Auxin inhibition of decapitation-induced branching is dependent on graft-transmissible signals regulated by genes Rms1 and Rms2

Decapitation-induced axillary bud outgrowth is a vital mechanism whereby shoots are able to continue normal growth and development. In many plants, including wild-type garden pea (Pisum sativum L.), this process can be inhibited by exogenous auxin. Using the ramosus (rms) increased branching mutants of pea, we present evidence that this response to auxin is dependent on graft-transmissible substance(s) regulated by the genes Rms1 and Rms2. The response to exogenous auxin is massively diminished in decapitated rms1 and rms2 mutant plants. However, basipetal auxin transport is not reduced in intact or decapitated mutants. Grafting rms1 or rms2 shoots onto wild-type rootstocks restored the auxin response, indicating that Rms1 and Rms2 gene action in the rootstock is sufficient to enable an auxin response in mutant shoots. We conclude that Rms1 and Rms2 act in the rootstock and shoot to control levels of mobile substance(s) that interact with exogenous auxin in the inhibition of bud outgrowth after decapitation. At least for rms1, the reduced auxin response is unlikely to be due to an inability of auxin to decrease xylem sap cytokinin content, as this is already low in intact rms1 plants. Consequently, we have genetic evidence that auxin action in decapitated plants depends on at least one novel long-distance signal.     

Mutational Analysis of Branching in Pea. Evidence That Rms1 and Rms5 Regulate the Same Novel Signal

The fifth increased branching ramosus (rms) mutant, rms5, from pea (Pisum sativum), is described here for phenotype and grafting responses with four other rms mutants. Xylem sap zeatin riboside concentration and shoot auxin levels in rms5 plants have also been compared with rms1 and wild type (WT). Rms1 and Rms5 appear to act closely at the biochemical or cellular level to control branching, because branching was inhibited in reciprocal epicotyl grafts between rms5 or rms1 and WT plants, but not inhibited in reciprocal grafts between rms5 and rms1 seedlings. The weakly transgressive or slightly additive phenotype of the rms1 rms5 double mutant provides further evidence for this interaction. Like rms1, rms5 rootstocks have reduced xylem sap cytokinin concentrations, and rms5 shoots do not appear deficient in indole-3-acetic acid or 4-chloroindole-3-acetic acid. Rms1 and Rms5 are similar in their interaction with other Rms genes. Reciprocal grafting studies with rms1, rms2, and rms5, together with the fact that root xylem sap cytokinin concentrations are reduced in rms1 and rms5 and elevated in rms2 plants, indicates that Rms1 and Rms5 may control a different pathway than that controlled by Rms2. Our studies indicate that Rms1 and Rms5 may regulate a novel graft-transmissible signal involved in the control of branching.     

Branching Mutant rms-2 in Pisum sativum (Grafting Studies and Endogenous Indole-3-Acetic Acid Levels)

Isogenic lines of pea (Pisum sativum L.) were used to determine the physiological site of action of the Rms-2 gene, which maintains apical dominance, and its effect on endogenous free indole-3-acetic acid (IAA) levels. In mutant rms-2 scions, which normally produce lateral branches below node 3 and above node 7, apical dominance was almost fully restored by grafting to Rms-2 (wild-type) stocks. In the reciprocal grafts, rms-2 stocks did not promote branching in wild-type shoots. Together, these results suggest that the Rms-2 gene inhibits branching in the shoot of pea by controlling the synthesis of a translocatable (hormone-like) substance that is produced in the roots and/or cotyledons and in the shoot. At all stages, including the stage at which aerial lateral buds commence outgrowth, the level of IAA in rms-2 shoots was elevated (up to 5-fold) in comparison with that in wild-type shoots. The internode length of rms-2 plants was 40% less than in wild-type plants, and the mutant plants allocated significantly more dry weight to the shoot than to the root in comparison with wild-type plants. Grafting to wild-type stocks did not normalize IAA levels or internode length in rms-2 scions, even though it inhibited branching, suggesting that the involvement of Rms-2 in the control of IAA level and internode length may be confined to processes in the shoot.     

Long-distance signaling and the control of branching in the rms1 mutant of pea

The ramosus (rms) mutation (rms1) of pea (Pisum sativum) causes increased branching through modification of graft-transmissible signal(s) produced in rootstock and shoot. Additional grafting techniques have led us to propose that the novel signal regulated by Rms1 moves acropetally in shoots and acts as a branching inhibitor. Epicotyl interstock grafts showed that wild-type (WT) epicotyls grafted between rms1 scions and rootstocks can revert mutant scions to a WT non-branching phenotype. Mutant scions grafted together with mutant and WT rootstocks did not branch despite a contiguous mutant root-shoot system. The primary action of Rms1 is, therefore, unlikely to be to block transport of a branching stimulus from root to shoot. Rather, Rms1 may influence a long-distance signal that functions, directly or indirectly, as a branching inhibitor. It can be deduced that this signal moves acropetally in shoots because WT rootstocks inhibit branching in rms1 shoots, and although WT scions do not branch when grafted to mutant rootstocks, they do not inhibit branching in rms1 cotyledonary shoots growing from the same rootstocks. The acropetal direction of transport of the Rms1 signal supports previous evidence that the rms1 lesion is not in an auxin biosynthesis or transport pathway. The different branching phenotypes of WT and rms1 shoots growing from the same rms1 rootstock provides further evidence that the shoot has a major role in the regulation of branching and, moreover, that root-exported cytokinin is not the only graft-transmissible signal regulating branching in intact pea plants.     

Feedback regulation of xylem cytokinin content is conserved in pea and Arabidopsis

Increased-branching mutants of garden pea (Pisum sativum; ramosus [rms]) and Arabidopsis (Arabidopsis thaliana; more axillary branches) were used to investigate control of cytokinin export from roots in relation to shoot branching. In particular, we tested the hypothesis that regulation of xylem sap cytokinin is dependent on a long-distance feedback signal moving from shoot to root. With the exception of rms2, branching mutants from both species had greatly reduced amounts of the major cytokinins zeatin riboside, zeatin, and isopentenyl adenosine in xylem sap compared with wild-type plants. Reciprocally grafted mutant and wild-type Arabidopsis plants gave similar results to those observed previously in pea, with xylem sap cytokinin down-regulated in all graft combinations possessing branched shoots, regardless of root genotype. This long-distance feedback mechanism thus appears to be conserved between pea and Arabidopsis. Experiments with grafted pea plants bearing two shoots of the same or different genotype revealed that regulation of root cytokinin export is probably mediated by an inhibitory signal. Moreover, the signaling mechanism appears independent of the number of growing axillary shoots because a suppressed axillary meristem mutation that prevents axillary meristem development at most nodes did not abolish long-distance regulation of root cytokinin export in rms4 plants. Based on double mutant and grafting experiments, we conclude that RMS2 is essential for long-distance feedback regulation of cytokinin export from roots. Finally, the startling disconnection between cytokinin content of xylem sap and shoot tissues of various rms mutants indicates that shoots possess powerful homeostatic mechanisms for regulation of cytokinin levels.     

Pea rms6 mutants exhibit increased basal branching

Our studies on two branching mutants of pea ( Pisum sativum L.) have identified a further Ramosus locus , Rms6, with two recessive or partially recessive mutant alleles: rms6-1 (type line S2-271) and rms6-2 (type line K586). Mutants rms6-1 and rms6-2 were derived from dwarf and tall cultivars, Solara and Torsdag, respectively. The rms6 mutants are characterized by increased branching from basal nodes. In contrast, mutants rms1 through rms5 have increased branching from both basal and aerial (upper stem) nodes. Buds at the cotyledonary node of wild-type (WT) plants remain dormant but in rms6 plants these buds were usually released from dormancy. Their growth was either subsequently inhibited, sometimes even prior to emergence above ground, or they grew into secondary stems. The mutant phenotype was strongest for rms6-1 on the dwarf background. Although rms6-2 had a weak single-mutant phenotype, the rms3-1 rms6-2 double mutant showed clear transgression and an additive branching phenotype, with a total lateral length almost 2-fold greater than rms3-1 and nearly 5-fold greater than rms6-2 . Grafting studies between WT and rms6-1 plants demonstrated the primary action of Rms6 may be confined to the shoot. Young WT and rms6-1 shoots had similar auxin levels, and decapitated plants had a similar magnitude of response to applied auxin. Abscisic acid levels were elevated 2-fold at node 2 of young rms6-1 plants. The Rms6 locus mapped to the R to Gp segment of linkage group V (chromosome 3). The rms6 mutants will be useful for basic research and also have possible agronomical value.     

STRUCTURE AND DEVELOPMENT OF THE DIMORPHIC BRANCH SYSTEM OF THEOBROMA CACAO

The vegetative morphology of Theobroma cacao, the cacao tree, was studied in order to provide a foundation for further investigations on the morphogenesis of the cacao dimorphic shoot system. The seedling of cacao has a determinate orthotropic shoot with a (2+3) phyllotaxis. Branch dimorphism is initiated after 1 to 2 years of growth at which time the apical meristem of the orthotropic shoot aborts and a pseudowhorl of plagiotropic branches is initiated from axillary positions in the shoot tip. The plagiotropic branches are characterized by a distichous phyllotaxis and indeterminate growth. Subsequently an axillary bud below the pseudowhorl develops into a new orthotropic shoot. The apical meristem of this shoot eventually aborts and another pseudowhorl is formed. The apical anatomy of the two types of shoots is similar. The developmental potentiality of the orthotropic shoot axillary buds to form one or the other type of shoot was investigated. The phyllotaxis of the axillary buds of the orthotropic shoot is spiral and that of the axillary buds of the plagiotropic branch is distichous. Pruning and apical puncture experiments showed that the axillary buds of a plagiotropic branch, and of an orthotropic seedling shoot which has not yet formed a pseudowhorl, always give rise to the parent type of shoot. However, the axillary buds of an orthotropic shoot which already bears a pseudowhorl give rise to either type of shoot for several nodes below the point of origin of the pseudowhorl. The type of shoot has no influence on the form of branch which develops from an axillary bud grafted to it. This evidence supports the hypothesis that the axillary buds are initiated as one or the other type of shoot, i.e., once initiated they are predestined.     

AtMYB2 regulates whole plant senescence by inhibiting cytokinin-mediated branching at late stages of development in Arabidopsis

Whole plant senescence of monocarpic plants consists of three major processes: arrest of shoot apical meristem, organ senescence, and permanent suppression of axillary buds. At early stages of development, axillary buds are inhibited by shoot apex-produced auxin, a mechanism known as apical dominance. How the buds are suppressed as an essential part of whole plant senescence, especially when the shoot apexes are senescent, is not clear. Here, we report an AtMYB2-regulated post apical dominance mechanism by which Arabidopsis (Arabidopsis thaliana) inhibits the outgrowth of axillary buds as part of the whole plant senescence program. AtMYB2 is expressed in the compressed basal internode region of Arabidopsis at late stages of development to suppress the production of cytokinins, the group of hormones that are required for axillary bud outgrowth. atmyb2 T-DNA insertion lines have enhanced expression of cytokinin-synthesizing isopentenyltransferases genes, contain higher levels of cytokinins, and display a bushy phenotype at late stages of development. As a result of the continuous generation of new shoots, atmyb2 plants have a prolonged life span. The AtMYB2 promoter-directed cytokinin oxidase 1 gene in the T-DNA insertion lines reduces the endogenous cytokinin levels and restores the bushy phenotype to the wild type.     

The gravity-regulated growth of axillary buds is mediated by a mechanism different from decapitation-induced release

When the upper part of the main shoot of the Japanese morning glory (Pharbitis nil or Ipomoea nil) is bent down, the axillary bud situated on the uppermost node of the bending region is released from apical dominance and elongates. Here, we demonstrate that this release of axillary buds from apical dominance is gravity regulated. We utilized two agravitropic mutants of morning glory defective in gravisensing cell differentiation, weeping (we) and weeping2 (we2). Bending the main shoots of either we or we2 plants resulted in minimal elongation of their axillary buds. This aberration was genetically linked to the agravitropism phenotype of the mutants, which implied that shoot bending-induced release from apical dominance required gravisensing cells. Previous studies have shown that basipetal translocation of auxin from the apical bud inhibits axillary bud growth, whereas cytokinin promotes axillary bud outgrowth. We therefore compared the roles of auxin and cytokinin in bending- or decapitation-induced axillary bud growth. In the wild-type and we plants, decapitation increased cytokinin levels and reduced auxin response. In contrast, shoot bending did not cause significant changes in either cytokinin level or auxin response, suggesting that the mechanisms underlying gravity- and decapitation-regulated release from apical dominance are distinct and unique.     

AXR1 acts after lateral bud formation to inhibit lateral bud growth in Arabidopsis

The AXR1 gene of Arabidopsis is required for many auxin responses. The highly branched shoot phenotype of mature axr1 mutant plants has been taken as genetic evidence for a role of auxin in the control of shoot branching. We compared the development of lateral shoots in wild-type Columbia and axr1-12 plants. In the wild type, the pattern of lateral shoot development depends on the developmental stage of the plant. During prolonged vegetative growth, axillary shoots arise and develop in a basal-apical sequence. After floral transition, axillary shoots arise rapidly along the primary shoot axis and grow out to form lateral inflorescences in an apical-basal sequence. For both patterns, the axr1 mutation does not affect the timing of axillary meristem formation; however, subsequent lateral shoot development proceeds more rapidly in axr1 plants. The outgrowth of lateral inflorescences from excised cauline nodes of wild-type plants is inhibited by apical auxin. axr1-12 nodes are resistant to this inhibition. These results provide evidence for common control of axillary growth in both patterns, and suggest a role for auxin during the late stages of axillary shoot development following the formation of the axillary bud and several axillary leaf primordia.     

In vitro clonal multiplication of an apple rootstock by culture of shoot apices and axillary buds

In vitro clonal multiplication of apple rootstock MM 111 using axillary buds and shoot apices were carried out. Vegetative axillary buds of the size of 0.2-2.0 cm and shoot apices measuring 4 mm in length were initiated to shoot proliferation on MS medium supplemented with BA (0.5 - 1.0 mgl(-1)), GA3(0.5 mgl(-1)), with or without IBA(0.05 - 0.1 mgl(-1)). Small size explants showed less phenol exudation and less contamination. Following establishment phase, the small shoots emerged from explants were subcultured on MS medium supplemented with different combinations and concentrations of growth regulators. BA (1.0 mgl(-1)) and GA3 (0.5 mgl(-1)) combination showed highest multiplication rate (1:5), andcl also produced longer shoots. Two step rooting was done by transferring microcuttings to auxin free solid medium after root initiation in dark on 1/2 strength MS liquid medium containing IBA (0.5 mgl(-1) ). Rooted plantlets were transferred to peat containing paper cups and resulting plants of MM 111 acclimated successfully for transfer to field.     

Floral determination in the terminal bud of the short-day plant Pharbitis nil

Temporal and spatial aspects of floral determination in seedling terminal buds of the qualitative short-day plant Pharbitis nil were examined using a grafting assay. Floral determination in the terminal buds of 6-day-old P. nil seedlings is rapid; by 9 hr after the end of a 14-hr inductive dark period more than 50% of the induced terminal buds grafted onto uninduced stock plants produced a full complement of flower buds. When grafted at early times after the end of the dark period the terminal buds of induced plants produced three discrete populations of plants: plants with no flowers, plants with two axillary flowers at nodes 3 and 4 and a vegetative terminal shoot apex, and plants with five to seven flowers including a terminal flower. The temporal relationship among these populations of plants produced by apices grafted at different times indicates that under our conditions, the region of the terminal bud that will form the axillary buds at nodes 3 and 4 becomes florally determined prior to floral determination of the region of the terminal bud giving rise to the nodes above node 4.     

Apical wilting and petiole xylem vessel diameter of the rms2 branching mutant of pea are shoot controlled and independent of a long-distance signal regulating branching

RMS2 (RAMOSUS2) affects the level or transport of a graft-transmissible signal produced in the shoot and root that controls axillary bud outgrowth in pea (Pisum sativum L.). The shoot apex of rms2 transiently wilts under high evaporative demand. The origin of this phenotype was investigated to determine whether it was involved in the regulation of branching. Wild-type (WT) and rms2 leaves showed a similar stomatal conductance at both low and high evaporative demand in vivo, indicating normal stomatal function. Leaves of both genotypes had similar ABA content and response to ABA. Although root hydraulic conductance (determined by pressure-induced flow) of rms2 plants was normal, more xylem vessels per vascular bundle were identified in cross-sections of fully expanded rms2 petioles compared with those of the WT. However, the diameter of these vessels was nearly half that of the WT. Since the conductance of each vessel is proportional to the fourth power of the vessel radius (according to the Hagen-Poiseulle law), the theoretical (calculated) petiole hydraulic conductance of rms2 was greatly decreased compared with WT plants. Under high evaporative demand, this would cause a temporary imbalance between water supply to, and demand from, rms2 shoots, directly resulting in the wilting phenotype of the mutant. Reciprocal grafting showed that xylem vessel development in rms2 shoots is strictly shoot controlled, probably via elevated auxin levels. This altered xylem vessel development, though causing wilting in rms2 shoot tips, does not appear to affect shoot branching.     

棉花花芽分化及部分内源激素变化规律的研究

棉花（Ｇｏｓｓｙｐｉｕｍ ｈｉｒｓｕｔｕｍ）的腋芽原基,有的将来发育成叶枝;有的将来发育成果枝。这２种不同命运的腋芽,在其刚分化的初期就表现出了不同的解剖学特征。将来发育为叶枝的腋芽,其生长锥呈圆锥形或扁圆球形,体积较小,原套层数为１－２层;而将来发育为果枝的腋芽,其生长锥为圆柱形,顶端表面平坦,体积较大,原套层数为２－３层。从子叶展平后到肉眼可见花芽（现蕾）,连续测茎尖的内源ＡＢＡ及ＩＡＡ的含量     

In vitro propagation of Clerodendrum colebrookianum Walp., a potential natural anti-hypertension medicinal plant

A rapid clonal propagation system for Clerodendrum colebrookianum Walp. (Verbenaceae), a anti-hypertension folk medicinal shrub has been developed. A range of cytokinins has been investigated for multiple shoot induction with shoot apex, axillary shoot, leaf, petiole and root explants. Optimum shoot induction occurred with axillary buds using 6-benzyladenine where an average of 21 shoots were produced per explant in 6 weeks. Subculturing the newly produced shoots, by separating into groups of five shoots, produced an average of 43 new shoots per culture within 4 weeks. In vitro rooting and weaning of over 200 plantlets was completely successful. Cytological studies revealed no visible abnormalities in chromosome number.Abbreviations 2iP 2-isopentenyladenine - BA 6-benzyladenine - LSD Least Significant Difference - NAA 1-naphthaleneacetic acid - TDZ thidiazuron - WPM Woody Plant Medium (Lloyd & McCown 1980) basal medium     

Endorhythmic development in Choisya ternata Kunth (Rutaceae), with a further elucidation of lammas shoots, as well as sylleptic and proleptic shoots

Most apical resting buds of Choisya tenata include inflorescence buds in the axils of their lower consecutive paired scales. These inflorescences develop as apical buds which burst in spring. The whole of the lateral inflorescence system on a shoot originating from an apical bud may be viewed as a single, proliferous inflorescence. After the spring flush there are usually two other flushes of the same shoot within the same season, each of which may be accompanied by the development of lateral inflorescences as in the spring flush. Each further flush produces an apical 'lammas shoot'. As an apical lammas shoot elongates, lateral lammas shoots may also develop from upper, previously resting, axillary buds on the underlying stem segment of the preceding flush. Lateral inflorescences on apical lammas shoots arise from axillary buds preformed within the briefly-dormant apical buds terminating the preceding flush. These inflorescences, as well as the spring ones, represent proleptic shoots. The production of resting apical buds between two intra-season flushes of a shoot may be fugacous, without the differentiation of perfect bud-scales, and with curtailmenl ol internode elongation. As no environmental influence seems to be responsible for intra-season rhythmicity in development, this is said to be endorhythmic. The interrelations of proleptic to sylleptic shoots are discussed.     

Control of plant growth resides in the shoot,and not in the root,in reciprocal grafts of flacca and wild-type tomato (Lysopersicon esculentum), in the presence and absence of salinity stress

Grafted plants of flacca, an ABA-deficient mutant of tomato (Lycopersicon esculentum), and the wild-type variety Rheinlands Ruhm were grown with and without salinity stress to test the roles of roots and shoots in the regulation of plant growth. Fourteen days after exposure to 200 mM NaCl, shoot and root fresh weight, endogenous ABA concentrations, nitrate concentration, activities of selected enzymes related to nitrogen assimilation, and cation accumulation were determined. Rootstock genotype had little influence on the growth of the grafted plants, whereas grafted plants having wild-type shoots (Ws) produced more biomass than those having flacca shoots (Fs), irrespective of the salinity level. Growth of flacca shoots grafted onto wild-type rootstock (Fs/Wr) was superior to that of flacca shoots grafted onto flacca rootstock (Fs/Fr). The improved growth correlated with enhanced levels of ABA in the flaccashoots of Fs/Wr. In all the graft combinations, ABA content was higher in wild-type shoots than in flacca shoots, with or without salinity. There were no significant differences in root ABA concentrations among the different grafted types. Enhanced growth correlated with higher nitrate levels and higher nitrate reductase activity in the roots and shoots of plants with wild-type shoots and with higher shoot concentrations of ABA in plants with wild-type shoots. There were no significant differences in glutamine synthetase and phosphoenol pyruvate carboxylase activities in the shoots and roots of all the grafted plants, regardless of the salinity level. While shoot genotype determined the accumulation of K⁺ and Na⁺ in grafted plants regardless of salinity, it had no influence on Ca²⁺ concentrations. Regardless of the salinity, the total concentration of cations was the same in all the plants, while salinity decreased Mg²⁺ concentration in roots and shoots of all grafts, with the exception of flacca grafted shoots. The scion genotype – and its ABA level – thus played the major role in the growth of grafted plants, regardless of the rootstock genotype and the salinity of the growth medium.     

Nauclea diderrichii: rooting of stem cuttings,clonal variation in shoot dominance,and branch plagiotropism

Summary Nauclea diderrichii (De Wild, and Th. Dur.) Merill (Rubiaceae), an indigenous hardwood of West Africa, is increasingly being grown commercially. This study investigates the potential for vegetative propagation and clonal selection, and raises some fundamental questions about the physiology of apical dominance and of plagiotropism. Rooting ability was high, with up to 100% rooting in 2–4 weeks, when different Indole-3-butyric acid (IBA) concentrations and leaf areas were tested. Auxin applications greatly increased the numbers of roots per cutting. The decapitation of unbranched plants revealed clonal variation in apical dominance and also in the establishment of outright dominance by the two shoots formed from the outgrowth of the axillary buds of the opposite leaves at the top node. Regression analysis of the Dominance Ratio (length of dominant: length of the sub-dominant shoot at the time of achieving dominance) against overall lateral bud activity (r = 0.82), showed that when the two top shoots co-dominate they provide a more powerful source of Correlative Inhibition than when one of the top shoots dominates the other. The imposition of plagiotropism in the axillary bud occurred over a period of a few days as the terminal and axillary buds emerged from the stipule. Growth of accessory buds on intact plants and debranched cuttings was orthotropic. These results are discussed with regard to the role of the leaf in root formation and the understanding of dominance relationships, branching and crown development in trees.     

Bud Structure in Relation to Shoot Morphology and Position on the Vegetative Annual Shoots of Juglans regia L. (Juglandaceae)

The length and basal diameter of all lateral and terminal budsof vegetative annual shoots of 7-year-oldJuglans regia treeswere measured. All buds were dissected and numbers of cataphylls,embryonic leaves and leaf primordia were recorded. Each axillarybud was ranked according to the position of its associated leaffrom the apex to the base of its parent shoot. Bud size andcontent were analysed in relation to bud position and were comparedwith the size and number of leaves of shoots in equivalent positionswhich extended during the following growing season. Length andbasal diameter of axillary buds varied according to their positionon the parent shoot. Terminal buds contained more embryonicleaves than any axillary bud. The number of leaves was smallerfor apical and basal axillary buds than for buds in intermediatepositions on the parent shoot only. All new extended shootswere entirely preformed in the buds that gave rise to them.Lateral shoots were formed in the median part of the parentshoot. These lateral shoots derived from buds which were largerthan both apical and basal ones. Copyright 2001 Annals of BotanyCompany Juglans regia L., Persian walnut tree, branching pattern, preformation, bud content, shoot morphology