JXB Advance Access originally published online on July 28, 2003
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Journal of Experimental Botany, Vol. 54, No. 390, pp. 2091-2104,
September 1, 2003
© 2003 Oxford University Press
Relationships among shoot sinks for resources exported from nodal roots regulate branch development of distal non-rooted portions of Trifolium repens L.
Received 20 February 2003; Accepted 23 May 2003
AgResearch Grasslands, Private Bag 11008, Palmerston North, New Zealand
* To whom correspondence should be addressed. Fax: +64 6 3518 138. E-mail: mike.hay{at}agresearch.co.nz
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Abstract |
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Two manipulative experiments tested hypotheses pertaining to the correlative control exerted by nodal roots on branch development of the distal non-rooted portion of Trifolium repens growing clonally under near-optimal conditions. The two experiments, differing in their pattern of excision to manipulate the number of branches formed at the first 9–10 phytomers distal to the youngest nodal root, each found that after 20 phytomers of growth the total number of lateral branches formed on the primary stolon remained between five and seven regardless of where the branches formed along the stolon. Additional treatments established that nodal roots influenced branch development via relationships among shoot sinks for the root-supplied resources rather than through variation in the supply of such resources induced by fluctuations in photosynthate supply to roots from branches. Regression analysis of data pooled from treatments of both experiments confirmed that shoot-sink relationships for root- supplied resources controlled the branching processes on the non-rooted portion of plants. A disbudding treatment, which removed all the apical and axillary buds present on basal branches, but left other branch tissues intact, increased branch development of the apical region in the same way as did complete excision of the basal lateral branches. The apical buds and the elongation processes occurring immediately proximal to the buds were thus identified as strong sinks for the root-supplied resources. Such results suggest that branch development on the non-rooted shoot portion distal to the youngest nodal root is regulated by competition among sinks for root-derived resources, of limited availability, necessary for the processes of elongation of axillary buds and the primary stolon apical bud.
Key words: Axillary bud outgrowth, branch development, clonal growth, correlative control, nodal roots, Trifolium repens.
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Introduction |
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Variation in the outgrowth of axillary buds is one of the key processes by which plants adapt phenotype to the local environment (Harper, 1977; Stearns and Koella, 1986) and has consequences for plant architecture (Hallé et al., 1978; Tomlinson, 1982; Bryan and Bell, 1991), reproductive potential, growth and vegetative persistence (Sackville-Hamilton et al., 1987; Watson et al., 1997). The physiological mechanisms controlling the outgrowth of axillary buds have therefore been a major investigative topic where the emphasis has been given to the role of apical dominance, although the part played by roots has been recognized (Cline, 1997; Napoli et al., 1999; Shimizu-Sato and Mori, 2001). These previous studies have commonly used orthotropic species such as bean, pea and Arabidopsis in which the terminal buds of the shoots become increasingly distanced from the root systems during development. By contrast, clonal species are characterized by having adventitious nodal roots close to their terminal buds (Klimes et al., 1997). Such species provide an alternative plant system for testing the nature of the relationship between roots and axillary bud development as they allow ready manipulation of the number of nodal roots and their distance from both the terminal buds and the individual axillary buds. This relationship is explored in the present study in an experimental system developed using the plagiotropic clonal herb, Trifolium repens. This species is easily grown in a wide range of environments, has its sites of individual nodal roots and axillary buds comfortably spatially separated, and has genotypes in which flowering can be prevented.
Lötscher and Nösberger (1996) and Thomas et al. (2002) showed for T. repens that a consistent, repeatable pattern of restriction of branch development occurred on a non-rooted region of a stolon distal to the youngest nodal root, irrespective of the number and age of any other nodal root systems proximal to that root. Hence the potential for buds to develop into branches on the non-rooted portions of stolons appears to be determined solely by the youngest actively growing nodal root proximal to them. Further more, excision of all elongating branches immediately distal to the youngest root stimulated the outgrowth of axillary buds distal to them that would otherwise have remained repressed (Thomas et al., 2003). Taken together these results suggest two hypotheses: (1) that the total number of axillary buds which are capable of forming elongating lateral branches in the region of the primary stolon distal to the youngest nodal root is constant; and (2) that the number of buds capable of forming elongating lateral branches in this region is independent of the number of phytomers between them and the youngest nodal root. A major objective of this study was to test these hypotheses using two manipulative experiments.
A second objective was to gain some insight into the mechanisms by which elongating lateral branches control the development of axillary buds at unrooted phytomers distal to them along the primary stolon axis. To test whether the repressive influence of the lateral branches was brought about by their actively growing buds (hypothesis 3) or by their leaves (hypothesis 4), a subset of three additional treatments was included within the second experiment.
As it was planned in both experiments to use manipulations involving excision of axillary buds, lateral shoots or leaves, which theoretically could stimulate the outgrowth of the remaining axillary buds through wound-induced ethylene production (Yeang and Hillman, 1981), preliminary tests were made to assess the response of axillary buds to wounding in the absence of tissue excision. These tests involved wounding the stolons by making 10 mm long longitudinal scalpel blade incisions to a depth of half the stolon diameter at each node bearing an axillary bud, lateral shoot or well-developed lateral branch. Each wound damaged an area at least equal to that of the surface exposed by removal of well-developed lateral branches, but in no case was there any stimulation of bud outgrowth at wounded nodes. Thus for T. repens, any response of axillary buds to treatments involving excision of shoot tissues is unlikely to result merely from the physiological responses to wounding and the differences in extent and timing of wounding proposed for Experiments 1 and 2 in the present investigation were considered unlikely to detract from the validity of comparisons made between the results obtained from them.
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Materials and methods |
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Definitions
Primary stolon: the main shoot stem axis developed from a cutting.
Phytomer: the repeated structural unit of a stolon differentiated from its apical meristem (Barlow, 1989). Each phytomer consists of a node, the internode proximal to it, an axillary bud and its subtending leaf, and two nodal root primordia.
Phytomer emergence: the stage of phytomer development at which its developing leaf has just reached 0.3 on Carlson’s scale of leaf development (Carlson, 1966).
Unemerged axillary bud: a bud still completely enclosed by the stipular sheath of its subtending leaf.
Emerged axillary bud: a bud in which one leaf is macroscopically visible beyond its stipular sheath and the average internode length of its stem axis is less than 10% that of the proximal internode of its parent stolon.
Lateral shoot: an outgrown axillary bud with average internode length greater than 10% of that of the proximal internode on the parent stolon and/or two or more emerged phytomers. Lateral shoots are classified either as lateral branches or short shoots (see below).
Lateral branch: a lateral shoot in which the average internode length exceeds 10% of that of the proximal internode on the parent stolon. Lateral branches growing directly from the primary stolon are designated first-order branches. These are further categorized as either unbranched laterals (lateral stolons on which no higher order branching is present) or secondarily branched laterals (lateral stolons on which one or more constituent phytomers have formed a higher order secondary lateral branch). Throughout this paper the terms ‘branch’ and ‘lateral branch’ are used to denote first-order lateral branches. Second-order branches growing from these are referred to as ‘secondary branches’.
Short shoot: a lateral shoot bearing two or more fully expanded leaves, but with an average internode length equal to or less than 10% of that of the proximal internode on the parent stolon.
Plant material
A genotype of Trifolium repens L. in which branching responses to nodal root treatments are typical for the species (Thomas et al., 2002) from a Spanish ecotypic collection (AgResearch Grasslands Accession number C1067) was selected for this study because of its tendency to remain fully vegetative under warm glasshouse conditions, irrespective of photoperiod (Thomas, 1982). A glasshouse-grown stock clone of the genotype, maintained at temperatures above 12 °C through the previous winter, supplied all cuttings for the experiments and these grew through the experimental period (spring–early summer) without flowering.
One hundred stolon tip cuttings, each with an apical bud and two emerged phytomers, were taken on 8 September 2000 and each planted into a 1.5 l polyethylene planter bag filled with a commercially obtained 60:40 peat moss:coarse sand mix supplemented with Osmocote fertilizer (1.5 kg m–3), dolomite (3 kg m–3), superphosphate (1 kg m–3), and trace elements (125 g m–3). As the plants grew, nodal root systems developed at each of the two original emerged phytomers and their primary stolons extended away from the planter bags. By 18 October 2000 each primary stolon had eight emerged phytomers beyond the planter bag and the youngest of these was labelled and designated phytomer 1. At this time the axillary buds/branches were excised from all nodes proximal to phytomer 1 and the leaves of the oldest four unrooted phytomers were removed so that plants had the appearance given in Fig. 1. Each planter bag was then placed so that the primary stolon of each plant grew horizontally out over a dry wooden grid. This prevented any further nodal root formation by ensuring that the relative humidity at the sites of the root primordia on stolons was below the threshold of 85% required for root outgrowth (Stevenson and Laidlaw, 1985). Plants were grown in a temperature-controlled glasshouse, allowing approximately 55% mean transmission of sunlight, with mean daily maximum and minimum temperatures of 28 °C and 12.5 °C and natural photoperiods.
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Experiment 1
On 18 October 2000, 48 of the rooted stolon cuttings were selected for uniformity of development and eight of these were randomly allocated to each of six treatments (A to F) and set out in eight randomized blocks in the glasshouse.
The treatments provided variation in both the number and placement of branches at phytomer 1 and the next eight phytomers (numbered 2–9) which subsequently emerged distal to it during the first phase of growth (Phase 1, Fig. 2). At each phytomer position on the primary stolon designated to have no branch, the axillary bud was excised as soon as the tip of its first leaf was visible beyond the subtending stipular sheath. The experiment thus had a treatment phase (Phase 1) during which the first nine phytomers (1–9) formed, and a response phase (Phase 2) during which plants received no further treatment and outgrowth of axillary buds at the next-formed ten phytomers (10–19) was permitted. Branches developing at phytomers formed on the primary stolon in Phase 1 are termed basal branches whereas those formed on phytomers emerging in Phase 2 are called apical branches in both this experiment and in Experiment 2. At the end of Phase 1, on 17 November 2000, when the primary stolon of every plant had produced a minimum of ten emerged phytomers (numbered 1–10), the length of each lateral shoot and the number of emerged phytomers along it was recorded. Plants were then grown on and an interim record made on 29 November 2000 of the number of phytomers along the primary stolon formed in Phase 2 (from position 10 onwards) and the length and phytomer number of any lateral shoots formed at these phytomer positions. After ten untreated Phase 2 phytomers (numbers 10–19) had emerged on a primary stolon, a record was again made of the length and phytomer number of each lateral shoot formed along it and of any secondary shoots formed on the lateral shoots. As the rate of phytomer emergence on the primary stolon of plants varied amongst treatments, individual plants reached the final assessment time at different times so the final assessments took place over the period inclusive of 8–19 December 2000.
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Experiment 2
On 18 October 2000, 35 rooted stolon cuttings, established as described above, were selected for Experiment 2.
These were then grown for a period of pretreatment (Phase 1) while 10 phytomers, distal to the marked phytomer 1, emerged on their primary stolons. At the end of this phase (19 November 2000) the number of phytomers and lengths of the primary stolons and lateral branches were assessed on a sample of 10 plants (subsequently used in treatments G and L). Immediately before the start of treatments on that same day, axillary buds or shoots were excised from phytomers 7–10 inclusive on all plants, leaving six well-developed branches on each. Plants were then randomly allocated, in groups of five, to each of seven treatments. These treatments are divisible into two partly overlapping subsets. One subset (treatments G, H, I, and J) was designed to test further the hypothesis that there is an upper limit to the number of elongating lateral branches which are able to form distal to the youngest nodal root, irrespective of where they arise along the primary stolon, by removing 0, 2, 4 or all 6 of the basal branches at the start of Phase 2 (Fig. 3) and recording the subsequent branching response of the next-emerging ten phytomers on the primary stolon. The other subset (treatments K, L and M, with G and J as controls) sought to shed light on the mechanism by which the presence of branches limits the development of further branches at distal nodes.
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Treatments K, L and M retained all lateral branches at phytomer positions 1–6, similar to treatment J, but, whereas J received no further treatment, K, L and M all received the following additional treatments at the start of Phase 2 (Fig. 3).
In treatment K the six basal lateral shoots were ‘disbudded’ by excising all apical buds, including those on secondary shoots, and all emerged axillary buds (buds being defined as all tissue distal to an expanding leaf at the Carlson (1966) developmental stage of 0.6). Thereafter, plants in treatment K were monitored every second day and any further emerged axillary buds appearing on the six basal lateral shoots excised. Thus treatment K had the same complement of mature (old) leaves on its basal branches as did treatment J at the start of Phase 2. Treatment L retained all axillary and apical buds, but was designed to have a similar complement of leaves to treatment K on its six basal branches throughout Phase 2. To achieve this, all newly emerged leaves, including those on any secondary branches present, were removed from these basal branches as they reached full expansion, leaving no more than one unfolded leaf plus unfolding leaves behind each apical bud. Treatment M plants also retained all axillary buds, but in these plants all fully expanded leaves, both young and old, and including those on secondary branches, were removed from the six basal branches at the start of Phase 2 and thereafter these basal branches were continually defoliated as in treatment L.
An interim record of the response of the apical region of each plant was made on 5 December 2000, 16 d after the start of treatments. Plants were then allowed to grow until their primary stolons had produced a total of ten emerged phytomers (numbers 11–20) during Phase 2. At the end of this phase, when phytomer 20 had emerged, each plant was assessed for phytomer number and length of all branches (inclusive of any secondary branches). As the rate of phytomer emergence on the primary stolon varied with treatment and from plant to plant within treatments, the time at which this stage was reached varied from plant to plant and, as a consequence, final assessments were spread over the period of 15–20 December 2000 inclusive. After assessment of branching, plants of treatments G, J, K, L, and M were harvested and dissected into roots, primary stolon stem and leaves, stem and leaves of basal branches (inclusive of those on their secondary branches) formed at primary stolon phytomers 1–10 (Phase 1), and of apical branches (similarly including those on any secondary branches present) formed at phytomers 11–20 (Phase 2). The dry weight of each component was then measured after drying for 48 h at 60 °C.
Analysis and presentation of data
Phytomers on the primary stolon were numbered acropetally with the eighth phytomer distal to the youngest of the two basal roots designated phytomer 1 (Fig. 1), on the 18 October 2000, and successively formed phytomers were termed phytomer 2, 3 etc. Branches were similarly identified by the position of their phytomer of origin on the primary stolon. As the phyllotaxy of T. repens is distichous, even numbered phytomers had roots and branches orientated to the same side of the primary stolon as the youngest basal root whereas odd numbered phytomers were orientated to the other side.
The effects of treatments on the number of lateral branches to form in the Phase 2 apical region and the total number along the primary stolon (Phases 1 and 2) were tested by Chi-square analysis using the overall mean as the expected value. The data from Experiment 1 for the effect of treatment A versus F on the relationship between branch length and phytomer position was tested by linear regression analysis (Genstat, 2001). The fitted curve of stolon diameter at internode 11 of the primary stolon plotted against number of basal branches permitted to form in Experiment 1 was derived from non-linear regression analysis (Genstat, 2001). The relationships were tested by regression analysis (Genstat, 2001) for the data from Experiment 2 recorded 16 d after application of branch excision treatments. At the conclusion of both experiments the relationship between the total number of elongating branches in the basal and apical regions was individually analysed by linear regression (Genstat, 2001) for each experiment and found to be similar. Then the data from both experiments were pooled, with the exception of the treatments K, L and M from Experiment 2 which received defoliation or disbudding treatments rather than branch removal, for further regression analysis.
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Results |
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Effect of number of basal branches present at the end of Phase 1 on number and length of apical branches formed in Phase 2
In Experiment 1 the number of lateral branches allowed to form in Phase 1 (basal branches) was prescribed by treatment, but the number formed in Phase 2 (apical branches) was not and this differed very significantly (P <0.001) with treatment (Fig. 4). Furthermore, the number of apical branches that formed in each treatment was such that for all treatments the total number of first-order branches (basal plus apical) did not differ significantly (Fig. 4). In treatments where branches were allowed to develop at any of phytomer positions 1–4 on the primary stolon, the lengths at a given phytomer position did not differ (Fig. 4). A two sample t-test showed the branch length at phytomer 7 of treatment D was greater than that of treatment F (P <0.01).
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In Experiment 2 the variation in basal branch number and position was achieved by branch excision at the end of Phase 1. Once again, the number of lateral branches formed in the Phase 2 apical region was inversely related to the number of basal branches retained and was such that the total number of lateral branches on the primary stolon did not differ significantly from treatment to treatment (Fig. 5). The length of the lateral branches present at any given phytomer position between 3 and 6 on the primary stolon did not differ between treatments H, I and J. Lengths of the apical branches decreased as the number of basal branches in a treatment increased.
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Effect of basal branch position on the number and length of the lateral branches forming from apical phytomers on the primary stolon
Comparisons of treatments within Experiment 1 (Fig. 4), provide tests for the effect of the phytomer position of lateral branches on the primary stolon on the total number of lateral branches formed. First, although treatments C and D differed in the phytomer positions at which the three basal branches were located (C had branches at positions 1–3, D at positions 1, 4 and 7), this had no significant effect on the number or length of the lateral branches formed in Phase 2. Second, the most extreme comparison of the effect of branch position, that between treatments A (no basal branches, all branches positioned at primary stolon phytomers formed in Phase 2) and F (unrestricted basal branching, all branches positioned at phytomers formed in Phase 1), showed a striking similarity in the lengths of the lateral branches developed in the basal and apical regions of the primary stolon (Fig. 6). The growth of basal branches at the end of Phase 1 in treatment F, after emergence of ten basal phytomers on the primary stolon from phytomer 1 onwards, was compared with that of apical branches at the end of Phase 2 in treatment A after the emergence of ten apical phytomers from phytomer 11 onwards. These two sets of branches were thus directly developmentally comparable, the oldest in each case having been growing for approximately 4 weeks during which nine further phytomers had emerged from the apical bud of the primary stolon to which it was attached. Tests by regression analysis found no evidence of difference between lateral branch lengths at equivalent phytomer positions in the two treatments although the effect of phytomer position was highly significant (P <0.001).
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Similar comparisons within Experiment 2 also indicated that the phytomer position of origin of branches along the primary stolon had no significant effect on the total number of lateral branches formed per primary stolon (Fig. 5). Treatments were designed so that, where the number of basal branches was restricted, those permitted to develop were positioned at the younger phytomers of the primary stolon (e.g. treatment H with two branches, positions 5 and 6; I with four branches, positions 3, 4, 5, and 6) and there was no evidence in the pattern of apical branching that branch number or length were any more or less limited than when the basal branches were positioned at the oldest phytomer positions on the primary stolon as in Experiment 1.
In Experiment 2 there was no manipulative treatment of branching until the end of Phase 1. Thus, in treatment G it was possible to obtain data for the same plants both on the development of basal branches at the end of Phase 1 and, also, after exactly the same developmental period (emergence of eleven phytomers on the primary stolon), on the development of apical branches at the end of Phase 2. Comparison of these two sets of data (Table 1) reveals no significant difference in the number or length of the lateral branches except for a reduction in length of the first Phase 2 branch (phytomer position 11) formed immediately after the plant had received the severe treatment of excision of all basal branches.
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Effect of number of basal branches on primary stolon development
The phytomer emergence rate of the primary stolon of plants in Experiments 1 and 2 during the first 12 and 16 d of Phase 2 was higher in treatments that reduced the number of lateral branches in the basal region of the primary stolon (Table 2). The maximum extent of the increase (the comparison between no basal branches and six basal branches) was greater in Experiment 1 (50%) than in Experiment 2 (20%). The treatments involving defoliation, K, L, and M in Experiment 2, were excluded from this analysis because they involved disbudding and defoliation as well as manipulation of the number of branches.
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In addition to showing a higher phytomer emergence rate, the youngest lateral shoots in the plants totally lacking basal branches (treatment A) exhibited precocious elongation of their stem axes. In these, the region of axillary stem proximal to the first-formed leaf on the bud was often as long as 10 mm or more at the stage when the lamina of the first-formed leaf was still only 5 mm or less in length (at stage 0.2 of Carlson’s scale of leaf development: Carlson 1966), whereas the equivalent region of an axillary bud typically failed to elongate in other treatments. Such precocious bud axis elongation only took place at phytomers which emerged during the first 12 d of Phase 2; buds at the three phytomers which emerged subsequently, towards the end of Phase 2, developed no differently from those on plants in the other treatments.
Measurement of internode diameters in the region of the primary stolon which emerged from the apical bud during the first 12 d after the start of Phase 2 in Experiment 1 revealed a strong inverse relationship (P <0.01) between primary stolon diameter at the midpoint of the internode distal to the node at phytomer 11 and the number of basal lateral branches (Fig. 7). In particular, apical internodes in plants with no basal branches (treatment A) were significantly thicker than those in the fully branched control plants (treatment F).
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Effect of disbudding of basal branches on number and length of the apical branches formed on the primary stolon
Control plants in treatment J, which possessed a full complement of intact basal branches, produced virtually no apical branches during Phase 2 (Fig. 8). By contrast, treatments G and K both stimulated branch outgrowth in the untreated apical region. Disbudding of the six basal branches in treatment K induced a branching response in the untreated apical region during Phase 2 which was very similar to, but slightly different from, that of treatment G (complete removal of basal branches). Complete removal of basal branches stimulated apical branch development more rapidly than did removal of their buds and actively growing apices alone. After the first 16 d of Phase 2, branch lengths at successively emerged apical phytomers were 55, 46, 41, 26, 14, 2.5, and 0 mm in treatment G and 27, 29, 22, 14, 10, 0, and 0 mm in treatment K. By the end of Phase 2, branches at the first five of these apical phytomers (numbers 11–15) had each elongated by about a further 100 mm in both treatments so that those in K remained shorter (Fig. 8), but those at the later-emerged phytomers (numbers 16–20) were markedly longer in the disbudded plants (K). In addition, more of the emerged apical phytomers had produced elongating lateral shoots in treatment K (9.2) than in G (7.0), the youngest one or two of which, in the disbudded plants, exhibited precocious elongation of their stem axes similar to that observed in treatment A in Experiment 1.
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Effect of defoliation of basal branches on shoot growth in the apical region of the primary stolon
The mean rate of leaf emergence from the primary stolon apical bud throughout the whole of Phase 2 was uninfluenced by defoliation of the basal branches, ranging from 0.386 leaves per day in the fully foliated control plants (treatment J) to 0.378 in plants with totally defoliated basal branches (M) and 0.369 in those with partially defoliated basal branches (L).
Neither the total defoliation of basal branches at the start of Phase 2 (treatment M) nor continued removal of newly expanded leaves from the apical region of extending basal branches during Phase 2 (treatment L) was found to have any stimulatory effect on the development of additional branches in the apical region of the primary stolon when the results of these treatments were compared with those from fully foliated controls (J) (Fig. 8). Defoliation did, though, reduce the elongation and secondary branching of basal branches, the mean lengths of first-order branches and the number of secondary branches on them being 347 and 11.9, respectively, in treatment L, 399 and 12.7 in treatment M and 428 and 21.5 in treatment J.
Dry weights of plant components
The dry weights of the nodal root systems in the planter bags at the bases of the plants at the end of Experiment 2 were 2–3 times greater (P <0.001) in treatments J and K than in treatments G, L and M (Table 3) while the weight of stem and leaf of apical branches in treatments G and K were similar, and both very much greater (P <0.001), than those of J, L and M. Relative to J, the defoliation treatments (L and M) and the bud excision treatment (K) significantly decreased basal branch weights (Table 3).
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The relationship between development of lateral shoots in apical and basal regions of primary stolons
This relationship was determined at two time intervals after the start of Phase 2: namely, after 16 d in Experiment 2 and at final harvest after 21–32 d in Experiment 1 and 26–31 d in Experiment 2.
After 16 d in Experiment 2, there was a direct linear relationship (P <0.001, r2=0.784) between axillary bud outgrowth (total number of emerged phytomers) in the apical region of the primary stolon and the number of basal branches removed (Fig. 9a). This relationship was independent of the large difference in total numbers of emerged phytomers (including those on secondary branches) on the basal branches: the two most basal branches removed in treatment I bore 27 emerged phytomers at the start of Phase 2, the two additionally removed distal to these in treatment H bore 10, and the two youngest, which were removed together with the other four in treatment G, bore only seven. Thus the total numbers of phytomers removed in treatments G, H, I, and J were 44 (namely, 27+10+7), 37 (27+10), 27, and 0, respectively (Fig. 9b). The apical branching response to removing the basal-most two branches (treatment J minus treatment I) was the same as the response to removing the most distal two (treatment H minus treatment G) despite there being four times as many emerged phytomers on the former than the latter (namely 27 versus 7) (Fig. 9a).
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A similar relationship was seen at the conclusion of the two experiments (Fig. 10). The strength of this relationship (P <0.001, r2=0.992) has been made clearer by combining the data for both Experiments 1 and 2 in this figure, a process considered justified because the experiments were carried out concurrently, side by side in the same glasshouse using the same genetic material. The data from all treatments involving branch removal (A, B, C, D, and E in Experiment 1 and G, H and I in Experiment 2) together with the two control treatments (F and J) were pooled for regression analyses testing the strength of the inverse relationships. In this figure, the number of lateral branches formed in the apical region of the primary stolon in response to basal branch removal treatments is plotted against the total number of elongating branches (including the lateral branches grown from the primary stolon together with all elongating secondary branches growing from them) present in the basal region at the end of Phase 2. Data from treatments K, L and M in subset 2 of Experiment 2 are shown in Fig. 10, but excluded from the regression analysis because, unlike plants in other treatments, their development had been additionally influenced by combinations of disbudding and defoliation.
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Discussion |
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This communication extends the present knowledge of nodal root–axillary bud interrelationships by showing that for any given conditions the total number of lateral branches formed distal to the youngest nodal root on a stolon is constant, regardless of the position of the branches relative to the nodal root, and by identifying the sinks in the shoot which compete for the limited resources available from the nodal root. These conclusions were reached as a result of tests of four hypotheses propounded at the outset of this investigation.
Hypothesis 1: that the total number of axillary buds forming elongating branches in the region of the primary stolon distal to the youngest root is constant
Although the techniques used to manipulate basal branch number differed substantially in the two experiments performed to test this hypothesis, the results were similar in that the total number of elongating lateral branches distal to the youngest nodal root did not differ significantly with treatment (Figs 4, 5) and so support the retention of the hypothesis. The tendency for plants of Experiment 2 to have slightly higher numbers of apical branches, as exemplified by the comparison of the two groups of plants which totally lacked basal branches (5.6 in treatment A, Experiment 1 versus 7.0 in treatment G, Experiment 2), may relate to the different techniques used to manipulate basal branch number and the effect this, in turn, may have had in limiting the mass of the basal root systems in Experiment 1 relative to Experiment 2.
Hypothesis 2: that the number of axillary buds on the primary stolon which form lateral branches distal to the youngest nodal root is independent of their phytomer position relative to that root
The results shown in Figs 4, 5 and 6 support this hypothesis. The position of the basal branches along the primary stolon had no effect on the total number of lateral stolons formed, as seen by comparing branching in treatments C and D in Experiment 1 (Fig. 4) and by comparing Phase 1 branching of treatment F with Phase 2 branching of treatment A (Fig. 6). Further supporting evidence is provided by the similarity of the branching of plants in treatment G in Phases 1 and 2 of Experiment 2 (Table 1).
Hypothesis 3: that excision of axillary and apical buds from basal lateral branches stimulates outgrowth of distal axillary buds
Comparison of the results of treatments G, J, K, and L in Experiment 2 (Fig. 8) shows that this hypothesis is correct. Distal bud growth was restricted in untreated plants in treatment J, whereas it was stimulated by complete removal of basal lateral branches (treatment G) and in plants that were just disbudded without removing their branches or the leaves on them (treatment K).
Treatment L was designed to overcome the problem that disbudding in treatment K also prevented the addition of new young leaves to the tips of the basal lateral branches by removing their sites of origin. Comparison of the effects of treatments K and L, in which the number of mature leaves on the basal branches was kept similar and the only major difference was the lack of buds and actively elongating stem tissue and young leaves immediately proximal to them in K, showed the stimulation of bud outgrowth in the apical region of the primary stolon induced by treatment K failed to occur in treatment L. Thus, stimulated bud outgrowth was solely the result of bud removal.
Furthermore, it was apparent, by consideration of the results from treatments G, H, I, and J, that this stimulation was a direct result of removal of actively elongating shoot apical buds rather than total number of buds removed. This is shown by comparison of the linear relationship in Fig. 9a with the exponential relationship in Fig. 9b. At the time the basal branches were removed in these treatments, each still possessed only one actively elongating shoot apical bud, none having yet developed actively elongating secondary branches. However, as was detailed in the Results, removal of the two oldest branches (treatment I) with buds at 27 emerged phytomers had the same effect as removal of the youngest pair which had buds at only seven phytomers. Each led to the emergence of approximately three new apical branch phytomers (Fig. 9a).
Thus, while treatment K involved the removal of all buds (ranging from actively elongating lateral shoot apical buds to quiescent axillary buds) from basal lateral branches, it is apparent that those buds undergoing rapid leaf expansion and internode elongation had by far the greatest restricting influence on distal bud activity. The process of leaf initiation in slowly growing non-elongating buds and in slowly elongating short shoots on lateral branches had little restrictive effect on bud outgrowth at phytomers distal to them on the primary stolon.
Hypothesis 4: that defoliation of basal lateral branches stimulates outgrowth of distal axillary buds
The results of Experiment 2 did not support this hypothesis. Neither of the two defoliation treatments, L and M, had a stimulatory effect on outgrowth or elongation of apical branches compared with the fully foliated control treatment, J (Fig. 8). They did, however, reduce the mass of the basal root systems 2–3-fold (Table 3). The possibility that the failure of defoliation to stimulate apical branch development was simply an indirect result of reduced root activity is unlikely, because apical branches grew strongly in plants from which basal branches were removed (treatment G) and which had, as a consequence, even lower root masses (Table 3). Reduction of the supply of photosynthate to the roots, as evidenced by lower root mass correlated with defoliation, clearly had little stimulatory effect on the growth of buds in the apical region of the primary stolon.
Treatment effects on root mass and its relationship to the acropetal transport of root-supplied resources
Overall, the effects of treatment on root mass during Phase 2 (Table 3) were those to be expected on the basis that increase in root mass during that phase would have been in direct response to the supply of carbon available from the foliage on the basal region of the shoot. Thus, for four of the five treatments, the ratios of basal leaf to root were similar. In those with reduced basal foliage, G, M and L, the ratios were 1.11, 1.20 and 1.29, respectively, and in the disbudded plants of treatment K, which retained their basal foliage, the ratio was 0.97. In all these four treatments, basal branch growth was weak (L and M) or lacking (G and K) and, in the absence of strong branch elongation, much or most of the carbon fixed would have been available for accumulation of root mass. The much higher leaf:root ratio of 2.13 recorded for treatment J, with actively growing fully foliated basal branches, is understandable on the premise that much of the carbon fixed would have been used in the growth of branches as well as roots.
By contrast, the amount of branching in the apical region of the shoot showed no relationship with root mass. Treatments J and K, with similar root masses of 4.06 and 4.29 g, developed apical branches with very different total dry weights of 0.07 and 2.61 g, respectively, and treatments G and K with high apical branch dry weights of 2.76 and 2.61 g had root masses of 1.34 and 4.29 g, respectively (Table 3). Such results demonstrate that availability of RSR for acropetal transport into the apical region of the primary stolon is not simply a function of root mass. It would appear that another factor, such as the presence of actively growing basal branch apices, must interact with root mass in such a way that a relatively constant supply of resources from root systems of differing mass is delivered to the apical regions under any given set of environmental conditions.
Intra-plant sink relationships: a putative mechanism controlling branching
In both experiments, plants comprised three sets of competitive sinks, namely basal roots and a single actively growing primary stolon apical bud at either end of the plant, together with several branches intercalated between them. These sinks establish a balance with each other for the supply of resources from the roots (RSR) and carbon from the leaves. A competitive relationship was found between the apical bud of the primary stolon and first-order lateral branches for a finite, but limited, supply of resources from roots which sufficed only to support the elongation processes of the apical bud and a small number of first-order branches. This was evident in the inverse relationships between characteristics reflecting the vigour of growth of the apical bud of the primary stolon (phytomer emergence rate, Table 2 and stolon diameter, Fig. 7) and the number of first-order lateral branches growing proximal to it, and between total number of elongating branches present in the basal and apical regions (Fig. 10). The strength of the latter relationship (r2=0.992) indicates that control of lateral branch formation in the apical region can be accounted for solely by the competitive allocation of RSR. A corollary is that the restriction in branch formation in these plants can be accounted for without the need to postulate the action of a repressive mechanism imposed from the apical bud.
In general, after the set of first-order lateral branches formed, their continued growth increased their sink strength with the result that the supply of RSR to the apical bud of the primary stolon decreased which, in turn, decreased its growth rate. Any manipulation to reduce the number of first-order lateral branches increased the growth and vigour of the apical bud of the primary stolon and promoted the outgrowth of distally positioned axillary buds, thereby replacing the lateral branches removed. The sensitivity of the phytomer emergence rate of the primary stolon apical bud to manipulations of lateral branch number (a 50% increase in Experiment 1 upon removal of all branches (treatment A) compared with control (F), Table 2) was unexpected, given the extensive literature emphasizing the stability of this characteristic in the face of fluctuations in external resource availability (Chapman, 1983; Hay and Newton, 1996) and the dominant influence of temperature in determining phytomer emergence rate (Beinhart, 1963; Chapman, 1983; Sackville-Hamilton and Harper, 1989). The present result indicates that correlative influences within a stolon system should be added to the list of variables that can influence phytomer emergence rate.
Results obtained from both experiments can be interpreted as the outcome of perturbations to the balance between the sinks for RSR within each plant. In Experiment 2 up to the end of Phase 1, plants in all treatments grew fully branched and intact, under the same set of conditions, so the balance between the sinks within each plant for RSR would have been the same and their root masses similar.
In treatment G, total removal of the basal branches would have resulted in an immediate increase in RSR availability to the apical region of the primary stolon. This would explain the observed burst of axillary bud outgrowth at the start of Phase 2. The massive loss of foliage incurred by basal branch removal would then be expected to have reduced carbon supply to the root system, and thereby reduced any gain in root mass (Table 3), without affecting distal transport of RSR in sufficient quantity to support a full suite of first-order elongating branches in the apical region (Fig. 8).
In treatment J, where all basal branches remained intact, the supply of carbon from their leaves would have stimulated increases both in root mass and in their actively growing buds, internodes and young leaves, which would have acted as strong sinks for RSR in competition with the apical and axillary buds in the apical region of the primary stolon. As a result, the latter would have received relatively little RSR and phytomer emergence rate and axillary bud outgrowth would have gradually declined (Table 2; Fig. 8).
In treatment K, disbudding would have reduced the overall sink strength of the basal branches, thereby allowing more acropetal transport of RSR to the primary stolon apical bud and stimulating axillary bud outgrowth. Disbudding did not at first totally remove all active sinks from the basal branches, as young expanding leaves and elongating internodes were left in place. As a result, there would have been a lag in the build-up of RSR available to the primary stolon apex and, as described earlier in the Results section, the initial boost to growth in the apical region would have been less than in treatment G. Gradually thereafter, with the maturation of all young leaves and internodes on the basal branches, the sink for RSR would have been lost from the basal branches and the apical region would have benefited from increased RSR. This could explain the observation that, by the end of the experiment, the number of lateral shoots formed in treatment K exceeded that in treatment G (9.2 versus 7.0; Fig. 8).
In Experiment 1 the equilibrium state established in the untreated control plants (F) was prevented in the other treatments by various degrees of bud removal during Phase 1. This initial imbalance was overcome by the cessation of bud removal in Phase 2. In the extreme treatment where all buds were removed (A), the immediate response to cessation of bud removal was a burst of growth in the apical region during the first 12 d of Phase 2, made manifest by increases in phytomer emergence rate (Table 2) and stolon diameter (Fig. 7) as well as by precocious elongation of axillary bud stem axes. By the end of the experiment, this enhanced apical activity had settled back to the equilibrium state. As in the case of Experiment 2, these responses can be interpreted as resulting from an excess of RSR being available to the apical region of the primary stolon in the absence of competing basal branches and a subsequent decline in this availability as branches developed proximal to the primary stolon apical bud.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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This study demonstrates that under near-optimal conditions there is a basic underlying requirement for an adequate supply of RSR for branch development to proceed without check. It is proposed that the development of a set number of branches distal to the youngest nodal root is the result of the establishment and maintenance of an equilibrium state which is brought about by a balance between the activity of the root system as a source of RSR and the relative sink strengths of the elongating apical regions of lateral branches and of the primary stolon distal to them. These processes of allocation of RSR accounted for the observed restriction in branch formation without any requirement for action of repressive influences emanating from the shoot apical bud. Questions remain as to how this mechanism is modified so as to restrict branch development when nutritional and light resources are sub-optimal and as to the nature of the signal or resource limitation driving the branching responses. The experimental system described in this study has considerable potential for use in investigations seeking answers to such questions.
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Acknowledgements |
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We thank Jocelyn Tilbrook and Fred Potter of AgResearch Grasslands for technical assistance and statistical advice, respectively. Constructive comments on an earlier draft by Harry Clark and Keith Widdup of AgResearch are appreciated. This work was funded from the New Zealand Foundation for Research and Technology, contracts C10X0021 and C10X0203.
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