JXB Advance Access originally published online on August 28, 2009
Journal of Experimental Botany 2009 60(15):4275-4285; doi:10.1093/jxb/erp258
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© 2009 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Axillary bud outgrowth potential is determined by parent apical bud activity
AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand
* To whom correspondence should be addressed: E-mail: mike.hay{at}agresearch.co.nz
Received 3 June 2009; Revised 5 August 2009 Accepted 7 August 2009
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Abstract |
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Axillary buds within a plant shoot system are known to differ in their ability to respond to treatments favouring their development. This ability is referred to as their outgrowth potential. Using two species of prostrate nodally-rooting herbs, dicotyledonous Trifolium repens and monocotyledonous Tradescantia fluminensis, grown throughout in a strictly vegetative state, this study tested two hypotheses. Hypothesis 1: that each axillary bud exhibits an outgrowth potential that is directly related to the growth rate of its parent apical bud, and Hypothesis 2: that the growth rate attained by an axillary bud depends upon both its outgrowth potential and the local supply of stimulatory root-derived signal (NRS) available to it. Activation levels (growth rates) of apical buds were varied by differential exposure to nodal roots and the outgrowth responses of axillary buds recently emerged from them were then measured under standardized conditions of NRS supply. Hypothesis 1 was shown to be correct for both species. Hypothesis 2, tested only in T. repens, was supported by results showing that an axillary bud's outgrowth potential and the NRS supply to it each independently influenced its growth rate, there being no significant interaction between the two. These results emphasize the significant role the physiological state/activity of apical buds has on the outgrowth potential of axillary buds formed within them. The fact that similar relationships were observed on axillary buds on stems of differing developmental maturity and branching hierarchy, and in two taxonomically diverse species, suggests they might be widespread among morphologically similar species.
Key words: Axillary bud outgrowth, branch development, bud activation, bud outgrowth potential, nodal roots, prostrate clonal herbs, root signals, Tradescantia fluminensis, Trifolium repens
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Introduction |
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Studies with nodally-rooting, prostrate-stemmed perennial species indicate that their shoot branching is regulated predominantly by fluctuations in stimulatory signal(s) transported acropetally from roots (Thomas et al., 2002, 2003a, b; Thomas and Hay, 2004, 2007, 2008a, b). This is in marked contrast to much of the reported work on the regulation of branching in the erect-stemmed annual species of Arabidopsis thaliana, Pisum sativum, and Petunia hybrida, where genetic and physiological evidence points to the dominance of common inhibitory influences from roots and apical buds (see reviews by Leyser, 2005; McSteen and Leyser, 2005; Beveridge, 2006; Dun et al., 2006; Ongaro and Leyser, 2008; and Johnson et al., 2006; Simons et al., 2007, Gomez-Roldan et al., 2008; Umehara et al., 2008). In these model systems, it has been demonstrated clearly that there is a network of shoot and root feedback and interacting signals that collectively operate to regulate branching (Beveridge, 2006; Dun et al., 2006; Aguilar-Martínez et al., 2007; Simons et al., 2007; Ongaro and Leyser, 2008; Ferguson and Beveridge, 2009) and, in the case of Petunia, evidence has been presented of a stimulatory influence of roots that is expressed in the presence of the inhibitory influence (Napoli, 1996; Simons et al., 2007). Thus it appears that in these model species root signals have both stimulatory and inhibitory components although the net effect is usually inhibitory. Alternatively, in prostrate-stemmed species, the balance appears to favour the stimulatory signal with the reduction in axillary bud development at nodes distanced from the basal root resulting from a decrease in effectiveness of the stimulus. Thomas and Hay (2008a) suggested that decreased effectiveness might arise as a consequence of a decrease in the stimulatory signal, an increase in supply of the inhibitor, decreasing sensitivity of the axillary buds to the stimulatory signal with distance from the root system, or a combination of these factors. In view of this, the stimulatory influence transported from nodal roots in the prostrate-stemmed plants of this study is likely to be the net result of both stimulatory and inhibitory influences and is referred to henceforth, for convenience, as the net root stimulus (NRS).
At present the nature of the stimulatory signal is unknown. As foliar spray application of mineral nutrient to sub-optimally supplied plants restored the growth of the shoot organs but not branching (Hay et al., 2003), nutrient supply by nodal roots is unlikely to be directly involved. However, in these prostrate-stemmed species, xylem transport of root-derived cytokinin cannot be ruled out as the possible stimulatory signal, with its level being regulated by polar auxin transport to roots (Li and Bangerth, 1999; Bangerth et al., 2000) although there is now strong evidence from erect-stemmed species indicating that the cytokinin promoting axillary bud outgrowth is locally biosynthesized in the nodal region of the stem (Tanaka et al., 2006; Ferguson and Beveridge, 2009) with auxin regulating both its biosynthesis and its degradation (Shimizu-Sato et al., 2009).
It was shown earlier (Thomas and Hay, 2007) in Trifolium repens that the rate of outgrowth of an axillary bud (its activation level) in response to a stimulus from a nodal root is cumulative and is maintained after excision of the nodal root. Using this species as being representative of the group of nodally-rooting, prostrate-stemmed species (Thomas and Hay, 2004, 2008b), Thomas and Hay (2008a) attempted to develop a predictive model of the rate of growth of axillary buds based on the level of activation each bud attained from its exposure to NRS at the time of its emergence from its parent apical bud. While this model was able to explain the decline in growth rate of successively produced axillary buds on a primary stem as its apical bud grows away from its basal root system, it failed to explain the rapid progressive decline in secondary branch development on successively formed lateral branches. They therefore suggested that there is a need to take into account an additional factor and were led to hypothesize that the ability of an emerging axillary bud to respond to a given level of local NRS might be, at least in part, directly influenced by the activation level (growth rate) of its parent apical bud during its early development within it. A similar association between the outgrowth of axillary buds and the developmental stage and physiological activity of the shoot terminal meristem has been suggested to occur in Pisum (Gould et al., 1987), Nicotiana (McDaniel and Hsu, 1976), and Arabidopsis (Grbic and Bleecker, 2000).
This intrinsic ability of an axillary bud to respond to NRS supply is conceptually similar to that previously described as its ‘outgrowth potential’ (Napoli et al., 1999) or ‘growth potential’ (Husain and Linck, 1966; Gould et al., 1987). In the present paper this ability is referred to as ‘outgrowth potential’.
To assess the validity of the concept that an axillary bud's potential to respond to NRS supply is related to the level of activity of the apical bud in which it formed, experiments were undertaken to test two specific hypotheses.
Hypothesis 1:
that each axillary bud exhibits an outgrowth potential that is directly related to the growth rate of its parent apical bud.
Hypothesis 2:
that the rate of growth attained by an axillary bud depends upon the combination of its outgrowth potential and the local supply of NRS available to it at the time of its emergence from its parent apical bud.
To achieve this, it was necessary to design experiments in which the activation levels (growth rates) of apical buds were varied while holding all other variables constant. The steps involved in the experiments were thus: firstly, the manipulation of shoot and root systems to provide apical buds with a range of different activation levels. Secondly, the prevention of possible variation in the inhibitory influence of parent apical buds on the outgrowth of axillary buds (test buds) newly emerged from them, by excising the apical bud tissues immediately distal to the test buds. Thirdly, ensuring, by appropriate excision procedures, that test buds from parent apical buds with different activation levels were exposed to a similar supply of NRS from the source root systems. Fourthly, in Trifolium, the manipulation of nodal roots to provide two different levels of NRS supply to test buds formed by parent apical buds with differing activation levels.
To broaden the relevance of the findings from this study, experiments were carried out on two phylogenetically widely distanced species; namely, the dicotyledonous Trifolium repens and a monocotyledonous species, Tradescantia fluminensis, with a similar growth habit and apical bud structure (Denne, 1966).
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Materials and methods |
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Plant material
Trifolium repens L. (white clover): experimental plants were derived from a greenhouse-grown stock clone of a single genotype of T. repens selected from a Spanish ecotype collection (AgResearch Accession number C1067) previously described (see Thomas et al., 2003b; Thomas and Hay, 2007, 2008a).
Tradescantia fluminensis Vell. (wandering jew): This monocotyledonous species (family Commelinaceae) is a prostrate perennial clonal herb the stems of which, as in T. repens, produce roots at nodes wherever they contact moist ground. Cuttings from a single genotype growing in a nearby garden were grown in a heated greenhouse to produce stock plants from which the experimental plants were derived. These remained vegetative throughout the experiment.
Culture of experimental plants of both species
Plants were grown from stem tip cuttings planted in a commercially obtained potting mix (Thomas et al., 2002) in 1.35 l planter bags. After about 3 weeks, the two or three basalmost branches formed by this time were trimmed off each plant to leave a single primary stem growing away from its basal root system. All lateral branches that grew out subsequently from this primary stem were retained. The oldest phytomer on the primary stem that retained a branch at its node was termed phytomer 1 (P1) and later-formed ones termed P2, P3, etc. Outgrowth of nodal roots was prevented by growing shoot systems out over a dry plastic mesh. Throughout the investigation, plants were grown in a heated greenhouse in natural photoperiods at average maximum/minimum temperatures of 25/12 °C.
Where required, outgrowth of a nodal root on a primary stem was stimulated by firmly pinning the youngest newly emerged node to a mound of moist potting mix contained in a 200 ml plastic pot.
Experimental design
Trifolium experiment: outgrowth potential of axillary buds on branches:
Plants were grown for a 10-week pretreatment phase (Fig. 1a) during which apical buds on successively formed branches attained successively lower activation levels (Thomas et al., 2002). They were then given one of four core treatments (Fig. 1b, A–D) or two additional treatments (Fig. 1b, E, F) and responses of axillary buds that had formed within branch apical buds with different activation levels were measured over a 5-week period.
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Pretreatment:
Thirty-six plants were grown from shoot tip cuttings until 5 May 2004, by which time ten phytomers had emerged from the apical buds of their primary stems (Fig. 1a(i)). At this stage the NRS supply to phytomer 10 (P10) from the basal root system was sufficient for continued growth of the primary stem apical bud but inadequate for strong outgrowth of newly emerging axillary buds. To produce branch apical buds of differing activation levels, nodal root outgrowth was stimulated at the youngest node on the primary stem (at P10) on 30 plants [Fig. 1a(ii)], and, 19 d later, at P14 on a further six plants [Fig. 1a(iv)]. Development of these nodal root systems stimulated axillary bud activation and subsequent outgrowth of branches at the phytomers at and distal to them, but the apical bud on the branch at the rooted node became more highly activated (grew faster) than those on successively more distal branches at non-rooted nodes. All plants were then grown on for 45 d until the start of treatments on 19 June 2004 when the branch at P14 had three emerged phytomers. At this time the apical bud on the P14 branch was either weakly activated when the nodal root was distanced four phytomers from it at P10 [Fig. 1a(ii)] or more strongly activated when the root was at P14 [Fig.1a(iv)]. Branches at P1–P9 were similar in all plants and were retained throughout the experiment.
Core treatments:
The core treatments (Fig. 1b, A–D) were set up to compare outgrowth rates of three test axillary buds (X, Y, and Z) on the P10 and P14 branches in response to NRS (Fig. 1b). These were the youngest three newly emerged buds immediately proximal to their parent branch apical bud, and therefore of similar developmental age in each treatment. They were also similar as to their phytomeric distance (number of phytomers) from the root at P10 (Fig. 1b), thereby ensuring that they were subjected to a similar supply of root-derived stimulus (NRS) during their development (Thomas and Hay, 2008a) up to the time that treatments were applied.
To avoid possible variation in competition for NRS from the branches and buds intercalated between the test buds and the node at P10, all these intercalated branches and buds were excised at the start of core treatments A–D. At the same time, all primary stem tissue was excised distal to the point of attachment of the branches at P10 (treatments A, C) or P14 (treatments B, D), and to avoid the possibility of differences between inhibitory influences from faster and slower growing apical buds on branches at P10 and P14, respectively, these apical buds were excised immediately distal to the test buds. These excisions resulted in all the NRS available at P10 being channelled solely to the test buds in each treatment. Some leaflets were removed as necessary to even up the total area of leaves subtending the test buds.
To assess the influence of NRS supply on test bud responses, the P10 nodal root was either repotted into a 1.0 l container and retained, in treatments A and B, or excised at the start of treatments, in treatments C and D (Fig. 1b). Thus in treatments A and B, distal buds continued to receive NRS from both the basal root system and the nodal root (high NRS), or, in treatments C and D, they became dependent solely on NRS from the basal root (low NRS). Differences brought about solely by differences in the supply of NRS are apparent by comparison of treatment A with C and treatment B with D. Differences between axillary bud outgrowth responses on branches at P10 and P14 resulting solely from differences between the activation levels of their parent apical buds (outgrowth potential) are discernible by comparing treatment A with B and treatment C with D.
Treatment E was included to verify that the activation levels of the apical buds of the P10 and P14 branches did differ as predicted, by measuring their growth rates at the start of the treatment period. This treatment was identical with treatments C and D with regard to excision of the nodal root at P10, but differed in that no excisions of shoot material were made (Fig. 1b).
Because P10 branches formed earlier on the primary stems than those at P14, one inevitable consequence of the core experimental design (treatments A–D) was that test buds differed in their positions on the branches bearing them, being at the sixth, seventh, and eighth nodes that formed on the larger P10 branch compared with the first, second, and third on the smaller, younger, branch at P14 (see Fig. 1b). Treatment F, in which six plants were stimulated to root at P14 [Fig. 1a(iv)] on 24 May 2004, 19 d after nodal root formation at P10 in treatments A–E, was therefore included. This treatment, in which the nodal root at P14 was excised after 26 d, was otherwise directly comparable to treatment D in which the nodal root at P10 was excised after 45 d. It was essential as a means of distinguishing whether differences found between the outgrowth responses of test buds on branches at P10 and P14 resulted simply from their positions on the branches rather than from differences in outgrowth potentials. As it was known that the presence of a nodal root for 3–4 plastochrons is sufficient to activate fully the axillary bud at a rooted phytomer (Thomas and Hay, 2007), it was predicted that the presence of nodal roots for 26 d in treatment F would raise the activation level of the apical buds of the branches at P14 so that the outgrowth rates of their test buds would be significantly increased relative to those in treatment D and would approach those of the test buds in treatment C. Such a response in bud outgrowth rates on P14 branches in treatment F would indicate that their outgrowth potential, rather than their position on the branches, was modifying their outgrowth rate.
The effects of all six treatments were monitored weekly for 5 weeks. In treatments A–D and F, assessments were made of leaf emergence from, and stem length of, branches developing from the test buds. In treatment E, measurements were made of rates of stem elongation of the branches at P10 and P14 and of leaf emergence from their apical buds.
Tradescantia experiment: outgrowth potential of axillary buds on the primary stem:
This experiment was aimed at establishing the relationship between the outgrowth potential of axillary buds on the primary stem and the activation level (growth rate) of their parent apical buds at the time the axillary buds were formed within them. To achieve this, the activation levels of primary stem apical buds were varied by differential durations of exposure to the stimulatory influence of nodal roots [Fig. 2b(i), Phase 1] (Thomas and Hay, 2007, 2008a) and the outgrowth rates of axillary buds that had newly emerged from them were then measured in response to excision of their parent apical buds under common levels of NRS supply from the basal root systems during Phase 2 [Fig. 2b(ii)].
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Pretreatment:
Thirty plants were established on 14 August 2006, such that each had a non-rooted primary stem extending away from a basal root system. By the end of the pretreatment period, the primary stems bore about 12 lateral branches, the axillary buds at their six youngest phytomers had failed to grow out into branches, and their 19th non-rooted phytomers were newly emerged from their apical buds (Fig. 2a).
Treatments:
Treatments were started on 1 October 2006 by pinning the node at P19 onto moist potting mix to stimulate nodal root development on 24 of the 30 plants [Fig. 2b(i)], the remaining six being controls in which nodal root development was prevented (treatment 0D). Phase 1 treatments differed as to the length of time nodal roots were permitted to develop by excising them after 8, 12, 18, or 24 d in treatments 8D, 12D, 18D, and 24D, respectively. All plants were then grown on for a further 9 weeks from the onset of stimulation of root development, by which time differential activation of primary stem apical buds was apparent. A further set of treatments (Phase 2) was given on 5 December 2006 (Day 64), by excising the apical bud on the primary stem of three plants in each treatment and leaving the remaining three plants intact to act as controls [Fig. 2b(ii)]. All plants were then grown on for a further 3 weeks, until 28 December 2006, to allow the three youngest axillary buds immediately proximal to the excised apical bud on the primary stem of the decapitated plants to respond to the treatments. In all treatments, all previously formed branches proximal to the test axillary buds were retained for the duration of the experiment.
The effects of the duration of rooting treatments during Phase 1were assessed by measuring the rates of elongation of the primary stem and of leaf emergence from its apical bud during the period from nodal root stimulation through to the time of excision of the apical bud (5 December 2006). Elongation of the three test axillary buds (X, Y, and Z) on the primary stem of each decapitated plant and the equivalent buds on the control plants was then monitored for 3 weeks during Phase 2 (5–28 December 2006). Lastly, at the end of the experiment each shoot was divided into a basal and an apical region by severing the primary stem immediately proximal to P19. These portions were then oven-dried at 60 °C for 3 d before weighing.
Analysis of data
A single mean value for leaf emergence and stem elongation of the test buds for each treatment was calculated from the independently assessed values of each of the test buds (X, Y, and Z) in each plant within each treatment.
In the Trifolium experiment, the number of nodes and the branch stem length data at Day 35 in Fig. 3 were analysed using the planned contrast option of analysis of variance in GenStat statistical software (Payne et al., 2007). The effects of root retention and branch position and their interaction were all compared at the between-plant level of variability. Means and least significant differences (LSD5%) for comparing them were obtained from the relevant interaction tables.
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In the Tradescantia experiment, the slopes of fitted linear regression lines of each plant in each treatment were tested by ANOVA (Payne et al., 2007) for the significance of treatment (duration of nodal root presence) on elongation of the primary stem during Phase 1 (Fig. 4). Appropriate diagnostic checks were performed after model fitting. ANOVA was used to test for the significance of the treatment on the stem length of test buds at 23 d (Fig. 5). The simple linear regression analyses reported in Fig. 6 were performed using the Microsoft Excel software package.
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Results |
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Trifolium experiment: bud outgrowth responses on branches
(a) Hypothesis 1: outgrowth potential of an axillary bud is directly related to the growth rate (activation level) of its parent apical bud
Growth rates of the apical buds of experimental branches at P10 and P14:
The results of treatment E (Fig. 1b) established that growth rates of the apical buds that were retained on the branches at P10 and P14 were, indeed, different. Leaf emergence rates from these buds, and the rates of elongation of the internodes emerging from them, were both substantially greater on the P10 than on the P14 branch (Table 1). Furthermore, their growth rates were constant; the rates of both leaf emergence and stem elongation being similar in weeks 1 and 3.
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Effect of parent apical bud growth rates on the outgrowth of test buds in the core treatments (A–D):
Comparison of responses of test buds formed on the faster growing branches at P10 with those formed on the slower growing P14 branches shows there to have been a clear delay in their start of outgrowth on the more distal branches (Fig. 3). This was so both at high NRS levels (nodal roots at P10 retained, A versus B) and at low levels (nodal roots at P10 excised, C versus D). For leaf emergence (Fig. 3a) this delay was about 7 d, but the delay in the start of bud stem elongation (Fig. 3b) was closer to 3 weeks. Once outgrowth had started, bud leaf emergence on branches at P14 was about 15% less than on P10 branches but, even 35 d after treatment, bud stem elongation on P14 branches was less than half that of equivalent buds on P10 branches.
Effect of test bud positions on P10 and P14 branches on their outgrowth:
Test buds on the branch at P10 in treatment C grew out faster than those at P14 in treatment D (Fig. 3). Treatment F [Fig.1a(iv) and 1b] was designed to test whether this difference was related to differences in ages of the branches at P10 and P14, and hence the position of test buds on them, rather than the activation levels (growth rates) of their apical buds.
Comparison of the results of treatments F, C and D, in which test bud outgrowth after nodal root excision occurred at low NRS levels (Fig. 3), shows, however, that the observed differences between C and D were not the result of the position on branches of the test buds. In treatment F (with P14 nodally rooted during the pretreatment phase) test bud outgrowth was markedly higher than in treatment D that never possessed a root at P14, even though the position of test buds was the same in both (Fig. 1b). Comparison of treatments F and C, in each of which the branch bearing the test buds was borne at a previously rooted phytomer (for 26 d at P14 in F, compared with 45 d at P10 in C), showed that outgrowth of test buds in F was much closer to that of C than of D, indicating that the effect of the position of buds on branches was minor.
(b) Hypothesis 2: outgrowth of an axillary bud depends upon both its outgrowth potential and the NRS supply to it
Effect of net root-derived stimulus (NRS):
Test buds either received a high supply of NRS when the nodal root system at P10 was retained (treatments A and B) or a lower supply, when the nodal root system at P10 was excised (treatments C and D) (Fig. 1b). Comparison of A with C and B with D (Fig. 3) shows that both leaf emergence and stem elongation of axillary buds were significantly higher, by 29% and 80%, respectively, by the end of the assessment period (P <0.001) when their NRS supply was higher.
The ANOVA found that the interaction between NRS supply and branch position (outgrowth potential) was non-significant (P >0.05) for both leaf emergence and stem elongation. Thus NRS and outgrowth potential each independently influence test bud growth in an additive fashion.
Tradescantia experiment: bud outgrowth responses on the primary stem
This experiment tested Hypothesis 1 only: that outgrowth potential of an axillary bud is directly related to the growth rate (activation level) of its parent apical bud.
Whole plant responses to rooting treatments:
The dry weights of the dissected parts of the shoot systems determined at the conclusion of the experiment (Table 2) indicate that plants receiving the shortest (0D) and longest (24D) exposures to nodal roots (Fig. 2) did not differ in the weights of their large basal portions and that the difference between total shoot dry weights, including distal portions, was less than 10%.
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Effect of nodal roots on activation of primary stem apical buds:
The duration of nodal root presence at node 19 had a significant (P <0.001) and continuing effect on primary stem elongation in the region immediately proximal to the apical buds (Fig. 4). Stem elongation was 50% greater in plants exposed to the influence of nodal roots for 24 d (treatment 24D) than in the non-rooted control plants (treatment 0D) by the end of Phase 1 (see Fig. 2 for details of Phases 1 and 2). Strong treatment effects were also observed on the rates of leaf emergence over the whole 9 week Phase 1 period and elongation of the primary stem during week nine (Table 3).
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Growth rates of test axillary buds during three weeks following decapitation of the primary stem:
At the time of decapitation all test buds were still completely enclosed within the sheaths of their subtending leaves in all treatments and the decapitation treatment stimulated their outgrowth to varying degrees (Fig. 5). Duration of previous exposure to nodal roots during Phase 1 had significant (P <0.001) effects on the elongation of the test bud stems. Test buds on plants that had retained their nodal roots for longer during Phase 1 grew out sooner and formed faster growing branches than on those exposed to the influence of a nodal root for shorter periods (Fig. 5).
In non-decapitated control plants that had been previously exposed to a root for 0, 8 or 12 d, there was no measurable outgrowth of the buds equivalent to X, Y, and Z by day 23 after treatment. In those that retained a nodal root for 18 d or 24 d during Phase 1, the three equivalent buds attained mean stem lengths of 1.7 (±1.56) and 6.9 (±3.11) mm, respectively, compared with 30.3 (±5.59) and 32.7 (±4.68) mm in the decapitated plants (Fig. 5).
Correlation of growth rates of test axillary buds and primary stem apical buds
There was a direct linear relationship (P <0.01) between the rates of elongation of primary stem apical buds during the week preceding decapitation and those of the test axillary buds during weeks two and three following decapitation (Fig. 6).
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Discussion |
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Central to this study was the hypothesis (Hypothesis 1) that, at the time axillary buds emerge from their parent apical bud, they have an outgrowth potential that is determined by the activation level (growth rate) of that bud. A key requirement of the experimental design was therefore to be able to vary the activation levels of apical buds while as far as possible holding all other variables constant. To achieve this, nodal rooting treatments were imposed that led to different growth rates of the apical buds on the P10 and P14 branches (Table 1) in the Trifolium experiment and on the primary stems (Table 3; Fig. 4) in the Tradescantia experiment. Outgrowth responses of test axillary buds that were newly emerged from these apical buds were then measured on plants that had been manipulated so as to (i) provide uniform NRS supply to comparable test buds and (ii) remove possible variation in the inhibitory influence of apical buds by excising these immediately distal to the test buds.
Uniformity of NRS supply for both experiments was attained by means of the shoot excision procedures detailed earlier. In Trifolium, these were designed to channel all the NRS available at P10 solely to the test axillary buds, either on the P10 or P14 branches (Fig. 1). Although these buds were of similar chronological age and phytomeric distance (number of nodes) from P10, the physical distances from the P10 node to the test buds varied appreciably, the stem lengths between the youngest test buds and the node at P10 being: 305 (±12.9) mm in treatments A and C, 202 (±5.0) mm in B and D, and 215 (±7.5) mm in F. However, the buds that responded most strongly were those on the branches at P10, in A and C, and these were actually further from their sources of NRS than those with the weakest responses on the branches at P14 in B and D. Thus, the effect of distance that NRS travels along stems was small relative to the effects of outgrowth potential on the growth of axillary buds, as can be seen by comparison of the responses of treatment F with B, C, and D (Fig. 3). In the Tradescantia experiment, in which no lateral branches were excised (Fig. 2), the branches at the base of the plants (P1–P18), that would have acted as the largest competing sinks for NRS, were similar for all treatments (Table 2). Variation induced in distal branch development by the differences in exposure to nodal roots at P19 amounted to <10%, and, moreover, it was the test buds in the treatment with longest exposure to nodal roots (24D), and greatest development of distal competitive sinks (Table 2), that grew most strongly. Thus variation in NRS supply to the test buds in Tradescantia was not a major factor driving the results obtained.
The design of the Trifolium experiment was based on the assumption that differences in the positions of the test buds on the branches at P10 and P14 would have little influence on their outgrowth. Those on the P10 branch were situated at the sixth, seventh, and eighth nodes that emerged on the branch whereas those on the P14 branch were at the first three formed on it, at the first, second, and third nodes (Fig. 1). Because the larger P10 branch bore eight emerged leaves and the smaller P14 branch only three, and because those on the P14 branch, being the first-formed on the branch, were smaller (Wilman and Simpson, 1988; Hay et al., 1993) than leaves at the sixth, seventh, and eighth phytomers on the P10 branch, this assumption required testing. Treatment F was designed as a means of doing this. Comparison of the results of treatments F (rooted at P14) and C and D (rooted at P10) support the above-stated assumption: outgrowth of the buds on the P14 branch in treatment F was markedly greater than that of D and was approaching that of C (Fig. 3). This was so, despite there being differences in branch sizes and developmental maturity and in the time for which nodal roots were present. Nodal roots were present for 45 d at P10 in treatments C and D compared with only 26 d at P14 in treatment F. Clearly the difference between the outgrowth of buds in response to a common NRS supply in treatments C and D was almost entirely, and perhaps wholly, the result of differences in their intrinsic outgrowth potentials rather than differences in their positions on branches.
The excision of the parent apical buds immediately distal to the test buds in both experiments ensured that any variations in outgrowth of test axillary buds, under the same conditions of NRS supply, could be ascribed solely to variation in intrinsic properties of the axillary buds themselves rather than to the continuing influence of their parent apical buds. Observed variations in outgrowth were thus clearly a result of a carry-over of the conditioning of axillary buds by the environment prevailing within their parent apical bud during their early development. This was particularly apparent in Tradescantia in which the youngest test buds (Z, Fig. 2) were not initiated within their parent apical buds in any treatment until after the excision of the nodal roots, at which time all buds became wholly dependent upon NRS supplied from comparable basal root systems.
The results of both experiments strongly support the retention of Hypothesis 1: that each axillary bud exhibits an outgrowth potential that is directly related to the growth rate of its parent apical bud. In both, there was a close correlation between outgrowth rates of test buds and the activation levels of their parent apical buds that held true in relation to the measured growth of axillary buds on stems of different size, developmental maturity, and hierarchy within plants. In Trifolium the test buds on the P10 branch grew more rapidly in response to treatment than those on the P14 branch (Fig. 3), while in Tradescantia, there was a progressively increasing rate of growth of test buds as duration of exposure to the influence of nodal roots increased from treatment 0D through to 24D (Fig. 5). Regression analyses (Fig. 6) indicate that the rates of stem elongation of test axillary buds were correlated in a positive linear fashion with those of the parent apical buds in which they formed.
A second hypothesis (Hypothesis 2), that the rate of outgrowth of an axillary bud depends upon the combination of its outgrowth potential and the supply of NRS available to it, was also strongly supported by the results of the Trifolium experiment. In this, supply of NRS during the assessment period was varied by either retaining the distal nodal root at P10 (high NRS) or excising it so that NRS was supplied solely from the basal roots (low NRS). Analysis of the results (Fig. 3) showed that both bud outgrowth potential and NRS supply significantly influenced leaf emergence and stem elongation of test axillary buds. These factors were additive in their effect, the interaction between the two being non-significant.
These results raise the question as to how widespread the regulatory role of apical bud activation level might be among other species. Previous studies on a range of annual and short-lived perennial dicotyledonous species have led to the suggestion that the outgrowth potential of axillary buds might be related to the developmental stage and physiological activity of their parent apical bud (McDaniel and Hsu, 1976; Gould et al., 1987; Grbic and Bleecker, 2000). Because the changes in apical bud activity observed in these cases largely arose in parallel with the transition to flowering, it is difficult to separate influences associated with the development of reproductive maturity from those directly related to apical bud growth rates. The present findings, though, using plants maintained throughout in a strictly vegetative ‘steady developmental state’, have demonstrated that there is, indeed, a direct relationship between parent apical bud growth rates and the outgrowth potential of axillary buds newly emerged from them, at least in the two species studied.
It remains unclear the degree to which such a relationship is widespread among angiosperms, but it does seem likely that it might be common among prostrate nodally-rooting perennial herbs. It is pertinent in this regard that, despite the evolutionarily wide separation of Trifolium repens and Tradescantia fluminensis, they behave similarly in response to nodal roots. In both, stimulatory signals derived from nodal roots dominate their control of branching. In both, also, full activation of axillary buds is only weakly reversible following exposure to the influence of nodal roots (Fig. 4; Thomas and Hay, 2007). Similar stimulatory responses to nodal roots have also been found to occur in all other prostrate nodally-rooting perennial herbs examined so far (Thomas and Hay, 2004).
The additional understanding provided by the present investigation, in combination with knowledge of the pattern of intra-plant NRS distribution previously reported (Thomas and Hay, 2008a), will now serve as a basis for future work developing an improved model of the physiological regulation of branching by basal roots that might pertain to prostrate nodal-rooting herbs as a group.
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Acknowledgements |
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We thank Jocelyn Tilbrook and Rachael Sheridan for technical assistance and John Koolaard for statistical advice. This work was funded by the MeriNet programme, New Zealand Foundation for Research, Science and Technology, contract C10X0404.
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