Cumulative activation of axillary buds by nodal roots in Trifolium repens L

In Trifolium repens the rate of outgrowth of an axillary bud was closely correlated with its duration of exposure to a nearby nodal root. The dose-dependent nature of this relationship, over 0-22 d, is consistent with the concept that axillary buds are cumulatively activated by a root signal (RS) such that the longer they receive the signal the higher is their level of activation and hence their rate of outgrowth. Furthermore, the activation level attained by a bud was subsequently retained following the excision of the nodal root providing the source of its activation: its rate of growth 3-6 weeks after root excision still reflected the initial level of activation of the bud. Thus, once activated, a bud required relatively little RS to maintain its rate of outgrowth, implying that activation involves the establishment of an autonomous control mechanism within the bud itself. This provides an explanation of how a strongly activated apical bud can continue growth at relatively low RS levels when it is distanced from its nearest root system, while at the same time the prevailing low RS environment leads to weak activation of the axillary buds emerging from it.     

Evidence suggests plagiotropic clonal species have evolved a branching physiology emphasizing regulation by nodal roots

In the plagiotropic nodally rooting clonal herb, Trifolium repens,the development of branches on stems is primarily controlled by the presence of nodal roots, and apical dominance is of secondary importance; only six to ten branches form distal to the youngest nodal root on a horizontal stem. We assessed the hypothesis that this phenomenon is general for clonal herbs with prostrate nodally rooting stems, and that they all have the same physiological system regulating branching, by testing a selection of species from diverse angiosperm families that exhibit either phalanx (Leptinella (Asteraceae), Hydrocotyle (Apiaceae), Acaena (Rosaceae)) or guerilla (Vinca (Apocynaceae), Glechoma and Lamiastrum (Lamiaceae)) growth strategies. In all these species the establishment of a single nodal root on a prostrate stem, otherwise prevented from nodally rooting, induced the outgrowth of a limited number of axillary buds (the number of which was species specific) at the nodes immediately distal to the newly established root, thereby indicating a phenotypic response similar to that in T. repens. Furthermore, their branching responses to manipulative treatments were also similar to those of T. repens, indicating that their regulatory physiology of axillary bud outgrowth from their prostrate stems is similar. We conclude that, for the group of prostrate nodally rooting clonal herbs as a whole, the apical dominance phenotype arises predominantly from variation in the supply of resources from nodal roots rather than from repression of axillary buds by apical tissues (apical dominance). We suggest that evolution of such a physiological mechanism enhances the exploration for patchily distributed favourable nodal rooting sites by regulating shoot development so as to efficiently utilise the diminishing intra-plant availability of root-supplied resources. For the species examined, inter-specific variation in intensity of branching response to a nodal root is shown to be linked to a trade off in foraging strategy, with the allocation of resources primarily to explorative growth (long internodes, few branches) in guerilla species or to exploitive growth (short internodes, many branches) in phalanx species.Co-ordinating editor: J. Tuomi     

Relative importance of nodal roots and apical buds in the control of branching in Trifolium repens L.

Clonal species are characterised by having a growth form in which roots and shoots originate from the same meristem so that adventitious nodal roots form close to the terminal apical bud of stems. The nature of the relationship between nodal roots and axillary bud growth was investigated in three manipulative experiments on cuttings of a single genotype of Trifolium repens. In the absence of locally positioned nodal roots axillary bud development within the apical bud proceeded normally until it slowed once the subtending leaf had matured to be the second expanded leaf on the stem. Excision of apical tissues indicated that while there was no apical dominance apparent within fully rooted stems and very little in stems with 15 or more unrooted nodes, the outgrowth of the two most distal axillary buds was stimulated by decapitation in stems with intermediate numbers of unrooted nodes. Excision of the basal branches from stems growing without local nodal roots markedly increased the length and/or number of leaves on 14 distally positioned branches. The presence of basal branches therefore prevented the translocation of root-supplied resources (nutrients, water, phytohormones) to the more distally located nodes and this caused the retardation in the outgrowth of their axillary buds. Based on all three experiments we conclude that the primary control of bud outgrowth is exerted by roots via the acropetal transport of root-supplied resources necessary for axillary bud outgrowth and that apical dominance plays a very minor role in the regulation of axillary bud outgrowth in T. repens.     

A developmentally based categorization of branching in Trifolium repens L.: influence of nodal roots

This study describes the successive stages of development of branches from axillary buds in fully rooted plants of Trifolium repens grown in near optimal conditions, and the way in which this developmental pathway differs when nodal root formation is prevented as plants grow out from a rooted base. Cuttings of a single genotype were established in a glasshouse with nodal root systems on the two basal phytomers and grown on so that nodal rooting was either permitted (+R) or prevented (-R). In +R plants, axillary tissues could be assigned to one of four developmental categories: unemerged buds, emerged buds, unbranched lateral branches or secondarily branched lateral branches. In -R plants, branch development was retarded, with the retardation becoming increasingly pronounced as the number of -R phytomers on the primary stolon increased. Retarded elongation of the internodes of lateral shoots on -R plants resulted in the formation of a distinct fifth developmental category: short shoots (defined as branches with two or more leaves but with mean internode length equal to, or less than, 10% of that of the immediately proximal internode on the parent stolon) which had reduced phytomer appearance rates but retained the potential to develop into lateral branches. Transfer of +R plants to -R conditions, and vice versa, after 66 d demonstrated that subsequent branch development was wholly under the control of the youngest nodal root present, regardless of the age and number of root systems proximal to it.     

Branch and root formation in Trifolium repens is influenced by the light environment of unfolded leaves

In plagiotropic plants, axillary buds on the stolon can be exposed to low red:far-red (R:FR) ratios, while the leaves may be positioned in the uppermost layer of the sward where they are exposed to a high R:FR ratio. We tested whether the light environment of unfolded leaves influences outgrowth of the axillary buds and the formation of nodal roots of Trifolium repens. Single plants were grown in a growth cabinet with high photosynthetic photon flux rate (PPFR) and a high R:FR ratio (F_HR_H, control), low PPFR and high R:FR (F_LR_H) or low PPFR and low R:FR (F_LR_L). In an additional treatment (SS), only stolons were shaded so that developing leaves grew into light conditions similar to the control treatment. Neutral shading (F_LR_H) had a minor effect on branching and did not influence root formation. A reduction in the R:FR ratio (F_LR_L) significantly delayed the outgrowth of axillary buds so that, compared to the control plants, the percentage of branched phytomers was reduced by 43% on the parent axis and by 75% on primary branches. Furthermore, the number of nodal roots per plant was reduced by about 30%. When only the stolons were shaded (SS), the percentage of branched and rooted phytomers was similar to that of the control plants. Extension of petioles and leaves was very variable, increasing the values in the F_LR_L treatment at least 2.5-fold compared with the control plants. It was concluded that the light environment of the unfolded leaves had a significant influence on the regulation of the outgrowth of axillary buds and that the high plasticity in petiole growth allows the positioning of the leaves in a light environment conducive to the stimulation of branch outgrowth. Received: 8 February 1997 / Accepted: 26 April 1997     

Spatiotemporal regulation of morphogenetic molecules during in vitro branching of the isolated ureteric bud: toward a model of branching through budding in the developing kidney

In search of guiding principles involved in the branching of epithelial tubes in the developing kidney, we analyzed branching of the ureteric bud (UB) in whole kidney culture as well as in isolated UB culture independent of mesenchyme but in the presence of mesenchymally derived soluble factors. Microinjection of the UB lumen (both in the isolated UB and in the whole kidney) with fluorescently labeled dextran sulfate demonstrated that branching occurred via smooth tubular epithelial outpouches with a lumen continuous with that of the original structure. Epithelial cells within these outpouches cells were wedge-shaped with actin, myosin-2 and ezrin localized to the luminal side, raising the possibility of a "purse-string" mechanism. Electron microscopy and decoration of heparan sulfates with biotinylated FGF2 revealed that the basolateral surface of the cells remained intact, without the type of cytoplasmic extensions (invadopodia) that are seen in three-dimensional MDCK, mIMCD, and UB cell culture models of branching tubulogenesis. Several growth factor receptors (i.e., FGFR1, FGFR2, c-Ret) and metalloproteases (i.e., MT1-MMP) were localized toward branching UB tips. A large survey of markers revealed the ER chaperone BiP to be highly expressed at UB tips, which, by electron microscopy, are enriched in rough endoplasmic reticulum and Golgi, supporting high activity in the synthesis of transmembrane and secretory proteins at UB tips. After early diffuse proliferation, proliferating and mitotic cells were mostly found within the branching ampullae, whereas apoptotic cells were mostly found in stalks. Gene array experiments, together with protein expression analysis by immunoblotting, revealed a differential spatiotemporal distribution of several proteins associated with epithelial maturation and polarization, including intercellular junctional proteins (e.g., ZO-1, claudin-3, E-cadherin) and the subapical cytoskeletal/microvillar protein ezrin. In addition, Ksp-cadherin was found at UB ampullary cells next to developing outpouches, suggesting a role in epithelial-mesenchymal interactions. These data from the isolated UB culture system support a model where UB branching occurs through outpouching possibly mediated by wedge-shaped cells created through an apical cytoskeletal purse-string mechanism. Additional potential mechanisms include (1) differential localization of growth factor receptors and metalloproteases at tips relative to stalks; (2) creation of a secretory epithelium, in part manifested by increased expression of the ER chaperone BiP, at tips relative to stalks; (3) after initial diffuse proliferation, coexistence of a balance of proliferation vs. apoptosis favoring tip growth with a very different balance in elongating stalks; and (4) differential maturation of the tight and adherens junctions as the structures develop. Because, without mesenchyme, both lateral and bifid branching occurs (including the ureter), the mesenchyme probably restricts lateral branching and provides guidance cues in vivo for directional branching and elongation as well as functioning to modulate tubular caliber and induce differentiation. Selective cadherin, claudin, and microvillar protein expression as the UB matures likely enables the formation of a tight, polarized differentiated epithelium. Although, in vivo, metanephric mesenchyme development occurs simultaneously with UB branching, these studies shed light on how (mesenchymally derived) soluble factors alone regulate spatial and temporal expression of morphogenetic molecules and processes (proliferation, apoptosis, etc.) postulated to be essential to the UB branching program as it forms an arborized structure with a continuous lumen.     

Intermediate growth forms as a model for the study of plant clonality functioning: an example with root sprouters

In this contribution we want to show that growth forms intermediate between non-clonal and clonal plants can be used to ask questions about the functional ecology of clonality. We discuss this idea on plants sprouting adventitiously from roots and accomplishing clonal growth via root spacers. Based on extensive literature dealing with growth forms of root sprouting plants, we characterise forms functionally intermediate between clonal root-sprouters and non-clonal plants. We delimit them according to their potential ability to form adventitious shoots and horizontal roots. By reviewing experimental work with root sprouters, we identify the most important triggering factors and developmental constraints influencing these intermediate forms plant age, life-history mode and life-history stage. Using this information we ask questions about the importance of root sprouting in (1) conditions of unpredictable disturbance, where root-sprouting ability may be viewed as a tool for vegetative regeneration, and in (2) temporarily and spatially heterogeneous environment, where foraging by roots may serve as a way of exploiting patchy resources.     

Regulation of cell length in the Arabidopsis thaliana root by the ethylene precursor 1-aminocyclopropane- 1-carboxylic acid: a matter of apoplastic reactions

Treatment of the Arabidopsis thaliana root with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC) immediately imposes a reduced maximal cell length beyond which further elongation is blocked. Here, we investigated possible apoplastic reactions involved in the inhibition of cell elongation. Five-day-old Arabidopsis seedlings were transferred to a growth medium supplemented with ACC and the effect on root cell length was recorded after 3 h of treatment. Altered characteristics in the apoplast of the nonelongating cells in the ACC-treated root, such as 'reactive oxygen species' (ROS) production and callose deposition, were detected using specific fluorochromes. The presence of functional hydroxyproline-rich glycoproteins (HRGPs) and the crosslinking of these cell-wall proteins are essential in limiting cell elongation. The ROS that drive the oxidative crosslinking of HRGPs, accumulate in the apoplast of cells in the zone where cell elongation stops. In the same cells, callose is deposited in the cell wall. The final cell length in the Arabidopsis root treated for a short period with ACC is determined in the zone of fast elongation. Both HRGPs crosslinking by ROS and callose deposition in the cell wall of this zone are suggested as causes for the reduced cell elongation.     

The non-specific inhibition of enzymes by environmental pollutants: a study of a model system towards the development of electrochemical biosensor arrays

Previous research has shown that lactate dehydrogenase (LDH) was competitively inhibited by pentachlorophenol (PCP) and a modified assay produced a detection limit of 1 μM (270 μg l⁻¹). This work used spectrophotometric rate-determination but in order to move towards biosensor development the selected detection method was electrochemical. The linkage of LDH to lactate oxidase (LOD) provided the electroactive species, hydrogen peroxide. This could be monitored using a screen-printed carbon electrode (SPCE) incorporating the mediator, cobalt phthalocyanine, at a potential of +300 mV (vs. Ag/AgCl). A linked LDH/LOD system was optimised with respect to inhibition by PCP. It was found that the SPCE support material, PVC, acted to reduce inhibition, possibly by combining with PCP. A cellulose acetate membrane removed this effect. Inhibition of the system was greatest at enzyme activities of 5 U ml⁻¹ LDH and 0.8 U ml⁻¹ LOD in reactions containing 246 μM pyruvate and 7.5 μM NADPH. PCP detection limits were an EC₁₀ of 800 nM (213 μg l⁻¹) and a minimum inhibition detectable (MID) limit of 650 nM (173 μg l⁻¹). The inclusion of a third enzyme, glucose dehydrogenase (GDH), provided cofactor recycling to enable low concentrations of NADPH to be incorporated within the assay. NADPH was reduced from 7.5 to 2 μM. PCP detection limits were obtained for an assay containing 5 U ml⁻¹ LDH, 0.8 U ml⁻¹ LOD and 0.1 U ml⁻¹ GDH with 246 μM pyruvate, 400 mM glucose and 2 μM NADPH. The EC₁₀ limit was 150 nM (39.9 μg l⁻¹) and the MID was 100 nM (26.6 μg l⁻¹). The design of the inhibition assays discussed has significance as a model for other enzymes and moves forward the possibility of an electrochemical biosensor array for pollution monitoring.