or whether it is a secondary consequence of the auxin-dependent xylem regeneration which usually accompanies it, phloem regeneration was measured in decapitated plants from which auxin-producing leaves and buds had been removed (i.e., in “plant stumps”). In these stumps, 1% IAA in lanolin completely restored phloem regeneration to the intact plant level. In such stumps from which roots had been excised, and in excised internodes, IAA failed to restore it to this level. However, zeatin or zeatin riboside in aqueous solution applied to the bases of excised internodes receiving IAA at their apical ends restored phloem regeneration to the level of that in whole plants. When similarly tested, other cytokinins (kinetin, kinetin riboside, 2iP, and 2iPA), gibberellic acid (GA₃), glutamine, proline, sucrose, and a mixture of mineral salts failed to promote phloem regeneration. Glutamic acid, tested only once, was slightly promotive of it.     

Rapid cellular responses to auxin and the regulation of growth

Abstract The cellular responses rapidly evoked by auxin are reviewed, and related to a consideration of how growth rate is regulated in excised segments and in whole dicotyledonous plants. Two processes, synthesis of proteins and of cell wall components, are both promoted by auxin and essential for auxin-stimulated growth, whereas other processes show little promotion by auxin or do not appear essential for growth. Current models for the cellular regulation of growth by auxin are briefly discussed, and a new model presented. Auxin is suggested to act by bringing about a transient increase in cytosolic Ca²⁺ levels, which through the stimulation of protein kinases converts a cytoplasmic protein factor to an active state capable of binding auxin. The protein-auxin complex induces mRNA synthesis, which effects the increased synthesis of cell wall components and their incorporation into the wall, resulting in wall loosening and growth. It is proposed that the factor limiting growth in floating excised segments may initially be cell wall pH, but that this is not the case in whole plants and growth is instead mediated by increased protein and matrix cell wall synthesis. Differences are noted between monocotyledonous coleoptiles and dicotyledonous stems in some metabolic processes possibly involved in auxin growth responses, and it is cautioned that observations made on one tissue may not necessarily be applicable to the other. Care should also be taken in applying conclusions drawn from studies on excised tissue to the interpretation of growth regulation in the whole plant.     

Auxin-Gibberellin Interactions in Pea: Integrating the Old with the New

Recent findings on auxin-gibberellin interactions in pea are reviewed, and related to those from studies conducted in the 1950s and 1960s. It is now clear that in elongating internodes, auxin maintains the level of the bioactive gibberellin, GA₁, by promoting GA₁ biosynthesis and by inhibiting GA₁ deactivation. These effects are mediated by changes in expression of key GA biosynthesis and deactivation genes. In particular, auxin promotes the step GA₂₀ to GA₁, catalyzed by a GA 3-oxidase encoded by Mendel’s LE gene. We have used the traditional system of excised stem segments, in which auxin strongly promotes elongation, to investigate the importance for growth of auxin-induced GA₁. After excision, the level of GA₁ in wild-type (LE) stem segments rapidly drops, but the auxin indole-3-acetic acid (IAA) prevents this decrease. The growth response to IAA was greater in internode segments from LE plants than in segments from the le-1 mutant, in which the step GA₂₀ to GA₁ is impaired. These results indicate that, at least in excised segments, auxin partly promotes elongation by increasing the content of GA₁. We also confirm that excised (light-grown) segments require exogenous auxin in order to respond to GA. On the other hand, decapitated internodes typically respond strongly to GA₁ application, despite being auxin-deficient. Finally, unlike the maintenance of GA₁ content by auxin, other known relationships among the growth-promoting hormones auxin, brassinosteroids, and GA do not appear to involve large changes in hormone level.     

Blockage by gibberellic Acid of phytochrome effects on growth, auxin responses, and flavonoid synthesis in etiolated pea internodes

Red light inhibits the growth of etiolated pea internodes, causes a shift toward higher indoleacetic acid (IAA) concentrations in the IAA dose-response curve of excised sections, and promotes the synthesis in intact internodes of kaempferol-3-triglucoside. Gibberellic acid (GA₃) prevents all 3 effects, the first effect substantially and the last 2 completely. This suggests GA₃ blockage of an early or basic event initiated by the active form of phytochrome. The red light-induced shift in the IAA dose-response curve of excised sections is consistent with a light-induced increase in the activity of an IAA destruction system, since the magnitude of the red light inhibition varied with IAA concentration. The red light and GA₃ effects on growth and on flavonoid synthesis are consistent with the view that phytochrome may control growth by regulating the synthesis of phenolic compounds which act as cofactors in an IAA-oxidase system. GA₃ reversal of the red light-induced shift in the IAA dose-response curve involves both growth promotion and inhibition by GA₃ at different IAA concentrations and this, together with the GA₃ reversal of light-induced flavonoid synthesis, supports the suggested regulatory role of phenolic compounds in growth.     

Indole-3-acetic acid levels after phytochrome-mediated changes in the stem elongation rate of dark- and light-grownPisum seedlings

The effect of red (R) and far-red (FR) light on stem elongation and indole-3-acetic acid (IAA) levels was examined in dwarf and tall Pisum sativum L. seedlings. Red light reduced the extension-growth rate of etiolated seedlings by 70–90% after 3 h, and this inhibition was reversible by FR. Inhibition occurred throughout the growing zone. After 3 h of R, the level of extractable IAA in whole stem sections from the growing zone of etiolated plants either increased or showed no change. By contrast, extractable IAA from epidermal peels consistently decreased 3 h after R treatments. Decreases of 40% were observed for epidermal peels from the top 1 cm of tall plants receiving 3 h R. Brief R treatments resulted in smaller decreases in epidermal IAA levels and these decreases were not as great when FR followed R. In lightgrown plants, end-of-day FR stimulated growth during the following dark period in a photoreversible manner. The uppermost 1 cm of expanding third internodes was most responsive to the FR. Extractable IAA from epidermal peels from the upper 1 cm of third internodes increased by 30% or more 5 h after FR. When R followed the FR the increases were smaller. Levels of IAA in whole stem sections did not change and were twofold greater than in dark-grown plants. In both dark- and light-grown tall plants, IAA levels were lower in epidermal peels than in whole stem segments. These results provide evidence that IAA is compartmentalized at the tissue level within the growing stem and that phytochrome regulation of stem elongation rates may be partly based on modulating the level of IAA within the epidermis.Abbreviations IAA indole-3-acetic acid - R red light - FR farred light We thank Yu-Xian Zhu for helping to develop methods for IAA analysis, James Reid for supplying the genetic lines of Pisum and Richard Cyr for the use of microscopy equipment. This work was supported by NSF grant DCB-8801880 and by Hatch funds from the College of Agriculture and Life Sciences at Cornell University. The gas chromatograph-mass spectrometer was funded by NSF grant DMB-8505974 and funds from the College of Agriculture and Life Sciences at Cornell University. A preliminary report of some of these experiments has appeared in Plant Growth Substances, 1991 (Behringer et al. 1992 b).     

The Elongation Response of Watermelon Hypocotyls to Indole-3-Acetic Acid: A Comparative Study of Excised Segments and Intact Plants

Carrington, C. M. S. and Esnard, J. 1988. The elongation responseof watermelon hypocotyls to indole-3-acetic acid: a comparativestudy of excised segments and intact plants.—J. exp. Bot39: 441–450. The auxin-growth response along the hypocotyl of Citrullus lanatus(Thumb.) Mansf. seedlings was studied. In excised segments,promotion of elongation was seen in all zones at the concentrationsof IAA used (10–4–10–2 mol m-3). In intactplants, only the most basal zone showed unequivocal IAA-extensionwhile in the most apical zone elongation was inhibited by auxin.This difference between segments and intact plants for apicalzones suggests a modifying effect of the apex and cotyledonson the growth response. Indeed, removal of the apex and colyledonsonly affected elongation in the zones adjacent to the excisionbut only in buffer-treated plants, not auxin-treated plants.Auxin supplied apically to the intact plant only resulted ina short-lived promotion of elongation whereas basally suppliedauxin gave a longer-lasting effect Zonal differences betweenauxin-promoted growth of excised segments suggests that sensitivityto auxin varies in the hypocotyl. The response of intact plantsto auxin was shown to be more complex than in segments. Thus,responses given by segments are poor indicators of auxin activityin intact plants. Key words: IAA, Citrullus lanatus, growth, plant hormone sensitivity     

Cuticle Biosynthesis in Rapidly Growing Internodes of Deepwater Rice

Submergence induces rapid elongation of deepwater rice (Oryza sativa L.) internodes. This adaptive feature allows deepwater rice to grow out of the water and to survive flooding. The growth response of submerged deepwater rice plants is, ultimately, elicited by gibberellin (GA). Little attention has been given to the synthesis and role of the cuticle during plant growth. We investigated two questions regarding the cuticle in rapidly elongating deepwater rice internodes: (a) how does cuticle formation keep pace with internodal growth, which can reach rates of up to 5 mm/h; and (b) does the cuticle contribute to tissue stress in rice internodes? Treatment with GA for 48 h caused an up to 60-fold increase in the incorporation of [14C]palmitic acid and an up to 6-fold increase in the incorporation of [14C]oleic acid into the cuticle of growing internodes. GA also caused a qualitative change in the incorporation pattern of palmitic acid into several cutin monomers, the most prominent of which was tentatively identified by thin-layer chromatography as a derivative of dihydroxyhexadecanoic acid. Rapidly growing plant organs exhibit longitudinal tissue stress: the epidermal cell layer is under tension with a tendency to contract, whereas the internal cells are under compression with a tendency to expand. As a result of tissue stress, longitudinally sliced sections of elongating internodes bend outward upon isolation from the plant. Treating rapidly growing rice internodes with cutinase reduced such outward bending, indicating that the cuticle contributes to tissue stress. Based on these results, we propose that rapidly elongating structures such as deepwater rice internodes constitute an excellent system to study cuticle formation at the biochemical and cellular level.     

Interactions of microtubule disorganizers, plant hormones, and red light in wheat coleoptile segment growth

Growth response of coleoptile segments excised from 3-day-old seedlings of wheat (Triticum vulgare cv. Baart) to gibberellic acid, indoleacetic acid, and 2,4-dichlorophenoxyacetic acid, to red light, and to several microtubule disorganizers depends on the initial position of the excised segment in the intact coleoptile. Red light, 660 nm, stimulates the growth of the apical cells, but inhibits markedly the growth of the cells in the basal region of the coleoptile. The effects of red light are independent of sucrose, gibberellic acid, indoleacetic acid, and 2,4-dichlorophenoxyacetic acid, even though these substances themselves markedly affect the growth of the coleoptile segments. Concentractions of the microtubule disorganizers, vinblastine sulfate, cupric chloride, urea, and colchicine, which do not alter significantly the growth of the dark control apical segments, substantially repress the promotive effects of red light or auxin on the increase in length of the apical cells of the coleoptile. This suggests that stimulation by red light and by auxin involves microtubule production. Microtubule disorganizers repress the growth of elongating cells of the coleoptile, yet on the other hand, auxin and irradiation do not alter significantly the response of basal cells to the microtubule disorganizing agents. We hypothesized that light and growth regulators induce polymerization of nonaggregated microtubule subunits, resulting in faster growth.     

Changes in cell wall polysaccharides in the internodes of submerged floating rice

In excised stem segments of floating rice (Oryza sativa L.), as well as in intact plants, submergence greatly stimulates the elongation of internodes. The differences in the composition of cell wall polysaccharides along the highest internodes of submerged and air-grown stem segments were examined. The newly elongated parts of internodes that had been submerged for two days contained considerably less cellulosic and noncellulosic polysaccharides than air-grown internodes, an indication that the cell walls of the newly elongated parts of submerged internodes are extremely thin. In the young parts of both air-grown and submerged internodes, the relative amounts of noncellulosic polysaccharides were equal to those of -cellulose, whereas the relative amounts of -cellulose were higher than those of noncellulosic polysaccharides in the upper, old parts. In the cell-elongation zones of both air-grown and submerged internodes, glucose was predominant among the noncellulosic neutral sugars of cell wall. The relative amount of glucose in noncellulosic neutral sugars decreased toward the upper, old parts of internodes, whereas that of xylose increased.     

ent-Kaurene Biosynthesis in Cell-Free Extracts of Excised Parts of Tall and Dwarf Pea Seedlings

Investigations on the sites of ent-kaur-16-ene (ent-kaurene) biosynthesis were conducted with cell-free extracts from several excised parts of 10-, 13-, and 16-d-old tall and dwarf pea (Pisum sativum L.) seedlings. [¹⁴C]Mevalonic acid was incorporated into ent-kaurene in cell-free extracts from young developing leaves and elongating internodes of tall (`Alaska') and dwarf (`Progress No.9') pea seedlings at all three stages of development. ent-Kaurene biosynthesis also occurred readily in cell-free extracts from shoot tips, petioles, and stipules near the young elongating internodes. The ent-kaurene-synthesizing activity found in young developing tissues declined as tissues matured. Little or no activity was detectable in enzyme extracts from cotyledons and root tips at different stages. In light grown tall pea internodes ent-kaurene-synthesizing activity was low as they began to elongate, reached a maximum when the internodes reached about 2 cm in length and declined as they matured. Activity in extracts of dwarf shoot tips and internodes was generally lower than in equivalent tall plants, but the activity in dwarf leaves and stipules was somewhat higher than in tall plants. With the exception of root tips, there is a strong correlation between growth potential of a tissue and the rate of ent-kaurene biosynthesis in extracts from that tissue.     

The role of endogenous gibberellin-like substances and inhibitors in the growth of pea internodes

Third internodes or whole stems of 7-days old etiolated pea plants were extracted and the content of gibberellin-like substances and inhibitors has been determined. Extracts were found to contain four or five different gibberellin-like substances, some of which are chromatographically similar to GA₃. The content of gibberellins has been high in young internodes and decreased along with the internodes elongation. Brief red light irradiation brings about quantitative changes in gibberellin content, depending also on the length of internodes. The extracts contain acidic and neutral inhibitors which interfere with the response to GA₃. The content of the inhibitors does not seem to be affected by the ageing of internodes or by the light treatment.     

Phytochromabhängige Veränderungen des Wachstums und der Ionenaufnahme etiolierter Erbsenkeimlinge

Summary Inhibition of internodial growth of pea seedlings by light is compensated for by increased growth of leaves. At a given time the sum of fresh weight of internodes plus the product of fresh weight of leaves times a certain factor is constant in darkness or with different periods of light. This correlation may reflect a competition of internodes and leaves for materials delivered at a lightindependent rate from the cotyledons. This hypothesis was tested by immersing roots of pea seedlings into ⁸⁶Rb labelled K-solutions for one day in darkness, removing the plants from the solutions, exposing the seedlings to near or far red light and measuring the radioactivity and fresh weights of leaves and internodes separately. Radioactivity and fresh-weight were both dependent on phytochrome; i.e. inhibition of ion uptake and of growth in internodes and promotion of both processes in leaves by near red light as compared to dark or far red controls are mediated by phytochrome.Short time experiments of ion uptake by the roots show that K transport into the shoot organs is promoted by light after a lag phase of somewhat more than one hour. This interval corresponds well to the lag phase of the light induced growth inhibition of internodes.Seedlings deprived of cotyledons and roots grow well in water but exhibit no difference in growth rate of leaves and internodes in light and darkness. Light dependence is restored if the seedlings are submersed in approximately 3% sucrose solutions. This result seems to indicate that the influence of light on growth rates of leaves and internodes is dependent on the uptake of material by the cell. It seems possible that in the etiolated pea seedling light promotes growth of leaves by promoting uptake and hampers growth of internodes by inhibiting uptake of essential growth material delivered from the cotyledons.     

Effect of Light Quality on the Growth and Growth-substance Content of Plants

The main effect of incandescent light on most of several speciesand varieties of plants grown in cabinets illuminated by fluorescentlight was to increase the length of internodes. Although leafarea was little affected, incandescent light increased the dryweight of most plants. In dwarf French bean plants, the longer internodes of plantsgrown with additional incandescent light was associated with,and perhaps a result of, the higher content of gibberellic acid-likeand indolylacetic acid-like growth substances.     

Downstream effectors of light- and phytochrome-dependent regulation of hypocotyl elongation in Arabidopsis thaliana

Arabidopsis, like most plants, exhibits tissue-specific, light-dependent growth responses. Cotyledon and leaf growth and the accumulation of photosynthetic pigments are promoted by light, whereas hypocotyl growth is inhibited. The identification and characterization of distinct phytochrome-dependent molecular effectors that are associated with these divergent tissue-specific, light-dependent growth responses are limited. To identify phytochrome-dependent factors that impact the photoregulation of hypocotyl length, we conducted comparative gene expression studies using Arabidopsis lines exhibiting distinct patterns of phytochrome chromophore inactivation and associated disparate hypocotyl elongation responses under far-red (FR) light. A large number of genes was misregulated in plants lacking mesophyll-specific phytochromes relative to constitutively-deficient phytochrome lines. We identified and characterized genes whose expression is impacted by light and by phyA and phyB that have roles in the photoregulation of hypocotyl length. We characterized the functions of several identified target genes by phenotyping of T-DNA mutants. Among these genes is a previously uncharacterized LHE (LIGHT-INDUCED HYPOCOTYL ELONGATION) gene, which we show impacts light- and phytochrome-mediated regulation of hypocotyl elongation under red (R) and FR illumination. We describe a new approach for identifying genes involved in light- and phytochrome-dependent, tissue-specific growth regulation and confirmed the roles of three such genes in the phytochrome-dependent photoregulation of hypocotyl length.     

Loss of the species distinguishing trait among regenerated Bambusa ventricosa McClure plants

All B. ventricosa plants propagated from excised shoot tips displayed loss of the species characteristic bulbous internodes. The plants were obtained by stimulating axillary shoot differentiation, followed by rooting of shoot separates. Cause of the variance has not been attributable to removal of bamboo mosaic virus infection or growth stress. Chimeral tissue seemed to be excluded, since a few regenerants, would have retained the bulbous trait.     

Correlative Growth in Young Terminalia superba in a Controlled Environment: Effect of the Leaves on Internode Elongation

Young Terminalia superba plants were cultivated in a controlledenvironment at the Phytotron. Effects of the excision of a youngleaf at definite elongation stages and at two given levels ofthe main axis were studied on the elongation of internodes inthese plants. Effects of the leaf did not seem to depend onits nodal position on the main axis but predominantly occurredon the immediate surrounding internodes. The excision of a youngleaf enhanced the growth rate of the internode located belowit and markedly decreased the elongation of the internode aboveit but slightly affected internode growth duration. This excisionenhanced the final length of the internode located below theleaf and decreased the final length of the internode locatedabove the removed leaf. Significant linear regressions werefound between the length of the excised leaf and the internodefinal lengths. Microscopic examination of epidermal cells ofcontrol and disrupted internodes revealed that the decreasedelongation after leaf excision could be attributed to reductionof cell divisions. The increased elongation after leaf excisioncould be attributed both to slight increase in the length ofcells (significant negative correlation was found with the lengthof the removed leaf) and to increase of cell divisions. Terminalia superba, leaf excision, cell division, internode elongation, correlative growth