Transport of exogenous auxin in two-branched dwarf pea seedlings (Pisum sativum L.)

Dwarf pea plants bearing two cotyledonary shoots were obtained by removing the epicotyl shortly after germination, and the patterns of distribution of ¹⁴C in these plants was investigated following the application of [¹⁴C]IAA to the apex of one shoot. Basipetal transport to the root system occurred, but in none of the experiments was ¹⁴C ever detected in the unlabelled shoot even after transport periods of up to 48 h. This was true both of plants with two equal growing shoots and of plants in which one shoot had become correlatively inhibited by the other, and in the latter case applied whether the dominant or subordinate shoot was labelled. In contrast, when [¹⁴C]IAA was applied to a mature foliage leaf of one shoot transfer of ¹⁴C to the other shoot took place, although the amount transported was always low. Transport of ¹⁴C from the apex of a subordinate shoot on plants bearing one growing and one inhibited shoot was severely restricted compared with the transport from the dominant shoot apex, and in some individual plants no transport at all was detected. Removal of the dominant shoot apex rapidly restored the capacity of the subordinate shoot to transport apically-applied [¹⁴C]IAA, and at the same time led to rapid cambial development and secondary vascular differentiation in the previously inhibited shoot. Applications of 1% unlabelled IAA in lanolin to the decapitated dominant shoot maintained the inhibition of cambial development in the subordinate shoot and its reduced capacity for auxin transport. These results are discussed in relation to the polarity of auxin transport in intact plants and the mechanism of correlative inhibition.Abbreviations IAA Indol-3-yl-acetic acid - TIBA 2,3,5-triiodobenzoic acid - 2,4D 2,4-dichlorophenoxyacetic acid - IAAsp Indol-3-yl-acetyl aspartic acid     

Stimulatory effect of cytokinins and interaction with IAA on the release of lateral buds of pea plants from apical dominance

Lateral buds of pea plants can be released from apical dominance and even be transformed into dominant shoots when repeatedly treated with synthetic exogenous cytokinins (CKs). The mechanism of the effect of CKs, however, is not clear. The results in this work showed that the stimulatory effects of CKs on the growth of lateral buds and the increase in their fresh weights in pea plants depended on the structure and concentration of the CKs used. The effect of N-(2-chloro-4-pyridyl)-N'-phenylurea (CPPU) was stronger than that of 6-benzylaminopurine (6-BA). Indoleacetic acid (IAA) concentration in shoot, IAA export out of the treated apex and basipetal transport in stems were markedly increased after the application of CPPU or 6-BA to the apex or the second node of pea plant. This increase was positively correlated with the increased concentration of the applied CKs. These results suggest that the increased IAA synthesis and export induced by CKs application might be responsible for the growth of lateral shoots in intact pea plants.     

Effects of the auxin-inhibiting substances raphanusanin and benzoxazolinone on apical dominance of pea seedlings

The effects of the auxin-inhibiting substances raphanusanin ((3R*,6S*)-3-[methoxy (methylthio) methyl]-2-pyrrolidinethione, raphanusanin B)and benzoxazolinone (6-methoxy-2-bezoxazolinone, MBOA) on apical dominance of pea(Pisum sativum L. cv. Alaska) seedlings were studied.Application of raphanusanin B or MBOA to the apical bud, internode, or lateralbud of pea seedlings released apical dominance in either intact orindole-3-acetic acid (IAA )-treated, decapitated plants. These results suggestthat the auxin-inhibiting substances raphanusanin B and MBOA have activity inreleasing apical dominance. Conversely, the auxin transport inhibitors2,3,4-triiodobenzoic acid (TIBA) and 1-naphthylphthalamic acid (NPA) did notstimulate lateral bud growth when they were applied directly to the lateralbud,although application to the apical bud or internode released apical dominance.Therefore, the mode of action of raphanusanin B and MBOA in apical dominance isclearly different from that of auxin transport inhibitors. Raphanusanin B andMBOA may suppress the synthesis of growth-inhibiting factor(s) of the lateralbud induced by endogenous auxin transported from the apical bud or exogenouslyapplied auxin, and/or the action of the factor(s).     

Dormancy-associated gene expression in pea axillary buds.

Pea (Pisum sativum L. cv. Alaska) axillary buds can be stimulated to cycle between dormant and growing states. Dormant buds synthesize unique proteins and are as metabolically active as growing buds. Two cDNAs, PsDRM1 and PsDRM2, were isolated from a dormant bud library. The deduced amino acid sequence of PsDRM1 (111 residues) is 75% identical to that of an auxin-repressed strawberry clone. PsDRM2 encodes a putative protein containing 129 residues, which includes 11 repeats of the sequence [G]-GGGY[H][N] (the bracketed residues may be absent). PsDRM2 is related to cold- and ABA-stimulated clones from alfalfa. Decapitating the terminal bud rapidly stimulates dormant axillary buds to begin growing. The abundance of PsDRM1 mRNA in axillary buds declines 20-fold within 6 h of decapitation; it quickly reaccumulates when buds become dormant again. The level of PsDRM2 mRNA is about three fold lower in growing buds than in dormant buds. Expression of PsDRM1 is enhanced in other non-growing organs (roots root apices; fully-elongated stems >elongating stems), and thus is an excellent “dormancy” marker. In contrast, PsDRM2 expression is not dormancy-associated in other organs. Received: 10 December 1997 / Accepted: 23 January 1998     

Endogenous abscisic acid levels in stems and axillary buds of intact or decapitated broad-bean plants (Vicia faba L.)

Endogenous levels of abscisic acid (ABA) were measured by gas-liquid chromatography (electron capture) in stems and axillary buds of intact or decapitated broad-bean plants ( Vicia faba L. cv. Aguadulce). Endogenous ABA was distributed in the main axis according to a concentration gradient from the apical part of the stem towards the base. Axillary buds contained ABA levels which were from 4 to 9 times higher than those in the corresponding nodes and internodes. Decapitation of the plant was followed within 6 h by a large decrease of ABA levels in all the parts of the main axis. The diminution of ABA content was the most important in axillary buds released from apical dominance. Twenty-four hours after the decapitation, the ABA concentration further decreased in the upper parts of the stem, while no modification was observed in the basal parts of the stem containing the smallest levels of ABA.     

Another evidence for inhibitory effect of auxin in adventitious bud formation of decapitated flax (Linum usitatissimum L.) seedlings

Adventitious buds were formed on the hypocotyls of decapitated flax seedlings. Scanning electron and light microscopic examinations of hypocotyls showed that epidermal cells divided to produce meristematic spots from which several leaf primordia were formed. Between leaf primordia and the original vascular tissues of hypocotyls, new xylem cells were formed which connected them. About 10, 30 and 60% of adventitious buds were formed on upper, middle and basal parts of hypocotyls of decapitated seedlings, respectively. Removal of apical meristem together with longer hypocotyl zero to four cm long below the apical meristem) induced higher percentage of adventitious bud formation in the remaining hypocotyl. When the entire hypocotyl was cut into 16 segments (0.25 cm each) and these segments were cultured on MS medium containing 3% sucrose and 0.8% agar, adventitious buds were mainly formed in the lowest five segments. These results suggested that there was a gradient of inhibitory factor(s) from apical to basal part of hypocotyl with respect to adventitious bud formation. Auxin transport inhibitors, morphactin and TIBA induced adventitious bud formation on intact seedlings by suppressing the basipetal movement of auxin.     

Changes in cytokinins during initiation and development of potato tubers

Cytokinin-like activity was assayed in stolons and tubers of Solanum tuberosum L. ssp. andigena (Juz. et Buk.) Hawkes cv. 165 grown in pots under controlled environment conditions. The plants were allowed to tuberise without the application of environmental or other external stimuli. The soluble sugar and starch contents of stolon tips and tubers were measured. Starch accumulation was a precise indicator of tuber initiation. Cytokinin-like activity began to increase in tubers with a diameter greater than 7.5 mm and, as assessed on a per tuber basis, was greatest in the largest size-category analysed. However, expressed as a function of fresh and dry weight, activity was greatest in tubers of 15–20 mm in diameter. Increases in cytokinin-like activity occurred subsequent to tuber formation, indicating that the tuberisation stimulus is unlikely to be cytokinin-like in nature.     

Genetic mapping in pea. 2. Identification of RAPD and SCAR markers linked to genes affecting plant architecture

 Random amplified polymorphic DNA (RAPD) markers linked to two morphological markers ( fa and det), three ramosus genes (rms2, rms3 and rms4) and two genes conferring flowering response to photoperiod in pea (sn, dne) were selected by bulk segregant analysis on F₂ populations. Two RAPD fragments were cloned and sequenced to generate the two SCAR markers V20 and S2 which are linked to rms3 and dne, respectively. All these genes, except rms2, were previously located on the pea classical linkage map. Rms2 mapped to linkage group IB which contains the afila gene. Precise genetic maps of the regions containing the genes were obtained and compared to the RAPD map generated from the recombinant inbred-lines population of the cross Térèse×K586. This cross was chosen because several mutants were obtained from cultivars Térèse and Torsdag (K586 was derived from Torsdag). This collection of isogenic lines was used for the construction of F₂ mapping populations in which polymorphic RAPD markers were already known and mapped. Moreover, the well-known problem in pea of variability in the linkage associations between crosses was avoided. This work contributes to the precise integration between the classical map and the molecular maps existing in pea. Received: 13 March 1998 / Accepted: 29 April 1998     

高表达水稻WRKY72基因影响拟南芥生长素信号传导

植物转录调控因子WRKY基因家族是一个拥有众多成员的超家族,功能涵盖了植物生长发育的控制与抗病耐逆的调节。我们主要分析了OsWRKY72基因在外源植物拟南芥中的生物学功能。通过转基因拟南芥(Arabidopsis thaliana)的遗传学研究发现外源高表达该基因不单明显地抑制转基因植株的顶端优势,增强植株侧枝的生长,还改变了转基因植株叶片和角果的发育。进一步分析证实,高表达OsWRKY72基因所导致转基因拟南芥植株的表型和其它生理现象都与生长素信号通路改变所导致的表型和生理变化极其相近。这些结果说明OsWRKY72基因在外源植物拟南芥体内高表达后很可能改变了其正常的生长素信号通路。     

Competitive canalization of PIN-dependent auxin flow from axillary buds controls pea bud outgrowth

Shoot branching is one of the major determinants of plant architecture. Polar auxin transport in stems is necessary for the control of bud outgrowth by a dominant apex. Here, we show that following decapitation in pea (Pisum sativum L.), the axillary buds establish directional auxin export by subcellular polarization of PIN auxin transporters. Apical auxin application on the decapitated stem prevents this PIN polarization and canalization of laterally applied auxin. These results support a model in which the apical and lateral auxin sources compete for primary channels of auxin transport in the stem to control the outgrowth of axillary buds.     

Involvement of carbohydrates and cytokinins in pathogenicity ofHelminthosporium carbonum

Significant stimulation of the number of appressoria, penetration and colonization by conidia ofHelminthosporium carbonum occurred on decolorized maize leaves when exogenous carbohydrates and leaf leachates were added. Germination and germ tube length, however, did not exhibit appreciable differences on decolorized or non-decolorized maize leaves. Lower germination was recorded by leached conidia on decolorized leaves; while appressoria, penetration and colonization were absent. Addition of exogenous nutrients (sucrose>leaf leachates>yeast extract>glucose) enabled conidia to accomplish appressoria, penetration and colonization. Optimum levels for various nutrients observed were 2% (w/v) sucrose/glucose or 0.1% (w/v) yeast extract. Higher concentrations inhibited the infection stages of the pathogen. Depletion of host carbohydrates from green islands/infection sites adversely affect appressoria formation, penetration and colonization; and the loss of carbohydrates from the spore affects germination. Cytokinin-like activity at the infection site/green islands increased with the period of incubation of the host as compared to the surrounding tissue or tissue under water drops. The culture filtrate extracts ofH. carbonum recorded cytokinin-like activity which increased with growth of the fungus. TLC (thin layer chromatography) of cytokinin-like substances (tissue extract and culture filtrate) revealed major activity was confined to Rf zones 0.6 to 0.8 which co-chromatographed with zeatin and zeatin riboside. These substances increase at infection sites by virtue of which carbohydrates accumulate at these sites ensuring a continuous supply to the growing pathogen.     

Patterns of protein synthesis in dormant and growing vegetative buds of pea

Lateral buds on intact pea plants (Pisum sativum L. cv. Alaska) remain dormant until they are stimulated to develop by decapitating the terminal bud. Using two-dimensional gel electrophoresis, we have examined the protein content of terminal and lateral buds from intact plants and from plants at various times after decapitation. Silver-staining and in-vivo-labeling demonstrated very different sets of proteins. The level of expression of 18 stained and 25 labeled proteins was altered when growth was stimulated; this represents 3.4% and 9.1% of the total proteins detected by each method, respectively. Within 24 h of being stimulated, lateral buds doubled in length and their protein content was qualitatively nearly the same as that of terminal buds. Six hours after decapitation, before the onset of detectable growth, the overall pattern of protein synthesis in lateral buds was more like that of growing lateral buds or of terminal buds than that of dormant lateral buds. Direct application of N⁶-furfurylaminopurine (kinetin) to buds on intact plants stimulated their growth and resulted in the same pattern of protein synthesis as did decapitation. Inhibition of bud growth by addition of indole-3-acetic acid to the stumps of decapitated plants resulted in the synthesis of dormancy-related proteins. Lateral buds at all stages of development incorporated labeled amino acids at similar rates, indicating that metabolic activity is not a component of dormancy in these buds.Abbreviations IAA indole-3-acetic acid - IEF isoelectric focusing - KIN kinetin (N⁶-furfurylaminopurine) - SDS sodium dodecylsulfate - TCA trichloroacetic acid - 2D-PAGE two-dimensional polyacrylamide gel electrophoresis     

The possible roles of nutrient deprivation and auxin repression in apical control

Apical control is the suppression of growth in lower branches by a higher dominant branch or leader shoot. We investigated possible mechanisms involved in this developmental response in three widely diverse species (Japanese morning glory, Ipomoea nil, hybrid poplar, Populus trichocarpa, × P. deltoides, and Douglas-fir, Pseudotsuga menziesii). The following two hypotheses were tested: (1) the mineral nutrient-deprivation hypothesis, which is that the continued growth of the lower branches is repressed by the diversion of nutrients to the upper dominating branch or shoot, and (2) the auxin-repression hypothesis, which is that auxin produced in the upper dominating branch or shoot moves down to the lower branches where continued growth is repressed. The results of experiments involving the manipulation of available nutrients by dominant branch removal and fertilization were consistent with the first hypothesis for morning glory, poplar, and for second- or third flushing of lateral branches in Douglas-fir. The results of the experiments involving auxin (NAA, 1-naphthalene acetic acid) replacement treatments on decapitated shoots bearing growing lateral branches were inconsistent with the second hypothesis in morning glory, poplar and in first-flushing Douglas-fir. However, despite concerns about possible NAA toxic effects, there was evidence of auxin repression of second flushing in Douglas-fir. Overall, the data supported a significant role for nutrient availability but not for auxin repression in apical control of morning glory and poplar. In Douglas-fir, apical control in first-flushing lateral branches from over-wintered buds was largely insensitive to both nutrient availability and auxin repression; however, second flushing was sensitive to both.     

Requirement for the gravity-controlled transport of auxin for a negative gravitropic response of epicotyls in the early growth stage of etiolated pea seedlings

Gravity-controlled transport of auxin was studied for a negative gravitropic response in the early growth stage of etiolated pea (Pisum sativum L. cv. Alaska) seedlings, in which epicotyl bending was observed near the cotyledon nodes of the seedlings grown continuously from seeds germinated in a horizontal or an inclined position. Increased expression of an auxin-inducible gene, PsIAA4/5, was observed in the elongated side of epicotyls grown in a horizontal or an inclined position. Regardless of the conditions of seed germination, polar auxin transport in the proximal side of the first internodes of the seedlings was significantly higher than in the distal side. Polar auxin transport in the proximal side of epicotyls grown in an inclined position was significantly lower than in those grown in a horizontal position. In contrast, lateral auxin distribution from the proximal to distal sides in epicotyls grown in an inclined position was significantly higher than in epicotyls grown in a horizontal position. Accumulation of PsPIN1 mRNA encoding a putative auxin efflux facilitator, which was observed in vascular tissue, cortex and epidermis in the proximal and distal sides of epicotyls, was markedly influenced by gravistimulation. These results strongly suggest that gravistimulation induces changeable polar auxin transport and one-way lateral auxin distribution in epicotyls as well as asymmetric auxin accumulation in the proximal and distal sides of epicotyls, resulting in a negative gravitropic response of epicotyls in the early growth stage of pea seedlings.     

Spatial and temporal changes in endogenous cytokinins in developing pea roots

Germination and seedling establishment follows a distinct pattern which is partly controlled by hormones. Roots have high levels of cytokinins. By quantifying the fluctuations in endogenous cytokinins over time, further insight may be gained into the role of cytokinins during germination and seedling establishment. Radicles were excised from sterile Pisum sativum L. seeds after 30 min and 5 h imbibition. Seedlings germinated on agar were harvested after 1, 3, 6 and 9 days. The roots were divided into the root tip, root free zone, secondary root zone and from day 6, the secondary roots. Samples were purified by various chromatographic methods and endogenous cytokinins detected by LC(+)ES-MS. Benzyladenine levels doubled after 5 h imbibition and then gradually decreased over time. Low concentrations of cis-Zeatin (cZ) type cytokinins were detected in the radicle after 30 min imbibition. After 5 h imbibition, cis-zeatin riboside-5′-monophosphate had greatly increased. The total cytokinin content of the roots increased over time with the ribotides being the predominant conjugates. From day 3 onwards, there was a gradual increase in the free bases, O-glucosides and their ribosylated forms. Mainly N ⁶ -(2-isopentenyl)adenine (iP)-type cytokinins were detected in the root tip, whereas trans-zeatin- (tZ), dihyrozeatin- (DHZ) and iP-type cytokinins were found in the secondary roots and root zone. Cytokinin biosynthesis was only detected after day 6. Biosynthesis of iP and tZ derivatives was quite rapid, whereas biosynthesis of cZ derivatives remained at a low basal level. These fluctuations in cytokinin types and concentrations suggest the cytokinins may be synthesized from various pathways in pea roots.     

Response of cytokinin concentration in the xylem exudate of bean (Phaseolus vulgaris L.) plants to decapitation and auxin treatment,and relationship to apical dominance

When xylem exudate of previously untreated Phaseolus vulgaris plants was analysed for cytokinins by radioimmunoassay, a low concentration (about 5 ng · ml^–1) was found. However, when the plants were decapitated about 16 h before the xylem exudate was collected, an almost 25-fold increase in cytokinin concentration was observed. Twenty-four hours after decapitation this increase even reached 4000 compared to control plants. Applying naphthaleneacetic acid (NAA) to the shoot of decapitated plants almost eliminated the effect of shoot tip removal on cytokinin concentration, suggesting that cytokinins in the xylem exudate of intact plants are under the control of the polar auxin transport system. Other xylem constituents, such as potassium or free amino acids did not show this strong increase after decapitation and did not respond to NAA application. It is concluded that the observed auxin/cytokinin interaction has an important regulatory role to play, not only in apical dominance but in many other correlative events as well.Abbreviations AD apical dominance - CKs cytokinin(s) - iAde/iAdo isopentenyladenine/iospentenyladenosine - NAA naphthaleneacetic acid - Z/ZR zeatin/zeatin riboside     

Mutual interaction of auxin and cytokinins in regulating correlative dominance

Correlative dominance requires correlative signals from a dominant to a dominated organ. Auxins, particularly IAA, and cytokinins are obviously important components of this correlative system. Using a vegetative pea shoot and a generative apple and tomato fruit system it can be demonstrated that dominant organs always export more IAA and have a higher ³H-IAA transport capacity and velocity compared to dominated organs. In both systems the dominant organ can be replaced by the application of auxin, e.g. NAA, which maintains the differences in IAA export. This is an indication that similar regulatory mechanisms control dominance in both of these diverse systems. The possibility of replacing a dominant organ by auxin also makes it unlikely that growth of that organ or allocation of nutrients regulates the correlative inhibition of the dominated organ.It is suggested that differences in IAA export from, and transport capacities of, dominant and dominated shoots, may be explained by a mechanism of auxin transport autoinhibition (ATA), whereby the earlier and stronger export of IAA from the dominant shoot inhibits auxin export from the dominated shoot at the point where the two auxin streams converge. This hypothesis was tested with explants of pea, apple and tomato. It was shown that the basal application of cold IAA significantly reduced endogenous as well as exogenous IAA transport through these explants.Since the reduced IAA transport of dominated organs was not followed by an accumulation of IAA in the auxin producing subtending organ, it was concluded that IAA biosynthesis was possibly reduced and/or IAA conjugation stimulated. This could have been one of the determinants of their growth inhibition. ATA might also explain how the unidirectional IAA signal may affect the growth rate of organs even lateral or acropetal to its transport pathway and thus polar IAA-transport becomes a ``multidirectional' signal. From the experiments demonstrated it seems that ATA is a sufficient mechanism to impose growth inhibition in the dominated organ, without the need of other regulators.However, to release dominated organs from dominance cessation of ATA may not be sufficient and cytokinins are obviously a powerful antagonist to auxins. Their repeated exogenous application turns dominated lateral buds into strongly growing organs which ultimately may even dominate the previously dominant apex. These lateral shoots finally gain a strong IAA export capacity and inhibit, by ATA, IAA export from the hitherto dominant apex.In other experiments it was shown that interruption of polar IAA transport leads to a strong increase in root derived cytokinins. This can largely be prevented, in a concentration dependent manner, by the application of auxin, indicating that basipolar auxin may control cytokinin production in the roots and its possible delivery to lateral buds. In turn, the increased delivery of cytokinins to the lateral buds promotes a strong increase in IAA production and export. Thus there is a strong mutual interaction between auxin production in the shoots and cytokinin production in the roots, which may be important in regulating the balance between root and shoot growth.     

Polypheolic auxin protectors in buds of juvenile and adult chestnut

Both qualitative and quantitative estimates of the relative amounts of several poly-phenols in buds of juvenile and adult plants of chestnut ( Castanea sativa x C. crenata ) were carried out. In buds of both types of plants, chesnatin, crenatin, cretanin and (+)-catechin were identified. Crenatin and cretanin showed inhibitory activity of peroxidase-catalysed indoleacetic acid oxidation. Auxin protection capacity was greater in extracts of the juvenile tissues which also were richer in active phenols. The phenolic content and its possible relationship to the easier establishment in vitro of juvenile tissue is discussed.     

Rooting, growth and ethylene evolution of pea cuttings in response to chloroindole auxins

In pea cuttings ( Pisum sativum L. cv. Alaska) we measured shoot and root growth and ethylene production in response to 4-chloroindole-3-acetic acid (4-CI-IAA) or 4,6-dichloroindole-3-acetic acid (4,6-Cl₂-IAA). Leafy cuttings treated basally with either of the chlorinated auxins in high concentrations showed permanent epinasty, loss of apical growth and dominance resulting in the outgrowth of laterals from the lower-most axillary bud. The naturally occurring 4-CI-IAA was a better root promoter than the synthetic 4,6-Cl₂-IAA which inhibited rooting. Both chloroindole auxins induced very high ethylene evolution, which lasted much longer than the ethylene evolution after IAA treatment.     

Effect of apex excision and replacement by 1-naphthylacetic acid on cytokinin concentration and apical dominance in pea plants

As known from literature lateral buds from pea ( Pisum sativum ) plants are released from apical dominance when repeatedly treated with exogenous cytokinins. Little is known, however, about the endogenous role of cytokinins in this process and whether they interact with basipolar transported IAA, generally regarded as the main signal controlling apical dominance. This paper presents evidence that such an interaction exists.
The excision of the apex of pea plants resulted in the release of inhibited lateral buds from apical dominance (AD). This could be entirely prevented by applying 1-naphthylacetic acid (NAA) to the cut end of the shoot. Removal of the apex also resulted in a rapid and rather large increase in the endogenous concentrations of zeatin riboside (ZR), isopentenyladenosine (iAdo) and an as yet unidentified polar zeatin derivative in the node and internode below the point of decapitation. This accumulation of ZR and iAdo, was strongly reduced by the application of NAA. The observed increase in cytokinin concentration preceded the elongation of the lateral buds, suggesting that endogenous cytokinins play a significant role in the release of lateral buds from AD. However, the effect of NAA on the concentration of cytokinins clearly demonstrated the dominant role of the polar basipetally transported auxin in AD. The results suggest a mutual interaction between the basipolar IAA transport system and cytokinins obviously produced in the roots and transported via the xylem into the stem of the pea plants.