- Email: [email protected]
Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots
Annals of Botany 78 : 255–266, 1996
Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots M O R R I S G. C L I N E The Ohio State Uniersity, Department of Plant Biology, Columbus, Ohio 43210, USA Received : 5 July 1995
Accepted : 11 March 1996
In 1933 Thimann and Skoog demonstrated exogenous auxin repression of lateral bud outgrowth in decapitated shoots of Vicia faba. This evidence has given strong support for a role of auxin in apical dominance. Most, but not all, investigators have confirmed Thimann and Skoog’s results. In the present study, auxin treatments were carried out on ten different species or plant types, many of which were treated with auxin in different forms, media and under different light conditions. The Thimann–Skoog experiment did work for most species (i.e. exogenous auxin did repress bud outgrowth) including the dgt tomato mutant which is known to be insensitive to auxin in certain responses. Toxic auxin symptoms were observed in some but not all species. The Thimann–Skoog experiment did not work for greenhouse-grown Coleus or for Arabidopsis. Light was shown to reduce apical dominance in Coleus and Ipomoea nil. # 1996 Annals of Botany Company Key words : Apical dominance, lateral bud outgrowth, axillary bud, auxin, IAA, decapitation, Vicia faba, Ipomoea nil, Pisum satium, Phaseolus ulgaris, Lycopersion esculentum, dgt, Coleus blumei, Arabidopsis thaliana, Helianthus annuus, Thimann–Skoog.
INTRODUCTION Apically derived auxin in shoots is generally thought to control apical dominance either directly via entry into lateral buds with subsequent repression of outgrowth or indirectly via some other mechanism, i.e. activation of a second inhibitor messenger, auxin-cytokinin ratio, secondary growth substances, nutrient diversion, etc. (Martin, 1987 ; Cline, 1991 ; Bangerth, 1994 ; Stafstrom, 1995 ; Tamas, 1995). As studies of control mechanisms of developmental responses in plants have progressed, particularly with the use of molecular and genetic approaches (Cline, 1994), there has been increased interest directed towards the role of auxin in apical dominance, and with the degree of branching serving as a possible indicator of auxin activity (Tepfer, 1984 ; Lincoln, Britton and Estelle, 1990 ; Shen et al., 1990 ; Estruch et al., 1991 ; Klee and Romano, 1994). Because of this increased attention concerning auxin in apical dominance and inasmuch as the precise mechanism of auxin action in apical dominance has yet to be elucidated, a closer scrutiny of the evidence supporting auxin involvement in the control of lateral bud outgrowth is needed. The strongest evidence supporting either a direct or an indirect role for apically-derived auxin in controlling apical dominance comes from the classical Thimann–Skoog experiment with Vicia faba (Thimann and Skoog, 1933). They demonstrated that auxin applied in agar blocks every 6 h to the stump of a decapitated Vicia faba stem inhibited the outgrowth of lateral buds situated lower on the stem. Thimann (1937) initially proposed the direct inhibition hypothesis which presumes that auxin enters the lateral bud and directly inhibits its outgrowth. Subsequently, he modified his views to include indirect auxin action via involvement with cytokinins (Sachs and Thimann, 1967) or 0305-7364}96}08025512 $18.00}0
ethylene (Russell and Thimann, 1988) as well as effects of light (Thimann, 1977). While many investigators have substantiated and extended the results of the Thimann–Skoog experiment (i.e. exogenous auxin repression of lateral bud outgrowth in decapitated shoots), others have reported anomalous effects and there have been questions about the direct role of auxin in apical dominance (Jacobs et al., 1959 ; Brown, McAlpine and Kormanik, 1967 ; Ali and Fletcher, 1970 ; Shein and Jackson, 1972 ; Hillman, 1984). Wareing and Phillips (1981) stated ‘… it is now generally considered that auxin does not exert its inhibitory effect on lateral buds in such a direct manner as that originally proposed by Thimann ’. Some workers (Gregory and Veale, 1957 ; McIntyre, 1977) have proposed nutrition to be the most important controlling factor in apical dominance. To date, no one has reported evidence of auxin restoration of apical dominance in Arabidopsis via the Thimann–Skoog experiment. The question has also been raised as to how the terminal bud can continue to grow while simultaneously generating inhibitory concentrations of auxin to the outgrowth of lateral buds below. Klee et al. (1987) have found that overproduction of auxin in petunia via transformation with IAA biosynthetic genes of Agrobacterium eliminates branching. With a similar transformed system, Romano, Cooper and Klee (1993) have reported an increase in apical dominance in Arabidopsis as measured by a reduction in fresh weight of secondary inflorescences. However, this auxin overproduction occurs in every cell of the organism rather than being localized in buds of adjacent nodes as might occur in a natural system. In addition, Lincoln et al. (1990) reported that ‘ total number of inflorescences arising from the rosette does not differ greatly between axr1 [the bushy auxin-resistant mutant] and wildtype ’ even though # 1996 Annals of Botany Company
256
Cline—Exogenous Auxin Effects in Decapitated Shoots
F. 1. Lateral bud outgrowth approximately 1 week following decapitation. A, Vicia faba L. (Broad Windsor Bean). B, Helianthus annuus L. (Mammoth Grey-striped Sunflower). C, Ipomoea nil L. Roth (Japanese Morning Glory). D, Pisum satium L. [Little Marvel Pea (dwarf )], and E, Thomas Laxton Pea (normal). F, Phaseolus ulgaris L. (Blue Lake Bean). The stem stumps of all decapitated plants shown here were treated with lanolin except for Japanese Morning Glory (C) which has a taped cotton swab for treatment with aqueous solution.
the number of lateral branches did greatly increase in the former. Beveridge, Ross and Murfet (1994) reported that grafting of non-branching wildtype stocks to the branching rms-2 pea mutant scions did not normalize endogenous IAA levels even though it did inhibit branching.
White (1976) stated that ‘… discrepancies in the results of different workers using apparently similar methods of application of IAA to decapitated plants of a single variety of Phaseolus obviously require some explanation …’. He carried out experiments, focusing on this single species with
Cline—Exogenous Auxin Effects in Decapitated Shoots variations in plant age, concentrations and quantity of IAA, type of lanolin, region of application and time of year. His results showed that the effectiveness of IAA applied did vary significantly with those conditions. He also found that in many instances, IAA did completely replace the main shoot with respect to correlative inhibition of lateral bud growth. The objective of the present study was similar to that of White’s except that instead of analysing a single species, the focus here was to compare responses to exogenous IAA in repressing lateral bud outgrowth in ten different species or plant types, including the mutant dgt tomato which is known to be insensitive to auxin promotion of certain responses, and Arabidopsis thaliana. In addition, the effects of auxin in different concentrations (both aqueous and in lanolin), forms (IAA, α-NAA and β-NAA) and under different light conditions were studied in a number of these species.
MATERIALS AND METHODS Seeds of Vicia faba L. (Broad Windsor Bean), Helianthus annuus L. (Mammoth Grey-striped Sunflower), Ipomoea nil L. Roth, strain violet (syn. Pharbitis nil ) (Japanese Morning Glory), Pisum satium L. [Little Marvel (dwarf ) and Thomas Laxton (normal) Pea], Phaseolus ulgaris L. (Blue Lake Bean) and Lycopersicon esculentum, Mill., strain VNF8 and the mutant diageotropica (dgt) tomato were germinated in Pro-mix, a general purpose peat-vermiculite growing medium. The seeds of Ipomoea were scarified in concentrated sulphuric acid for 40 min and soaked overnight in running water and germinated in Petri dishes before planting. Coleus blumei Benth. was propagated from stem cuttings. All the plants except the normal pea were grown in a greenhouse (16–32 °C) during the winter}spring of 1993–94 and the spring}summer of 1995 with supplementary General Electric 400 watt mercury vapour lamps (total irradiance : up to 3300 µmol m−# s−"). All the above plants, except Coleus, were also grown in growth rooms (27–29 °C) under continuous light (General Electric, Power Groove cool white fluorescent and incandescent sources 25–450 µmol m−# s−"). The ages of the plants at the times of decapitation and the beginning of the auxin treatments varied with the growing conditions (i.e. in greenhouse and growth rooms) and the developmental status of the lateral buds. The ages (in d) ranged as follows : Vicia faba, 12–13 ; Helianthus annuus, 19–27 ; Ipomoea nil, 12–32 ; Pisum satium, dwarf, 10–21, normal, 15–16 ; Phaseolus, 10–18 ; Lycopersicon esculentum, VNF8, 27–54 ; dgt, 39–68 ; Coleus (from time of stem cutting), 21–37 ; Arabidopsis thaliana, 35–52. Intact plants with elongating lateral buds were excluded. Shoots were decapitated with a razor blade about 1 cm above the base of the lateral bud at the node indicated for each species in the Results section (Fig. 1). Excluding the cotyledons, nodes were counted upward from the base of the plant. There was some variation in this distance depending upon the species and the relevant internode lengths. It was necessary that there be sufficient distance between the lateral bud and the decapitated stem stump above so that auxin could be
257
applied to the stump without contact with the bud. Auxin was applied to the stem stump twice daily beginning immediately after decapitation in approximately 150 µl doses as IAA, α- or β-NAA at concentrations ranging from 10−' to 10−$ in 0±05 % Tween 20 aqueous solution in a taped cotton swab (Fig. 1 C) or as IAA in lanolin, 0±1, 0±5, 1 %. Treatments normally extended from 4 to 10 d with daily measurements of the lateral bud situated at the highest node below the point of decapitation. Two exceptions were Vicia faba and Pisum satium (normal) where the largest rather than highest lateral bud was used to determine bud outgrowth. Each treatment was usually carried out with four to nine plants. At least two experiments with each of the plant types were carried out in both the greenhouse and the growth room with the exceptions indicated above. For certain light experiments (as indicated in the Results section), Ipomoea seedlings were moved from growth rooms to out-of-doors (irradiance under shade screens, up to 210 µmol m−# s−" and in the open, up to 6900 µmol m−# s−") during the summers of 1992 and 1994 and Coleus plants propagated from stem cuttings were grown in growth rooms with greatly reduced irradiance from General Electric cool white fluorescent lamps (25–30 µmol m−# s−"). There were usually four to ten Coleus and Ipomoea plants in each of the auxin treatments. In the outside shade screen experiments, there were eight to 11 Ipomoea plants per treatment. Seeds of Arabidopsis thaliana (strain CS 1072 Chi-O from the Ohio State University Arabidopsis Biological Resource Center) were germinated in water-saturated Magik-moss potting soil containing perlite and vermiculite in greenhouse following a 2-d treatment in the refrigerator (0–3 °C). After several weeks, some seedlings were moved to the growth room (27–29 °C) under continuous light while the remaining were kept in the greenhouse.
RESULTS Auxin significantly inhibited lateral bud outgrowth following decapitation when applied to the stem stump of most of the species tested [Ipomoea nil, Helianthus annuus, Lycopersicon esculentum (VNF8), Pisum satium (Little Marvel and Thomas Laxton), and Vicia faba] at concentrations of 10−& and}or 10−% in aqueous solution or 0±1 and}or 1 % in lanolin (Figs 2, 4 ; Table 1). Inhibition was also found in the tomato mutant dgt (Fig. 4) which is insensitive to auxin with regard hypocotyl elongation and ethylene production (Kelly and Bradford, 1986). In Ipomoea, Helianthus and dgt tomato there were no obvious toxic effects of exogenous auxin applications observed in any experiments. In Vicia faba and Pisum, occasional auxininduced aberrations in stem and leaf growth were observed whereas in the VNF8 tomato such aberrations were more common. Auxin had no effect on restoring apical dominance in decapitated greenhouse-grown Coleus (Fig. 5) or Arabidopsis (Fig. 3, Tables 2 and 3) and only a partial effect on bean (Fig. 2 F). α-NAA was generally more potent than IAA in inhibiting lateral bud outgrowth. β-NAA, the inactive auxin
258
Cline—Exogenous Auxin Effects in Decapitated Shoots
Bud length (mm)
30
Decap control
A
B
Decap control
20 15
20
IAA –4 10 M
10 0.1% IAA
10
5
–5 α-NAA 10 M
1% IAA 1
2
3
20
Decap control
Bud length (mm)
C
10
16
–6
M
IAA
12 8
10
–5
IAA
4
10
–4
IAA
10
–3
IAA
25
12
2
Intact
3
D
4
Decap control
8
1
Bud length (mm)
1
4
2
M
4 M M
0.1% IAA Intact control 1
3
2
3
Decap control
30 Decap control
E
4
F
20 20 15
1% IAA
10
10
0.1% IAA
5
1% IAA Intact 1
2
3
4
Day
Intact control 1
2
3
4
Day
F. 2. Auxin repression of lateral bud outgrowth over 3 or 4 d following decapitation. A, Vicia faba (growth room, n ¯ 4). B, Helianthus annuus (growth room, n ¯ 5). C, Ipomoea nil (growth room, n ¯ 3–4). D, Pisum satium (dwarf ) (growth room, n ¯ 5–6). E, Pisum satium (normal) (growth room, n ¯ 7). F, Phaseolus ulgaris (greenhouse, n ¯ 4). Decap, Decapitation. Vertical lines represent³s.d.
analogue, which was tested on seven of the ten plant types, usually had little or no effect on apical dominance in most experiments. With some species [Vicia faba ; Pisum (Little Marvel), Helianthus annuus], the lack of effect of auxin in aqueous solution may have been due to a penetration or metabolism problem. Auxin in lanolin was always effective. In the following section are descriptions of apical dominance in the ten plant types, and their responses to decapitation and to auxin treatments under growth room and greenhouse conditions. The term ‘ strong apical dominance ’ as used here signifies little or no lateral bud outgrowth in intact plants. ‘ Medium ’ signifies some bud growth and ‘ weak ’ indicates substantial and continuing lateral bud growth in intact plants.
Vicia faba (Broad Windsor Bean) The intact plant had weak to medium apical dominance. Lateral buds on nearly half of the greenhouse plants and on a quarter of the growth room plants had grown out before the start of the auxin treatment. These plants had to be excluded. Decapitation above the third node resulted, most often, in the outgrowth of the lateral buds at the basal node but sometimes at the upper nodes (Fig. 1 A). All three lateral buds were repressed by the auxin treatment with the top (third) bud (closest to the auxin application) inhibited the most. Consistent repressive effects on bud growth were found with 1 % IAA in lanolin and partially at 0±1 % (Fig. 2 A). There was some swelling and abnormal curving of
Cline—Exogenous Auxin Effects in Decapitated Shoots T 1. Outgrowth of lateral buds (mm³s.d.) oer 3 or 4 d following decapitation and treatment of shoot stump with IAA, α-NAA, β-NAA in aqueous solution or IAA in lanolin. Decap., decapitation Days after decapitation 0
1
2
Vicia faba, growth room, n ¯ 4 Intact 0 0 0 Decap. control 0 1³2 8³3 IAA 10−% 0 5³5 14³10 α-NAA 10−% 0 1³2 5³6 Helianthus annuus, growth room, n ¯ 4–5 Intact 0 0 0 Decap. control 0 1³1 3³2 IAA 0±1 % lanolin 0 0 0 IAA 0±5 % lanolin 0 0 0 Ipomoea nil, growth room, n ¯ 10 Decap. control 1³0 3³1 8³1 IAA 0±1 % lanolin 2³0 2³1 3³2 IAA 1 % lanolin 1³0 2³0 2³1 Pisum satium (dwarf ), greenhouse, n ¯ 4 Intact 3³1 3³1 3³1 Decap. Control 2³1 3³1 5³1 3³1 4³1 5³2 IAA 10−% α-NAA 10−% 2³1 2³1 2³1 β-NAA 10−% 2³1 3³1 4³2 Phaseolus ulgaris, growth room, n ¯ 4 Intact 3³0 4³1 4³0 Decap. control 4³0 7³2 11³3 IAA 10−% 3³1 5³2 8³2 α-NAA 10−% 3³0 4³1 6³2 β-NAA 10−% 3³1 5³2 9³2
3
4
0 21³4 27³13 16³11
0 35³4 43³14 23³14
0 9³4 0 0
0 20³7 1³2 0
25³5 8³6 4³2 3³1 8³3 8³2 3³1 5³2
4³1 13³6 11³3 3³1 7³2
4³1 45³11 23³8 11³5 25³9
259
solution (10−& to 10−$ , Fig. 2 C) and IAA in lanolin (0±1 and 1 %, Table 1) significantly inhibited lateral bud outgrowth following decapitation with no toxic auxin symptoms observed except at 10−$ IAA. As Fig. 2 C indicates, although there was no effect at 10−' IAA, there was increasing repression of axillary bud outgrowth from 10−& to 10−$ . Pisum sativum [Little Marel (dwarf ) and Thomas Laxton (normal) Pea] The intact plants had moderate apical dominance. Following decapitation above one of the higher nodes (usually the fifth), many of the buds at the lower nodes would simultaneously grow out to considerable lengths (Fig. 1 D, E). In the dwarf pea, the bud at the highest node usually grew out most rapidly followed by the buds at the second and third highest nodes (Fig. 1 D). In the normal pea, it was the bud at the second or third node from the base which grew out more rapidly followed by those of the fourth and fifth nodes, respectively (Fig. 1 E). Auxin did significantly restore apical dominance in both the dwarf and the normal pea plants following decapitation. Significant inhibition of bud outgrowth was obtained in dwarf pea with 10−% α-NAA but not with 10−% IAA in aqueous solution (Table 1). IAA in lanolin at 0±1 % was effective in both dwarf (Fig. 2 D) and normal (Fig. 2 E) types although some swelling in the stem stump and abnormal stem curvature were observed in the normal peas and in one experiment with the dwarf peas. Phaseolus vulgaris (Blue Lake Bean)
stems which accompanied the auxin treatment. Neither IAA nor α-NAA in aqueous solution were very effective in repressing axillary bud outgrowth (Table 1).
The intact plant had very strong apical dominance with absolutely no sprouting of lateral buds. Outgrowth of buds in the greenhouse was not observed until nearly a week following decapitation above the first node, whereas it was observed in the growth room within 1 or 2 d (Fig. 1 B). Release from apical dominance could be inhibited by 10−& α-NAA (Fig. 2 B) or by 0±1 % IAA in lanolin (Table 1). IAA in aqueous solution was ineffective at 10−% (Fig. 2 B). No toxic auxin effects were observed.
The intact plant had weak to medium apical dominance. Its apical dominance has been described as ‘ incomplete ’ (Tamas, 1987). The axillary buds subtended by the primary leaves (the first node, above which we decapitated) were more inhibited than the buds at the second node subtended by the first trifoliate leaf. The bean plant grows rapidly, the internodes are large and the plant is easy to work with. Decapitation definitely accelerated lateral bud outgrowth (Fig. 1 F) but the inhibitory effect of IAA (1 % in lanolin, Fig. 2 F and 10−% in solution, Table 1) applied to the stem stump was only partial. α-NAA at 10−% had a stronger effect (Table 1). No toxic effects of auxin were observed except for a little swelling and bleaching of the stem stumps upon which the auxin was directly applied.
Ipomoea nil (Japanese Morning Glory)
Coleus
The intact plant had medium to strong apical dominance. There was no axillary bud outgrowth in the growth room or in winter-grown greenhouse plants. There was slight outgrowth of cotyledonary buds of greenhouse plants in the spring (data not shown). Decapitation above the second node nearly always resulted in the rapid outgrowth of the bud at the node (Fig. 1 C). The bud at the first node also often exhibited a small amount of temporary growth. Both IAA and α-NAA (data not shown) in aqueous
The intact Coleus plant had weak apical dominance. It had short internodes, grew slowly and was bushy. The basal branches were of greater length than the higher branches because of their greater age. These plants were propagated from cuttings. Many intact plants had to be excluded from the study because of extensive axillary bud development beginning at the basal nodes. Only plants with very small lateral buds (! 3–4 mm) were selected. At the time of decapitation, the lateral buds of the lowest of the top three
Helianthus annuus (Mammoth Grey-striped Sunflower)
260
Cline—Exogenous Auxin Effects in Decapitated Shoots
F. 3. Upper left and right, VNF8 (wildtype) and dgt (mutant) tomatoes about a week following decapitation showing lateral bud outgrowth. The stem stumps have been treated with lanolin. Lower left, Arabidopsis, 1 week following decapitation ; A, decapitation above the first node ; B, intact control ; C, decapitation above first node with 1 % IAA on stump of main stem ; Lower right, Coleus showing the branch-promoting effect of greenhouse high light (B) as compared with indoor low light (A).
nodes were removed. Decapitation above the third node of these greenhouse plants had only a small effect on accelerating bud outgrowth. Auxin (0±1 % and 1 % in lanolin) application to the stem stumps had no repressing effect on lateral bud outgrowth of greenhouse plants (Fig. 5). Lycopersicon esculentum [VNF8 (normal ) and dgt (mutant) tomato] The intact plants of both dgt and VNF8 had medium apical dominance with little or no lateral branching. Neither the dgt nor the VNF8 in our growth room or greenhouse conditions were categorized as having reduced apical dominance or as being bushy. In a few experiments, some
lateral bud development was noticed in intact plants. The VNF8 plants were larger than the dgt plants. The shoot of the young dgt seedling initially grows more or less vertically. After several months, the shoot gradually assumes a horizontal orientation. The seedling shoots of dgt, although not yet at horizontal growth stage, were floppy and hence were staked in an upright position for convenience in auxin treatment. Since horizontal positioning tends to weaken apical dominance (Prasad and Cline, 1985 b), such staking, if anything, would have a countering effect to anomalous bud outgrowth. Following decapitation, which was usually carried out above the fifth node, several of the lateral buds on any given dgt or VNF8 plant vigorously sprouted more or less simultaneously (Fig. 3). This most often occurred at the
261
Cline—Exogenous Auxin Effects in Decapitated Shoots B
Bud length (mm)
A
β-NAA 10–5M
16
Decap control
12
15
IAA
Decap control
–5
10
8
M
α-NAANA A10–5M Intact
4
1
3
2
10 5
4
1
α-NAA 10–5M IAA 10–4M α-NAA 10–4M 3 Intact 2
D
C Bud length (mm)
IAA 10–5M
20
20
Decap control
25
Decap control
15 IAA 1%
20
0.1% IAA 10
IAA 0.1% 10
5
0.5% IAA 1% IAA
Intact 2
4 Day
6
2
4 Day
6
F. 4. Auxin repression of tomato [VNF8 (A, C) and dgt (B, D)] lateral bud outgrowth in growth room following decapitation. Auxin in aqueous solution, A, B (n ¯ 4–5, 4). Auxin in lanolin, C, D (n ¯ 3–4, 4). Decap, decapitation. Vertical lines represent³s.d.
Bud length (mm)
10
1% IAA Decap control
8
6
Intact control
4 2
4 Day
6
F. 5. Lateral bud growth in Coleus blumei over 6 d following decapitation and stem stump treatment with 1 % IAA (greenhouse, n ¯ 9–10). Vertical lines represent ³s.d.
upper-most nodes but sometimes at one of the lower nodes. The points on the curves in the graphs represent the mean length of the highest lateral bud on each plant. The young axillary buds of tomato were difficult to observe and to measure because of their small size and narrow shape. The treatment with exogenous auxin [10−& α-NAA and}or 10−% IAA in aqueous solution (Fig. 4 A, B) and 0±1 % IAA in lanolin, Fig. 4 C, D] did, for the most part, significantly inhibit axillary bud outgrowth in both plant types. IAA in aqueous solution had variable effects on VNF8 but α-NAA, down to 10−& , had consistent repressive effects on bud outgrowth (Fig. 4 A). β-NAA had
no inhibitory effect. In several instances it appeared even to promote bud growth. IAA at 10−% and 10−& α-NAA generally restored apical dominance in dgt (Fig. 4 B and data not shown). IAA in lanolin (both at 0±1 and 1 %) was quite effective in repressing bud outgrowth in both VNF8 and dgt (Fig. 4 C, D). Hence, no essential difference in sensitivity could be positively detected between dgt and VNF8 in the response to auxin in repressing lateral bud outgrowth except for the toxic auxin effect mentioned previously for VNF8. Arabidopsis thaliana Arabidopsis is a facultative long-day plant with weak apical dominance consisting of a rosette from which arises a single branching floral shoot or inflorescence followed by the generation of several surrounding axillary or secondary inflorescences also arising from the rosette. The measurement of changes in apical dominance status is complex because of the two types of branching, lateral shoots developing from the main shoot and axillary inflorescences developing from the rosette and the rapidity with which they occur. The lateral buds (shoots) of the main shoot begin emerging almost as soon as the main shoot begins to bolt. In the many mutants and strains of Arabidopsis now available, there exists a wide range of branching habits. The CS 1072 Chi-O strain used in the present study has stronger apical dominance than most Arabidopsis types inasmuch as it generally lacks axillary inflorescences. Decapitation of the main floral shoot resulted in increased
262
Cline—Exogenous Auxin Effects in Decapitated Shoots
T 2. Effect of 1 % IAA on lanolin on Arabidopsis. Outgrowth of first lateral bud (mm³s.d.) and axillary inflorescence 1 week following decapitation of the main shoot and treatment of the shoot stump with 1 % IAA in lanolin. Decapitation was either 3–5 mm aboe the first node or 3–5 mm aboe the base of the main shoot (below the first node) as indicated. Axillary inflorescence deelopment is gien as a percentage of the plants sprouting one or more axillary inflorescences. *Main shoot length Decap above first node Decap below first node Intact plant
Control
1 % IAA
Control
1 % IAA
Decap above first node Decap below first node Intact plant
Control
Growth room n First Lateral Bud Axial Inflorescences
1 % IAA
Control
1 % IAA
Greenhouse
7
7
6
7
7
5
5
5
5
5
235³21* 0
145³24 71 %
132³29 33 %
— 100 %
— 100 %
124³57* 0
87³29 60 %
78³52 60 %
— 100 %
— 100 %
T 3. Effect of 10−% M IAA spray on Arabidopsis. Outgrowth of axillary inflorescences 1 week following decapitation of the main shoot 3–5 mm aboe the base of the plant which was sprayed eery other day beginning 15 d before decapitation with 10−% M IAA. Axillary inflorescence deelopment is gien as a percentage of plants sprouting one or more axillary inflorescences
Growth room Intact control Intact IAA 10−% Decap. control Decap. IAA 10−% Greenhouse Intact control Intact IAA 10−% Decap. control Decap. IAA 10−%
n
Main shoot
Axillary inflorescence length (mm³s.d.)
Axillary inflorescence percentage
7 7 7 8
235³21 214³20 — —
— 38³23 71³43 81³41
0 38 100 100
5 9 5 10
124³57 109³46 — —
— 0 25³31 21³24
0 0 100 60
outgrowth of lateral (buds) shoots from the main shoot as well as the subsequent generation of axillary inflorescences from the base of the plant (Fig. 3). If the point of decapitation of the main shoot was above the lowest lateral emerging shoot (bud), then that shoot grew out to a much greater extent than in the intact plant and took over as the main shoot. If the decapitation of the main shoot was below the lowest lateral emerging shoot (bud), then several axillary inflorescences grew out from the base of the plant. The greenhouse plants exhibited somewhat stronger apical dominance with respect to axillary inflorescence outgrowth than did the growth room plants. In essentially no plant did auxin, either as 1 % IAA in lanolin applied to the decapitated stump of main shoot or as 10−% IAA spray applied every other day to the whole plant, have any effect in restoring apical dominance, i.e., in inhibiting lateral branching of the main shoot or the outgrowth of the axillary inflorescences (Fig. 3, Tables 2 and 3). In the case of decapitation above the first node, there appeared to be some auxin repression in the percentage of axillary inflorescence development in the growth room (Table 2) but this was not confirmed in the greenhouse nor in the more direct test when IAA was applied following decapitation below the first node.
Attempts to restore apical dominance with auxin spray applied to the small and extremely bushy trp 1-1 tryptophanrequiring auxotrophic mutant (suggestive of an auxin defect, Last et al., 1992) were also unsuccessful (data not shown). Influence of light Past observation (Hosokawa et al., 1990) indicated that when Ipomoea nil was propagated in growth rooms under a mixture of cool white fluorescent and incandescent sources (irradiance : 25–450 µmol m−# s−"), a 30-d-old plant was characteristically tall (40–70 cm) without branching. However, when the plant was grown outdoors (irradiance : up to 6900 µmol m−# s−"), the plant was short, often with such a proliferation of branching that the study of auxin effects on bud outgrowth was impossible to carry out since most buds had substantially elongated at a very early developmental stage. Therefore, Ipomoea plants were grown indoors until the age of 14 d, at which time they were moved outdoors (still in pots), decapitated and stem stumps treated with auxin in lanolin for 4 d. There appeared to be little or no difference in sensitivity in auxin repression of axillary bud outgrowth between indoor plants at low irradiance and indoor plants moved outdoors at high irradiance at the time
263
Cline—Exogenous Auxin Effects in Decapitated Shoots T 4. Outgrowth of lateral buds (mm³s.d.) of Ipomoea nil plants oer 3 or 4 d following decapitation and treatment of shoot stump with IAA in lanolin either in growth room (25–450 µmol m−# s−") or outdoors (up to 6900 µmol m−# s−")
±32.1 100
200
±37.6
1
2
3
4
1³0 2³0 1³0
3³1 2³1 2³0
8³1 3³2 2³1
25³5 8³6 4³2
— — —
2³1 3³0 3³1
3³1 3³1 8³1
6³1 3³1 3³1
12³3 6³1 3³1
23³6 11³3 5³2
75 Weight (g)
Growth room, n ¯ 10 Decap. control IAA 0±1 % IAA 1 % Outdoors, n ¯ 4–8 Decap. control IAA 0±1 % IAA 1 %
0
50
100
±3.7
0
50
Weight (g)
0
Height (cm)
F. 7. Bar graphs comparing total fresh weights and heights of Ipomoea nil plants grown outside in heavy shade (+) and in the open (*). n ¯ 8–11.
that they were hardly recognizable as Ipomoea nil plants. Both the lateral bud length and the total fresh weights of the shaded plants were very much reduced as compared to those of unshaded plants (Figs 6 and 7). At the fifth, sixth and
±2.6
±22.6
±16.7
±35.3
±22.8
25
of decapitation and auxin treatment (Table 4). Hence, there was no immediate effect of high irradiance on exogenous auxin restoration of apical dominance in decapitated plants. When 16}17-d-old Ipomoea plants in pots were transplanted to outdoor plots under heavy shading (irradiance : up to 210 µmol m−# s−"), they appeared, after 19–34 d, very similar in overall form and height (i.e. tall without branching) to indoor plants of approximately the same age (no data given). Whereas those plants transplanted to outdoor plots under full sunlight had, in time, such an extensive proliferation of lower branches together with reduced height
50
150
Height (cm)
Days following decapitation
±20.5 ±17.9
30
±25.5
±27.3
Bud length (cm)
40
±0.3 ±0.1
±0.2 ±0
13
±0.4 ±0.06
±2.1 ±0.2
±2.6
9 10 11 12 Node number
±0.9 ±0.06
8
±0.7 ±0.7
7
±0.9 ±0.4
6
±0.2
±3.5 ±1.3
±24.2
5
±1.4 ±0.2
4
±0.4
3
±0.3
2
±15.7
±0.5
1
±4.9
±
0
±0.1
10
±2.5
±18.2
±9.6
±1.8
20
14
15
16
17
18
19
20
F. 6. Bar graphs comparing lateral bud length (cm³s.d.) of Ipomoea nil plants grown outside in heavy shade (+) and in the open (*) at different nodes numbering up from the base of the shoot. n ¯ 8–11.
264
Cline—Exogenous Auxin Effects in Decapitated Shoots
Length (mm)
Decap control 12 0.1% IAA 8 1% IAA 4 Intact 2
4
6 Day
8
10
F. 8.Partial auxin repression of lateral bud outgrowth in Coleus at reduced irradiance (25–30 µmol m−# s−") following decapitation over 10 d. Vertical lines represent³s.d. n ¯ 10.
seventh nodes of the shade plants (Fig. 6), there was some anomalous branching due to the fact that the shoot tips had impinged against the overhanging shade screen, had bent over and had released the buds at those nodes. Floral buds were found to be much more prevalent in the sun plants than in the shade plants. Since the spectral quality of sunlight in both groups of plants presumably was the same (i.e., the plastic shade screens would not be expected to cause any such changes), the pronounced weakening of apical dominance as exhibited by the proliferation of branching in the sun plants could be attributed to increases in irradiance (fluence) and not to changes in light quality. When Coleus plants were grown indoors at greatly reduced light levels (irradiance : 25–30 µmol m−# s−"), branching in intact plants was much decreased compared to that of greenhouse plants (Fig. 3) but increased lateral bud outgrowth did occur at the highest node following decapitation. Definite but partial inhibitory effects of 0±1 and 1 % IAA in lanolin were detected in one out of five experiments carried out at the reduced light level (Fig. 8). DISCUSSION The results of this study corroborated the Thimann-Skoog (1933) experiment, that exogenously applied auxin to the stem stump of a decapitated plant does restore apical dominance for many plants under most conditions. This is a classic example wherein the results one obtains depend upon the particular plant system used and upon the conditions employed. There were exceptions where the Thimann-Skoog experiment did not work. In those plants with weak apical dominance such as Coleus or Arabidopsis, auxin had little or no effect on repressing bud outgrowth in decapitated plants. Hence, it is understandable why Jacobs et al. (1959) were not able to detect repressive auxin effects on the release apical dominance with greenhouse-grown Coleus plants. In plants with incomplete apical dominance such as Phaseolus, auxin had only a partially inhibiting effect on axillary bud growth. Hence, it is understandable why Hillman (1984) questioned the role of auxin in apical dominance in this species. Certain environmental conditions can proliferate branching to an extent which makes it impossible to carry out the Thimann-Skoog experiment.
Increased irradiance levels can greatly weaken apical dominance (Gregory and Veal, 1957 ; Anderson, 1976). High wind velocity (via thigmomorphic effects) or alterations in the direction of gravity can also release apical dominance (Prasad and Cline, 1985 a, b). Although Thimann, Sachs and Mathur (1971) reported auxin in aqueous solution to be more effective than in lanolin with Coleus and Pisum, in this study it was found otherwise. This might have been due to different methods used for applying the aqueous solution. Thimann employed a fine piece of tubing over the end of the stem for continuous flow whereas two doses of approximately 150 µl were applied daily by pipette to a cotton swab taped to the end of the stem in the present study (Fig. 1 C). The auxin concentration (1400–1700 units) which Thimann and Skoog (1933) used to completely inhibit bud outgrowth in Vicia faba is presumed to be equivalent to approximately 1 % in lanolin or to 6¬10−# in aqueous solution (Stafstrom, 1993). In retrospect, Vicia faba, the plant system which Thimann and Skoog (1933) used in their classic study, probably was not the best for studying apical dominance. First, it has weak apical dominance and many plants had to be excluded in the present study because they were already branching before the time designated for decapitation, particularly in the greenhouse plants. Second, following decapitation, it was usually the bud at the basal node instead of the highest node which sprouted first. This complicated the situation because of the greater distance between the site of auxin application (the decapitated stem stump) and the potentially active axillary buds than when the highest bud sprouted first. It was observed in some plants where several Vicia buds sprouted simultaneously that the higher buds located closer to the site of auxin application were repressed the most. This suggested that the auxin concentration probably decreased gradually during transport away from its original source. However, V. faba was sensitive to auxin and its apical dominance was restored by auxin application. Those concentrations of exogenous auxin (0±1–1 % IAA) which inhibited apical dominance release also exhibited toxic auxin effects. The Thimann-Skoog experiment is carried out with the greatest facility in a fast growing plant with large internodes, moderate to strong apical dominance, easily observed axillary buds, and where only the highest lateral bud grows out following decapitation. Of the ten plants tested, Ipomoea nil conformed most closely to the expectations of the Thimann-Skoog experiment, followed by Helianthus annuus which, however, was somewhat hindered by its slow growth in our greenhouse conditions. Pea (Thomas Laxton) worked well in many ways except for the uncertainty in which lateral bud would sprout following decapitation. It was usual for several buds to sprout simultaneously, which complicated both the execution of the experiment and the interpretation of the data. In Arabidopsis, the complete lack of effect of auxin on restoring apical dominance following decapitation may be due in part to the fact that the upright shoot of Arabidopsis is a floral shoot. All experiments done with other species in
Cline—Exogenous Auxin Effects in Decapitated Shoots this study were carried out with vegetative shoots, or at least shoots without the presence of visible floral buds. For reasons which are not entirely clear, aging and reproduction often have a weakening effect on apical dominance (Tamas, 1987). dgt mutant tomato If one accepts the auxin inhibition hypothesis as the explanation for the mechanism of action of apical dominance then the lack of outgrowth of the axillary buds is presumed to be due to repression imposed by apicallyproduced}basipetally transported auxin in the shoot. A mutant such as the dgt tomato which has been demonstrated to be insensitive to auxin-induced hypocotyl elongation and ethylene production (Kelly and Bradford, 1986) might also be expected to be insensitive to auxin repression of lateral bud outgrowth and, hence, would exhibit heavy branching. This was not observed in the present study in growth room or greenhouse plants up to an age of 4–5 weeks. It was noticed in older plants (8–10 weeks or more) that high soil fertilization and exposure of plants to open sunlight usually did promote axillary bud growth in both dgt and VNF8. Interpretation of data (not shown) which indicated insensitivity of dgt to the auxin transport inhibitors, TIBA (2,3,5-triiodobenzoic acid) and NPA (N-1 naphthylphthalamic acid), with respect to releasing apical dominance was unclear due to significant non-specific effects (abnormal stem and leaf growth) commonly observed in VNF8 in cases of lateral bud outgrowth. Auxin did inhibit lateral bud outgrowth in decapitated dgt plants following application to the stem stump. Hence, dgt is sensitive to auxin with respect to apical dominance. It has been suggested for apical dominance as well as for other responses to auxin that there may be separate auxin receptors (M. O. Kelly and M. G. Cline, unpub. res. ; Palm et al., 1991). Light When irradiance is high, as outdoors, the lateral buds of many species (except for those with very strong apical dominance such as sunflower) will begin to grow out and to proliferate, thus excluding the execution of the ThimannSkoog experiment which requires lateral buds to be in a repressed state before decapitation and auxin treatment. This proliferation of branching in outdoor-grown plants was observed in the present study of Ipomoea nil, a plant with moderate to strong apical dominance. From previous indoor tests, it was clear that once vigorous axillary bud growth had commenced, it was very difficult to repress, even with high concentrations of auxin (data not shown). It is also possible that other factors in the outdoor environment besides light may have a weakening effect on apical dominance. When the irradiance is low, the lateral buds of many species (except for those with very weak apical dominance such as Arabidopsis) will be in a repressed state, thus allowing for the execution of the Thimann–Skoog experiment with subsequent inhibition of lateral bud outgrowth. In Coleus, a plant with weak apical dominance
265
where exogenous auxin is known to have no repressive effect on lateral bud outgrowth following decapitation at high irradiance in greenhouse environment (Jacobs et al., 1959), marginal evidence was found in the present study to support Thimann’s contention (Thimann et al., 1971 ; Thimann, 1977) that auxin did inhibit bud growth when the irradiance level was reduced (Fig. 8). The fact that outdoor light control of apical dominance in Ipomoea could be manipulated wholly by the use of shade screens suggested that the response under the present conditions was dependent upon changes in irradiance and not in spectral differences. This result, while not excluding the interaction with indirect light quality effects or with auxin, is also consistent with the nutritional hypothesis of apical dominance via increased photosynthate availability for bud growth at high irradiance. The possibility that the plastic shade screens could affect light quality and, hence, other physiological processes cannot be ruled out. Light is one of many stimuli (e.g. gravity, CO , nutrients, # etc.) which often will promote axillary bud outgrowth (Hillman, 1984). The mechanism by which light induces such outgrowth is unknown. Field and Jackson (1975) point out some of the complexities involved in interpreting light effects on apical dominance. Gregory and Veale (1957) reported that auxin repression of axillary bud outgrowth increased with decreasing light in Linum particularly at low nitrogen levels. Thimann et al. (1971) suggested that high light may promote the synthesis of cytokinins which in turn may reverse the auxin effect. Accumulating evidence in the literature for an indirect role of auxin in apical dominance might also indicate the involvement of certain phytochromemediated processes, interaction with other hormones and second messengers or by decreasing sensitivity to auxin. CONCLUSION The results of this study confirm that the Thimann–Skoog experiment does work for most species and suggests a controlling role for auxin in apical dominance. Most likely the role is indirect, perhaps involving cytokinins (Bangerth, 1994 ; Sandberg et al., 1995). It is hoped that the present accelerating research involving both traditional physiological and molecular approaches with mutants will soon be able to resolve these problems and more fully elucidate this classic developmental phenomenon. A C K N O W L E D G E M E N TS Appreciation is expressed to Daniel Repicz for his diligent efforts with the statistical data and graphs, Annabelle Chern and Liang Shi for their competent assistance with the preliminary dgt tomato experiments, to the Livingston Seed Co. of Columbus, Ohio for their gracious donation of Vicia faba seeds and to the Ohio State University Arabidopsis Biological Resource Center for their generous help in the donation of seeds and propagation of seedlings. LITERATURE CITED Ali A, Fletcher RA. 1970. Hormonal regulation of apical dominance in soybeans. Canadian Journal of Botany 48 : 1989–1994.
266
Cline—Exogenous Auxin Effects in Decapitated Shoots
Anderson AS. 1976. Regulation of apical dominance by ethephon, irradiance and CO . Physiologia Plantarum 37 : 303–308. # Bangerth F. 1994. Response of cytokinin concentration in the xylem exudate of bean plants (Phaseolus ulgaris L.) to decapitation and auxin treatment and relationship of apical dominance. Planta 194 : 439–442. Beveridge CA, Ross JJ, Murfet IC. 1994. Branching mutant rms-2 in Pisum satium. Grafting studies and endogenous indole-3-acetic acid levels. Plant Physiology 104 : 953–959. Brown CL, McAlpine RG, Kormanik PP. 1967. Apical dominance and form in woody plants : a reappraisal. American Journal of Botany 54 : 153–162. Cline MG. 1991. Apical dominance. The Botanical Reiew 57 : 318–358. Cline MG. 1994. The role of hormones and apical dominance. New approaches to an old problem in plant development. Physiologia Plantarum 90 : 230–237. Estruch JJ, Chriqui D, Grossmann K, Spena A. 1991. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates EMBO Journal 10 : 2889–2895. Field RJ, Jackson DI. 1975. Light effects on apical dominance. Annals of Botany 39 : 369–374. Gregory FC, Veale JA. 1957. A reassessment of the problem of apical dominance. Symposia of Society of Experimental Biology 11 : 1–20. Hillman JR. 1984. Apical dominance. In : Wilkins M, ed. Adanced plant physiology. London : Pitman Publishing, 127–148. Hosokawa Z, Shi, L, Prasad TK, Cline MG. 1990. Apical dominance control in Ipomoea nil : the influence of the shoot apex, leaves and stem. Annals of Botany 65 : 547–556. Jacobs WP, Danielson J, Hurst V, Adams P. 1959. What substance normally controls a given biological process ? II. The relation of auxin to apical dominance. Deelopmental Biology 1 : 534–554. Kelly MO, Bradford KJ. 1986. Insensitivity of the diageotropica tomato mutant to auxin. Plant Physiology 82 : 713–717. Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffman NL. 1987. The effects of over production of two Agrobacterium tumefaciens TDNA auxin biosynthetic gene products in transgenic petunia plants. Genes and Deelopment 1 : 86–96. Klee HJ, Romano C-P. 1994. The roles of phytohormones in development as studied in transgenic plants. Critical Reiews in Plant Sciences 13 : 311–324. Last RL, Borczak JL, Pruitt KD, Radwanski ER, Rose AB. 1992. The genetics of tryptophan biosynthesis in Arabidopsis. Journal of Experimental Botany Supplement 43 : 18. Lincoln C, Britton J, Estelle M. 1990. Growth and development of the axr1 mutants of Arabidopsis. The Plant Cell 2 : 1071–1080. McIntyre GI. 1977. The role of nutrition in apical dominance. In : Jennings DH, ed. Integration of actiity in the higher plants. Symposium of Society for Experimental Biology, Cambridge : Cambridge University Press, 251–273. Martin GC. 1987. Apical dominance. HortScience 22 : 824–833. Palm K, Hesse T, Moore I, Campos N, Feldwisch J, Garbers C, Hesse F, Snell J. 1991. Hormonal modulation of plant growth : the role of auxin perception. Mechanisms of Deelopment 33 : 97–106. Prasad TK, Cline MG. 1985 a. Mechanical perturbation-induced ethylene releases apical dominance in Pharbitis nil by restricting shoot growth. Plant Science 41 : 217–222.
Prasad TK, Cline MG. 1985 b. Gravistimulus direction, ethylene production and shoot elongation in the release of apical dominance in Pharbitis nil. Journal of Experimental Botany 36 : 1969–1975. Romano C, Cooper ML, Klee HJ. 1993. Uncoupling auxin and ethylene effects in transgenic tobacco and Arabidopsis plants. Plant Cell 5 : 181–189. Russell W, Thimann KV. 1988. The second messenger in apical dominance controlled by auxin. In : Phares RP, Hood SB, eds. Plant growth substances, 1988. New York : Springer-Verlag, 419–427. Sachs T, Thimann KV. 1967. The role of auxins and cytokinins in the release of buds from dominance. American Journal of Botany 54 : 136–144. Sandberg G, Sitbon F, Eklof S, Edlund A, Astot C, Blackwell J, Moritz T, Sundberg B, Olsson O. 1995. Regulation of auxin and cytokinin turnover in plants. 15th International Conference on Plant Growth Substances. Minneapolis. Program-Abstracts 015. Shein T, Jackson DI. 1972. Interaction between hormones, light and nutrition on extension of lateral buds in Phaseolus ulgaris L. Annals of Botany 36 : 791–800. Shen WH, Davioud E, David C, Barbier-Brygoo H, Tempe! J, Guern J. 1990. High sensitivity to auxin is a common feature of hairy root. Plant Physiology 94 : 554–560. Stafstrom J. 1993. Axillary bud development in pea : apical dominance, growth cycles, hormonal regulation and plant architecture. In : Amasino RM, ed. Cellular communication in plants. New York : Plenum Publishing Corp., 75–86. Stafstrom J. 1995. Developmental potential of shoot buds.In : Gartner BL, ed. Plant stems : Physiology and functional morphology. San Diego : Academic Press, 257–279. Tamas IA. 1987. Hormonal regulation of apical dominance. In : Davies PJ, ed. Plant hormones and their role in plant growth and deelopment. Dordrecht : Martinus Nijhoff Publishers, 393–410. Tamas IA. 1995. Hormonal regulation of apical dominance. In : Davies PJ, ed. Plant hormones, physiology, biochemistry and molecular biology 2nd edn. Boston : Kluwer Academic Publishers, 572–597. Tepfer D. 1984. Transformation of several species of higher plants by Agrobacterium rhizogenes : sexual transmission of the transformed genotype and phenotype. Cell 37 : 959–967. Thimann KV. 1937. On the nature of inhibitions caused by auxin. American Journal of Botany 24 : 407–412 Thimann KV. 1977. Hormone action is the whole life of plants. Amherst : University of Massachusetts Press. Thimann KV, Sachs T, Mathur KN. 1971. The mechanism of apical dominance in Coleus. Physiologia Plantarum 24 : 68–72. Thimann KV, Skoog F. 1933. Studies on the growth hormones of plants. III. The inhibition action of growth substance on bud development. Proceedings of the National Academy of Science, USA 19 : 714–716. Wareing PF, Phillips IDJ. 1981. The control of growth and differentiation in plants, 3rd edn. New York : Pergamon Press. White JC. 1976. Correlative inhibition of lateral bud growth in Phaseolus ulgaris L. Effect of application of indol-3yl-acetic acid to decapitated plants. Annals of Botany 40 : 521–529.
Exogenous Auxin Effects on Lateral Bud Outgrowth in Decapitated Shoots M O R R I S G. C L I N E The Ohio State Uniersity, Department of Plant Biology, Columbus, Ohio 43210, USA Received : 5 July 1995
Accepted : 11 March 1996
In 1933 Thimann and Skoog demonstrated exogenous auxin repression of lateral bud outgrowth in decapitated shoots of Vicia faba. This evidence has given strong support for a role of auxin in apical dominance. Most, but not all, investigators have confirmed Thimann and Skoog’s results. In the present study, auxin treatments were carried out on ten different species or plant types, many of which were treated with auxin in different forms, media and under different light conditions. The Thimann–Skoog experiment did work for most species (i.e. exogenous auxin did repress bud outgrowth) including the dgt tomato mutant which is known to be insensitive to auxin in certain responses. Toxic auxin symptoms were observed in some but not all species. The Thimann–Skoog experiment did not work for greenhouse-grown Coleus or for Arabidopsis. Light was shown to reduce apical dominance in Coleus and Ipomoea nil. # 1996 Annals of Botany Company Key words : Apical dominance, lateral bud outgrowth, axillary bud, auxin, IAA, decapitation, Vicia faba, Ipomoea nil, Pisum satium, Phaseolus ulgaris, Lycopersion esculentum, dgt, Coleus blumei, Arabidopsis thaliana, Helianthus annuus, Thimann–Skoog.
INTRODUCTION Apically derived auxin in shoots is generally thought to control apical dominance either directly via entry into lateral buds with subsequent repression of outgrowth or indirectly via some other mechanism, i.e. activation of a second inhibitor messenger, auxin-cytokinin ratio, secondary growth substances, nutrient diversion, etc. (Martin, 1987 ; Cline, 1991 ; Bangerth, 1994 ; Stafstrom, 1995 ; Tamas, 1995). As studies of control mechanisms of developmental responses in plants have progressed, particularly with the use of molecular and genetic approaches (Cline, 1994), there has been increased interest directed towards the role of auxin in apical dominance, and with the degree of branching serving as a possible indicator of auxin activity (Tepfer, 1984 ; Lincoln, Britton and Estelle, 1990 ; Shen et al., 1990 ; Estruch et al., 1991 ; Klee and Romano, 1994). Because of this increased attention concerning auxin in apical dominance and inasmuch as the precise mechanism of auxin action in apical dominance has yet to be elucidated, a closer scrutiny of the evidence supporting auxin involvement in the control of lateral bud outgrowth is needed. The strongest evidence supporting either a direct or an indirect role for apically-derived auxin in controlling apical dominance comes from the classical Thimann–Skoog experiment with Vicia faba (Thimann and Skoog, 1933). They demonstrated that auxin applied in agar blocks every 6 h to the stump of a decapitated Vicia faba stem inhibited the outgrowth of lateral buds situated lower on the stem. Thimann (1937) initially proposed the direct inhibition hypothesis which presumes that auxin enters the lateral bud and directly inhibits its outgrowth. Subsequently, he modified his views to include indirect auxin action via involvement with cytokinins (Sachs and Thimann, 1967) or 0305-7364}96}08025512 $18.00}0
ethylene (Russell and Thimann, 1988) as well as effects of light (Thimann, 1977). While many investigators have substantiated and extended the results of the Thimann–Skoog experiment (i.e. exogenous auxin repression of lateral bud outgrowth in decapitated shoots), others have reported anomalous effects and there have been questions about the direct role of auxin in apical dominance (Jacobs et al., 1959 ; Brown, McAlpine and Kormanik, 1967 ; Ali and Fletcher, 1970 ; Shein and Jackson, 1972 ; Hillman, 1984). Wareing and Phillips (1981) stated ‘… it is now generally considered that auxin does not exert its inhibitory effect on lateral buds in such a direct manner as that originally proposed by Thimann ’. Some workers (Gregory and Veale, 1957 ; McIntyre, 1977) have proposed nutrition to be the most important controlling factor in apical dominance. To date, no one has reported evidence of auxin restoration of apical dominance in Arabidopsis via the Thimann–Skoog experiment. The question has also been raised as to how the terminal bud can continue to grow while simultaneously generating inhibitory concentrations of auxin to the outgrowth of lateral buds below. Klee et al. (1987) have found that overproduction of auxin in petunia via transformation with IAA biosynthetic genes of Agrobacterium eliminates branching. With a similar transformed system, Romano, Cooper and Klee (1993) have reported an increase in apical dominance in Arabidopsis as measured by a reduction in fresh weight of secondary inflorescences. However, this auxin overproduction occurs in every cell of the organism rather than being localized in buds of adjacent nodes as might occur in a natural system. In addition, Lincoln et al. (1990) reported that ‘ total number of inflorescences arising from the rosette does not differ greatly between axr1 [the bushy auxin-resistant mutant] and wildtype ’ even though # 1996 Annals of Botany Company
256
Cline—Exogenous Auxin Effects in Decapitated Shoots
F. 1. Lateral bud outgrowth approximately 1 week following decapitation. A, Vicia faba L. (Broad Windsor Bean). B, Helianthus annuus L. (Mammoth Grey-striped Sunflower). C, Ipomoea nil L. Roth (Japanese Morning Glory). D, Pisum satium L. [Little Marvel Pea (dwarf )], and E, Thomas Laxton Pea (normal). F, Phaseolus ulgaris L. (Blue Lake Bean). The stem stumps of all decapitated plants shown here were treated with lanolin except for Japanese Morning Glory (C) which has a taped cotton swab for treatment with aqueous solution.
the number of lateral branches did greatly increase in the former. Beveridge, Ross and Murfet (1994) reported that grafting of non-branching wildtype stocks to the branching rms-2 pea mutant scions did not normalize endogenous IAA levels even though it did inhibit branching.
White (1976) stated that ‘… discrepancies in the results of different workers using apparently similar methods of application of IAA to decapitated plants of a single variety of Phaseolus obviously require some explanation …’. He carried out experiments, focusing on this single species with
Cline—Exogenous Auxin Effects in Decapitated Shoots variations in plant age, concentrations and quantity of IAA, type of lanolin, region of application and time of year. His results showed that the effectiveness of IAA applied did vary significantly with those conditions. He also found that in many instances, IAA did completely replace the main shoot with respect to correlative inhibition of lateral bud growth. The objective of the present study was similar to that of White’s except that instead of analysing a single species, the focus here was to compare responses to exogenous IAA in repressing lateral bud outgrowth in ten different species or plant types, including the mutant dgt tomato which is known to be insensitive to auxin promotion of certain responses, and Arabidopsis thaliana. In addition, the effects of auxin in different concentrations (both aqueous and in lanolin), forms (IAA, α-NAA and β-NAA) and under different light conditions were studied in a number of these species.
MATERIALS AND METHODS Seeds of Vicia faba L. (Broad Windsor Bean), Helianthus annuus L. (Mammoth Grey-striped Sunflower), Ipomoea nil L. Roth, strain violet (syn. Pharbitis nil ) (Japanese Morning Glory), Pisum satium L. [Little Marvel (dwarf ) and Thomas Laxton (normal) Pea], Phaseolus ulgaris L. (Blue Lake Bean) and Lycopersicon esculentum, Mill., strain VNF8 and the mutant diageotropica (dgt) tomato were germinated in Pro-mix, a general purpose peat-vermiculite growing medium. The seeds of Ipomoea were scarified in concentrated sulphuric acid for 40 min and soaked overnight in running water and germinated in Petri dishes before planting. Coleus blumei Benth. was propagated from stem cuttings. All the plants except the normal pea were grown in a greenhouse (16–32 °C) during the winter}spring of 1993–94 and the spring}summer of 1995 with supplementary General Electric 400 watt mercury vapour lamps (total irradiance : up to 3300 µmol m−# s−"). All the above plants, except Coleus, were also grown in growth rooms (27–29 °C) under continuous light (General Electric, Power Groove cool white fluorescent and incandescent sources 25–450 µmol m−# s−"). The ages of the plants at the times of decapitation and the beginning of the auxin treatments varied with the growing conditions (i.e. in greenhouse and growth rooms) and the developmental status of the lateral buds. The ages (in d) ranged as follows : Vicia faba, 12–13 ; Helianthus annuus, 19–27 ; Ipomoea nil, 12–32 ; Pisum satium, dwarf, 10–21, normal, 15–16 ; Phaseolus, 10–18 ; Lycopersicon esculentum, VNF8, 27–54 ; dgt, 39–68 ; Coleus (from time of stem cutting), 21–37 ; Arabidopsis thaliana, 35–52. Intact plants with elongating lateral buds were excluded. Shoots were decapitated with a razor blade about 1 cm above the base of the lateral bud at the node indicated for each species in the Results section (Fig. 1). Excluding the cotyledons, nodes were counted upward from the base of the plant. There was some variation in this distance depending upon the species and the relevant internode lengths. It was necessary that there be sufficient distance between the lateral bud and the decapitated stem stump above so that auxin could be
257
applied to the stump without contact with the bud. Auxin was applied to the stem stump twice daily beginning immediately after decapitation in approximately 150 µl doses as IAA, α- or β-NAA at concentrations ranging from 10−' to 10−$ in 0±05 % Tween 20 aqueous solution in a taped cotton swab (Fig. 1 C) or as IAA in lanolin, 0±1, 0±5, 1 %. Treatments normally extended from 4 to 10 d with daily measurements of the lateral bud situated at the highest node below the point of decapitation. Two exceptions were Vicia faba and Pisum satium (normal) where the largest rather than highest lateral bud was used to determine bud outgrowth. Each treatment was usually carried out with four to nine plants. At least two experiments with each of the plant types were carried out in both the greenhouse and the growth room with the exceptions indicated above. For certain light experiments (as indicated in the Results section), Ipomoea seedlings were moved from growth rooms to out-of-doors (irradiance under shade screens, up to 210 µmol m−# s−" and in the open, up to 6900 µmol m−# s−") during the summers of 1992 and 1994 and Coleus plants propagated from stem cuttings were grown in growth rooms with greatly reduced irradiance from General Electric cool white fluorescent lamps (25–30 µmol m−# s−"). There were usually four to ten Coleus and Ipomoea plants in each of the auxin treatments. In the outside shade screen experiments, there were eight to 11 Ipomoea plants per treatment. Seeds of Arabidopsis thaliana (strain CS 1072 Chi-O from the Ohio State University Arabidopsis Biological Resource Center) were germinated in water-saturated Magik-moss potting soil containing perlite and vermiculite in greenhouse following a 2-d treatment in the refrigerator (0–3 °C). After several weeks, some seedlings were moved to the growth room (27–29 °C) under continuous light while the remaining were kept in the greenhouse.
RESULTS Auxin significantly inhibited lateral bud outgrowth following decapitation when applied to the stem stump of most of the species tested [Ipomoea nil, Helianthus annuus, Lycopersicon esculentum (VNF8), Pisum satium (Little Marvel and Thomas Laxton), and Vicia faba] at concentrations of 10−& and}or 10−% in aqueous solution or 0±1 and}or 1 % in lanolin (Figs 2, 4 ; Table 1). Inhibition was also found in the tomato mutant dgt (Fig. 4) which is insensitive to auxin with regard hypocotyl elongation and ethylene production (Kelly and Bradford, 1986). In Ipomoea, Helianthus and dgt tomato there were no obvious toxic effects of exogenous auxin applications observed in any experiments. In Vicia faba and Pisum, occasional auxininduced aberrations in stem and leaf growth were observed whereas in the VNF8 tomato such aberrations were more common. Auxin had no effect on restoring apical dominance in decapitated greenhouse-grown Coleus (Fig. 5) or Arabidopsis (Fig. 3, Tables 2 and 3) and only a partial effect on bean (Fig. 2 F). α-NAA was generally more potent than IAA in inhibiting lateral bud outgrowth. β-NAA, the inactive auxin
258
Cline—Exogenous Auxin Effects in Decapitated Shoots
Bud length (mm)
30
Decap control
A
B
Decap control
20 15
20
IAA –4 10 M
10 0.1% IAA
10
5
–5 α-NAA 10 M
1% IAA 1
2
3
20
Decap control
Bud length (mm)
C
10
16
–6
M
IAA
12 8
10
–5
IAA
4
10
–4
IAA
10
–3
IAA
25
12
2
Intact
3
D
4
Decap control
8
1
Bud length (mm)
1
4
2
M
4 M M
0.1% IAA Intact control 1
3
2
3
Decap control
30 Decap control
E
4
F
20 20 15
1% IAA
10
10
0.1% IAA
5
1% IAA Intact 1
2
3
4
Day
Intact control 1
2
3
4
Day
F. 2. Auxin repression of lateral bud outgrowth over 3 or 4 d following decapitation. A, Vicia faba (growth room, n ¯ 4). B, Helianthus annuus (growth room, n ¯ 5). C, Ipomoea nil (growth room, n ¯ 3–4). D, Pisum satium (dwarf ) (growth room, n ¯ 5–6). E, Pisum satium (normal) (growth room, n ¯ 7). F, Phaseolus ulgaris (greenhouse, n ¯ 4). Decap, Decapitation. Vertical lines represent³s.d.
analogue, which was tested on seven of the ten plant types, usually had little or no effect on apical dominance in most experiments. With some species [Vicia faba ; Pisum (Little Marvel), Helianthus annuus], the lack of effect of auxin in aqueous solution may have been due to a penetration or metabolism problem. Auxin in lanolin was always effective. In the following section are descriptions of apical dominance in the ten plant types, and their responses to decapitation and to auxin treatments under growth room and greenhouse conditions. The term ‘ strong apical dominance ’ as used here signifies little or no lateral bud outgrowth in intact plants. ‘ Medium ’ signifies some bud growth and ‘ weak ’ indicates substantial and continuing lateral bud growth in intact plants.
Vicia faba (Broad Windsor Bean) The intact plant had weak to medium apical dominance. Lateral buds on nearly half of the greenhouse plants and on a quarter of the growth room plants had grown out before the start of the auxin treatment. These plants had to be excluded. Decapitation above the third node resulted, most often, in the outgrowth of the lateral buds at the basal node but sometimes at the upper nodes (Fig. 1 A). All three lateral buds were repressed by the auxin treatment with the top (third) bud (closest to the auxin application) inhibited the most. Consistent repressive effects on bud growth were found with 1 % IAA in lanolin and partially at 0±1 % (Fig. 2 A). There was some swelling and abnormal curving of
Cline—Exogenous Auxin Effects in Decapitated Shoots T 1. Outgrowth of lateral buds (mm³s.d.) oer 3 or 4 d following decapitation and treatment of shoot stump with IAA, α-NAA, β-NAA in aqueous solution or IAA in lanolin. Decap., decapitation Days after decapitation 0
1
2
Vicia faba, growth room, n ¯ 4 Intact 0 0 0 Decap. control 0 1³2 8³3 IAA 10−% 0 5³5 14³10 α-NAA 10−% 0 1³2 5³6 Helianthus annuus, growth room, n ¯ 4–5 Intact 0 0 0 Decap. control 0 1³1 3³2 IAA 0±1 % lanolin 0 0 0 IAA 0±5 % lanolin 0 0 0 Ipomoea nil, growth room, n ¯ 10 Decap. control 1³0 3³1 8³1 IAA 0±1 % lanolin 2³0 2³1 3³2 IAA 1 % lanolin 1³0 2³0 2³1 Pisum satium (dwarf ), greenhouse, n ¯ 4 Intact 3³1 3³1 3³1 Decap. Control 2³1 3³1 5³1 3³1 4³1 5³2 IAA 10−% α-NAA 10−% 2³1 2³1 2³1 β-NAA 10−% 2³1 3³1 4³2 Phaseolus ulgaris, growth room, n ¯ 4 Intact 3³0 4³1 4³0 Decap. control 4³0 7³2 11³3 IAA 10−% 3³1 5³2 8³2 α-NAA 10−% 3³0 4³1 6³2 β-NAA 10−% 3³1 5³2 9³2
3
4
0 21³4 27³13 16³11
0 35³4 43³14 23³14
0 9³4 0 0
0 20³7 1³2 0
25³5 8³6 4³2 3³1 8³3 8³2 3³1 5³2
4³1 13³6 11³3 3³1 7³2
4³1 45³11 23³8 11³5 25³9
259
solution (10−& to 10−$ , Fig. 2 C) and IAA in lanolin (0±1 and 1 %, Table 1) significantly inhibited lateral bud outgrowth following decapitation with no toxic auxin symptoms observed except at 10−$ IAA. As Fig. 2 C indicates, although there was no effect at 10−' IAA, there was increasing repression of axillary bud outgrowth from 10−& to 10−$ . Pisum sativum [Little Marel (dwarf ) and Thomas Laxton (normal) Pea] The intact plants had moderate apical dominance. Following decapitation above one of the higher nodes (usually the fifth), many of the buds at the lower nodes would simultaneously grow out to considerable lengths (Fig. 1 D, E). In the dwarf pea, the bud at the highest node usually grew out most rapidly followed by the buds at the second and third highest nodes (Fig. 1 D). In the normal pea, it was the bud at the second or third node from the base which grew out more rapidly followed by those of the fourth and fifth nodes, respectively (Fig. 1 E). Auxin did significantly restore apical dominance in both the dwarf and the normal pea plants following decapitation. Significant inhibition of bud outgrowth was obtained in dwarf pea with 10−% α-NAA but not with 10−% IAA in aqueous solution (Table 1). IAA in lanolin at 0±1 % was effective in both dwarf (Fig. 2 D) and normal (Fig. 2 E) types although some swelling in the stem stump and abnormal stem curvature were observed in the normal peas and in one experiment with the dwarf peas. Phaseolus vulgaris (Blue Lake Bean)
stems which accompanied the auxin treatment. Neither IAA nor α-NAA in aqueous solution were very effective in repressing axillary bud outgrowth (Table 1).
The intact plant had very strong apical dominance with absolutely no sprouting of lateral buds. Outgrowth of buds in the greenhouse was not observed until nearly a week following decapitation above the first node, whereas it was observed in the growth room within 1 or 2 d (Fig. 1 B). Release from apical dominance could be inhibited by 10−& α-NAA (Fig. 2 B) or by 0±1 % IAA in lanolin (Table 1). IAA in aqueous solution was ineffective at 10−% (Fig. 2 B). No toxic auxin effects were observed.
The intact plant had weak to medium apical dominance. Its apical dominance has been described as ‘ incomplete ’ (Tamas, 1987). The axillary buds subtended by the primary leaves (the first node, above which we decapitated) were more inhibited than the buds at the second node subtended by the first trifoliate leaf. The bean plant grows rapidly, the internodes are large and the plant is easy to work with. Decapitation definitely accelerated lateral bud outgrowth (Fig. 1 F) but the inhibitory effect of IAA (1 % in lanolin, Fig. 2 F and 10−% in solution, Table 1) applied to the stem stump was only partial. α-NAA at 10−% had a stronger effect (Table 1). No toxic effects of auxin were observed except for a little swelling and bleaching of the stem stumps upon which the auxin was directly applied.
Ipomoea nil (Japanese Morning Glory)
Coleus
The intact plant had medium to strong apical dominance. There was no axillary bud outgrowth in the growth room or in winter-grown greenhouse plants. There was slight outgrowth of cotyledonary buds of greenhouse plants in the spring (data not shown). Decapitation above the second node nearly always resulted in the rapid outgrowth of the bud at the node (Fig. 1 C). The bud at the first node also often exhibited a small amount of temporary growth. Both IAA and α-NAA (data not shown) in aqueous
The intact Coleus plant had weak apical dominance. It had short internodes, grew slowly and was bushy. The basal branches were of greater length than the higher branches because of their greater age. These plants were propagated from cuttings. Many intact plants had to be excluded from the study because of extensive axillary bud development beginning at the basal nodes. Only plants with very small lateral buds (! 3–4 mm) were selected. At the time of decapitation, the lateral buds of the lowest of the top three
Helianthus annuus (Mammoth Grey-striped Sunflower)
260
Cline—Exogenous Auxin Effects in Decapitated Shoots
F. 3. Upper left and right, VNF8 (wildtype) and dgt (mutant) tomatoes about a week following decapitation showing lateral bud outgrowth. The stem stumps have been treated with lanolin. Lower left, Arabidopsis, 1 week following decapitation ; A, decapitation above the first node ; B, intact control ; C, decapitation above first node with 1 % IAA on stump of main stem ; Lower right, Coleus showing the branch-promoting effect of greenhouse high light (B) as compared with indoor low light (A).
nodes were removed. Decapitation above the third node of these greenhouse plants had only a small effect on accelerating bud outgrowth. Auxin (0±1 % and 1 % in lanolin) application to the stem stumps had no repressing effect on lateral bud outgrowth of greenhouse plants (Fig. 5). Lycopersicon esculentum [VNF8 (normal ) and dgt (mutant) tomato] The intact plants of both dgt and VNF8 had medium apical dominance with little or no lateral branching. Neither the dgt nor the VNF8 in our growth room or greenhouse conditions were categorized as having reduced apical dominance or as being bushy. In a few experiments, some
lateral bud development was noticed in intact plants. The VNF8 plants were larger than the dgt plants. The shoot of the young dgt seedling initially grows more or less vertically. After several months, the shoot gradually assumes a horizontal orientation. The seedling shoots of dgt, although not yet at horizontal growth stage, were floppy and hence were staked in an upright position for convenience in auxin treatment. Since horizontal positioning tends to weaken apical dominance (Prasad and Cline, 1985 b), such staking, if anything, would have a countering effect to anomalous bud outgrowth. Following decapitation, which was usually carried out above the fifth node, several of the lateral buds on any given dgt or VNF8 plant vigorously sprouted more or less simultaneously (Fig. 3). This most often occurred at the
261
Cline—Exogenous Auxin Effects in Decapitated Shoots B
Bud length (mm)
A
β-NAA 10–5M
16
Decap control
12
15
IAA
Decap control
–5
10
8
M
α-NAANA A10–5M Intact
4
1
3
2
10 5
4
1
α-NAA 10–5M IAA 10–4M α-NAA 10–4M 3 Intact 2
D
C Bud length (mm)
IAA 10–5M
20
20
Decap control
25
Decap control
15 IAA 1%
20
0.1% IAA 10
IAA 0.1% 10
5
0.5% IAA 1% IAA
Intact 2
4 Day
6
2
4 Day
6
F. 4. Auxin repression of tomato [VNF8 (A, C) and dgt (B, D)] lateral bud outgrowth in growth room following decapitation. Auxin in aqueous solution, A, B (n ¯ 4–5, 4). Auxin in lanolin, C, D (n ¯ 3–4, 4). Decap, decapitation. Vertical lines represent³s.d.
Bud length (mm)
10
1% IAA Decap control
8
6
Intact control
4 2
4 Day
6
F. 5. Lateral bud growth in Coleus blumei over 6 d following decapitation and stem stump treatment with 1 % IAA (greenhouse, n ¯ 9–10). Vertical lines represent ³s.d.
upper-most nodes but sometimes at one of the lower nodes. The points on the curves in the graphs represent the mean length of the highest lateral bud on each plant. The young axillary buds of tomato were difficult to observe and to measure because of their small size and narrow shape. The treatment with exogenous auxin [10−& α-NAA and}or 10−% IAA in aqueous solution (Fig. 4 A, B) and 0±1 % IAA in lanolin, Fig. 4 C, D] did, for the most part, significantly inhibit axillary bud outgrowth in both plant types. IAA in aqueous solution had variable effects on VNF8 but α-NAA, down to 10−& , had consistent repressive effects on bud outgrowth (Fig. 4 A). β-NAA had
no inhibitory effect. In several instances it appeared even to promote bud growth. IAA at 10−% and 10−& α-NAA generally restored apical dominance in dgt (Fig. 4 B and data not shown). IAA in lanolin (both at 0±1 and 1 %) was quite effective in repressing bud outgrowth in both VNF8 and dgt (Fig. 4 C, D). Hence, no essential difference in sensitivity could be positively detected between dgt and VNF8 in the response to auxin in repressing lateral bud outgrowth except for the toxic auxin effect mentioned previously for VNF8. Arabidopsis thaliana Arabidopsis is a facultative long-day plant with weak apical dominance consisting of a rosette from which arises a single branching floral shoot or inflorescence followed by the generation of several surrounding axillary or secondary inflorescences also arising from the rosette. The measurement of changes in apical dominance status is complex because of the two types of branching, lateral shoots developing from the main shoot and axillary inflorescences developing from the rosette and the rapidity with which they occur. The lateral buds (shoots) of the main shoot begin emerging almost as soon as the main shoot begins to bolt. In the many mutants and strains of Arabidopsis now available, there exists a wide range of branching habits. The CS 1072 Chi-O strain used in the present study has stronger apical dominance than most Arabidopsis types inasmuch as it generally lacks axillary inflorescences. Decapitation of the main floral shoot resulted in increased
262
Cline—Exogenous Auxin Effects in Decapitated Shoots
T 2. Effect of 1 % IAA on lanolin on Arabidopsis. Outgrowth of first lateral bud (mm³s.d.) and axillary inflorescence 1 week following decapitation of the main shoot and treatment of the shoot stump with 1 % IAA in lanolin. Decapitation was either 3–5 mm aboe the first node or 3–5 mm aboe the base of the main shoot (below the first node) as indicated. Axillary inflorescence deelopment is gien as a percentage of the plants sprouting one or more axillary inflorescences. *Main shoot length Decap above first node Decap below first node Intact plant
Control
1 % IAA
Control
1 % IAA
Decap above first node Decap below first node Intact plant
Control
Growth room n First Lateral Bud Axial Inflorescences
1 % IAA
Control
1 % IAA
Greenhouse
7
7
6
7
7
5
5
5
5
5
235³21* 0
145³24 71 %
132³29 33 %
— 100 %
— 100 %
124³57* 0
87³29 60 %
78³52 60 %
— 100 %
— 100 %
T 3. Effect of 10−% M IAA spray on Arabidopsis. Outgrowth of axillary inflorescences 1 week following decapitation of the main shoot 3–5 mm aboe the base of the plant which was sprayed eery other day beginning 15 d before decapitation with 10−% M IAA. Axillary inflorescence deelopment is gien as a percentage of plants sprouting one or more axillary inflorescences
Growth room Intact control Intact IAA 10−% Decap. control Decap. IAA 10−% Greenhouse Intact control Intact IAA 10−% Decap. control Decap. IAA 10−%
n
Main shoot
Axillary inflorescence length (mm³s.d.)
Axillary inflorescence percentage
7 7 7 8
235³21 214³20 — —
— 38³23 71³43 81³41
0 38 100 100
5 9 5 10
124³57 109³46 — —
— 0 25³31 21³24
0 0 100 60
outgrowth of lateral (buds) shoots from the main shoot as well as the subsequent generation of axillary inflorescences from the base of the plant (Fig. 3). If the point of decapitation of the main shoot was above the lowest lateral emerging shoot (bud), then that shoot grew out to a much greater extent than in the intact plant and took over as the main shoot. If the decapitation of the main shoot was below the lowest lateral emerging shoot (bud), then several axillary inflorescences grew out from the base of the plant. The greenhouse plants exhibited somewhat stronger apical dominance with respect to axillary inflorescence outgrowth than did the growth room plants. In essentially no plant did auxin, either as 1 % IAA in lanolin applied to the decapitated stump of main shoot or as 10−% IAA spray applied every other day to the whole plant, have any effect in restoring apical dominance, i.e., in inhibiting lateral branching of the main shoot or the outgrowth of the axillary inflorescences (Fig. 3, Tables 2 and 3). In the case of decapitation above the first node, there appeared to be some auxin repression in the percentage of axillary inflorescence development in the growth room (Table 2) but this was not confirmed in the greenhouse nor in the more direct test when IAA was applied following decapitation below the first node.
Attempts to restore apical dominance with auxin spray applied to the small and extremely bushy trp 1-1 tryptophanrequiring auxotrophic mutant (suggestive of an auxin defect, Last et al., 1992) were also unsuccessful (data not shown). Influence of light Past observation (Hosokawa et al., 1990) indicated that when Ipomoea nil was propagated in growth rooms under a mixture of cool white fluorescent and incandescent sources (irradiance : 25–450 µmol m−# s−"), a 30-d-old plant was characteristically tall (40–70 cm) without branching. However, when the plant was grown outdoors (irradiance : up to 6900 µmol m−# s−"), the plant was short, often with such a proliferation of branching that the study of auxin effects on bud outgrowth was impossible to carry out since most buds had substantially elongated at a very early developmental stage. Therefore, Ipomoea plants were grown indoors until the age of 14 d, at which time they were moved outdoors (still in pots), decapitated and stem stumps treated with auxin in lanolin for 4 d. There appeared to be little or no difference in sensitivity in auxin repression of axillary bud outgrowth between indoor plants at low irradiance and indoor plants moved outdoors at high irradiance at the time
263
Cline—Exogenous Auxin Effects in Decapitated Shoots T 4. Outgrowth of lateral buds (mm³s.d.) of Ipomoea nil plants oer 3 or 4 d following decapitation and treatment of shoot stump with IAA in lanolin either in growth room (25–450 µmol m−# s−") or outdoors (up to 6900 µmol m−# s−")
±32.1 100
200
±37.6
1
2
3
4
1³0 2³0 1³0
3³1 2³1 2³0
8³1 3³2 2³1
25³5 8³6 4³2
— — —
2³1 3³0 3³1
3³1 3³1 8³1
6³1 3³1 3³1
12³3 6³1 3³1
23³6 11³3 5³2
75 Weight (g)
Growth room, n ¯ 10 Decap. control IAA 0±1 % IAA 1 % Outdoors, n ¯ 4–8 Decap. control IAA 0±1 % IAA 1 %
0
50
100
±3.7
0
50
Weight (g)
0
Height (cm)
F. 7. Bar graphs comparing total fresh weights and heights of Ipomoea nil plants grown outside in heavy shade (+) and in the open (*). n ¯ 8–11.
that they were hardly recognizable as Ipomoea nil plants. Both the lateral bud length and the total fresh weights of the shaded plants were very much reduced as compared to those of unshaded plants (Figs 6 and 7). At the fifth, sixth and
±2.6
±22.6
±16.7
±35.3
±22.8
25
of decapitation and auxin treatment (Table 4). Hence, there was no immediate effect of high irradiance on exogenous auxin restoration of apical dominance in decapitated plants. When 16}17-d-old Ipomoea plants in pots were transplanted to outdoor plots under heavy shading (irradiance : up to 210 µmol m−# s−"), they appeared, after 19–34 d, very similar in overall form and height (i.e. tall without branching) to indoor plants of approximately the same age (no data given). Whereas those plants transplanted to outdoor plots under full sunlight had, in time, such an extensive proliferation of lower branches together with reduced height
50
150
Height (cm)
Days following decapitation
±20.5 ±17.9
30
±25.5
±27.3
Bud length (cm)
40
±0.3 ±0.1
±0.2 ±0
13
±0.4 ±0.06
±2.1 ±0.2
±2.6
9 10 11 12 Node number
±0.9 ±0.06
8
±0.7 ±0.7
7
±0.9 ±0.4
6
±0.2
±3.5 ±1.3
±24.2
5
±1.4 ±0.2
4
±0.4
3
±0.3
2
±15.7
±0.5
1
±4.9
±
0
±0.1
10
±2.5
±18.2
±9.6
±1.8
20
14
15
16
17
18
19
20
F. 6. Bar graphs comparing lateral bud length (cm³s.d.) of Ipomoea nil plants grown outside in heavy shade (+) and in the open (*) at different nodes numbering up from the base of the shoot. n ¯ 8–11.
264
Cline—Exogenous Auxin Effects in Decapitated Shoots
Length (mm)
Decap control 12 0.1% IAA 8 1% IAA 4 Intact 2
4
6 Day
8
10
F. 8.Partial auxin repression of lateral bud outgrowth in Coleus at reduced irradiance (25–30 µmol m−# s−") following decapitation over 10 d. Vertical lines represent³s.d. n ¯ 10.
seventh nodes of the shade plants (Fig. 6), there was some anomalous branching due to the fact that the shoot tips had impinged against the overhanging shade screen, had bent over and had released the buds at those nodes. Floral buds were found to be much more prevalent in the sun plants than in the shade plants. Since the spectral quality of sunlight in both groups of plants presumably was the same (i.e., the plastic shade screens would not be expected to cause any such changes), the pronounced weakening of apical dominance as exhibited by the proliferation of branching in the sun plants could be attributed to increases in irradiance (fluence) and not to changes in light quality. When Coleus plants were grown indoors at greatly reduced light levels (irradiance : 25–30 µmol m−# s−"), branching in intact plants was much decreased compared to that of greenhouse plants (Fig. 3) but increased lateral bud outgrowth did occur at the highest node following decapitation. Definite but partial inhibitory effects of 0±1 and 1 % IAA in lanolin were detected in one out of five experiments carried out at the reduced light level (Fig. 8). DISCUSSION The results of this study corroborated the Thimann-Skoog (1933) experiment, that exogenously applied auxin to the stem stump of a decapitated plant does restore apical dominance for many plants under most conditions. This is a classic example wherein the results one obtains depend upon the particular plant system used and upon the conditions employed. There were exceptions where the Thimann-Skoog experiment did not work. In those plants with weak apical dominance such as Coleus or Arabidopsis, auxin had little or no effect on repressing bud outgrowth in decapitated plants. Hence, it is understandable why Jacobs et al. (1959) were not able to detect repressive auxin effects on the release apical dominance with greenhouse-grown Coleus plants. In plants with incomplete apical dominance such as Phaseolus, auxin had only a partially inhibiting effect on axillary bud growth. Hence, it is understandable why Hillman (1984) questioned the role of auxin in apical dominance in this species. Certain environmental conditions can proliferate branching to an extent which makes it impossible to carry out the Thimann-Skoog experiment.
Increased irradiance levels can greatly weaken apical dominance (Gregory and Veal, 1957 ; Anderson, 1976). High wind velocity (via thigmomorphic effects) or alterations in the direction of gravity can also release apical dominance (Prasad and Cline, 1985 a, b). Although Thimann, Sachs and Mathur (1971) reported auxin in aqueous solution to be more effective than in lanolin with Coleus and Pisum, in this study it was found otherwise. This might have been due to different methods used for applying the aqueous solution. Thimann employed a fine piece of tubing over the end of the stem for continuous flow whereas two doses of approximately 150 µl were applied daily by pipette to a cotton swab taped to the end of the stem in the present study (Fig. 1 C). The auxin concentration (1400–1700 units) which Thimann and Skoog (1933) used to completely inhibit bud outgrowth in Vicia faba is presumed to be equivalent to approximately 1 % in lanolin or to 6¬10−# in aqueous solution (Stafstrom, 1993). In retrospect, Vicia faba, the plant system which Thimann and Skoog (1933) used in their classic study, probably was not the best for studying apical dominance. First, it has weak apical dominance and many plants had to be excluded in the present study because they were already branching before the time designated for decapitation, particularly in the greenhouse plants. Second, following decapitation, it was usually the bud at the basal node instead of the highest node which sprouted first. This complicated the situation because of the greater distance between the site of auxin application (the decapitated stem stump) and the potentially active axillary buds than when the highest bud sprouted first. It was observed in some plants where several Vicia buds sprouted simultaneously that the higher buds located closer to the site of auxin application were repressed the most. This suggested that the auxin concentration probably decreased gradually during transport away from its original source. However, V. faba was sensitive to auxin and its apical dominance was restored by auxin application. Those concentrations of exogenous auxin (0±1–1 % IAA) which inhibited apical dominance release also exhibited toxic auxin effects. The Thimann-Skoog experiment is carried out with the greatest facility in a fast growing plant with large internodes, moderate to strong apical dominance, easily observed axillary buds, and where only the highest lateral bud grows out following decapitation. Of the ten plants tested, Ipomoea nil conformed most closely to the expectations of the Thimann-Skoog experiment, followed by Helianthus annuus which, however, was somewhat hindered by its slow growth in our greenhouse conditions. Pea (Thomas Laxton) worked well in many ways except for the uncertainty in which lateral bud would sprout following decapitation. It was usual for several buds to sprout simultaneously, which complicated both the execution of the experiment and the interpretation of the data. In Arabidopsis, the complete lack of effect of auxin on restoring apical dominance following decapitation may be due in part to the fact that the upright shoot of Arabidopsis is a floral shoot. All experiments done with other species in
Cline—Exogenous Auxin Effects in Decapitated Shoots this study were carried out with vegetative shoots, or at least shoots without the presence of visible floral buds. For reasons which are not entirely clear, aging and reproduction often have a weakening effect on apical dominance (Tamas, 1987). dgt mutant tomato If one accepts the auxin inhibition hypothesis as the explanation for the mechanism of action of apical dominance then the lack of outgrowth of the axillary buds is presumed to be due to repression imposed by apicallyproduced}basipetally transported auxin in the shoot. A mutant such as the dgt tomato which has been demonstrated to be insensitive to auxin-induced hypocotyl elongation and ethylene production (Kelly and Bradford, 1986) might also be expected to be insensitive to auxin repression of lateral bud outgrowth and, hence, would exhibit heavy branching. This was not observed in the present study in growth room or greenhouse plants up to an age of 4–5 weeks. It was noticed in older plants (8–10 weeks or more) that high soil fertilization and exposure of plants to open sunlight usually did promote axillary bud growth in both dgt and VNF8. Interpretation of data (not shown) which indicated insensitivity of dgt to the auxin transport inhibitors, TIBA (2,3,5-triiodobenzoic acid) and NPA (N-1 naphthylphthalamic acid), with respect to releasing apical dominance was unclear due to significant non-specific effects (abnormal stem and leaf growth) commonly observed in VNF8 in cases of lateral bud outgrowth. Auxin did inhibit lateral bud outgrowth in decapitated dgt plants following application to the stem stump. Hence, dgt is sensitive to auxin with respect to apical dominance. It has been suggested for apical dominance as well as for other responses to auxin that there may be separate auxin receptors (M. O. Kelly and M. G. Cline, unpub. res. ; Palm et al., 1991). Light When irradiance is high, as outdoors, the lateral buds of many species (except for those with very strong apical dominance such as sunflower) will begin to grow out and to proliferate, thus excluding the execution of the ThimannSkoog experiment which requires lateral buds to be in a repressed state before decapitation and auxin treatment. This proliferation of branching in outdoor-grown plants was observed in the present study of Ipomoea nil, a plant with moderate to strong apical dominance. From previous indoor tests, it was clear that once vigorous axillary bud growth had commenced, it was very difficult to repress, even with high concentrations of auxin (data not shown). It is also possible that other factors in the outdoor environment besides light may have a weakening effect on apical dominance. When the irradiance is low, the lateral buds of many species (except for those with very weak apical dominance such as Arabidopsis) will be in a repressed state, thus allowing for the execution of the Thimann–Skoog experiment with subsequent inhibition of lateral bud outgrowth. In Coleus, a plant with weak apical dominance
265
where exogenous auxin is known to have no repressive effect on lateral bud outgrowth following decapitation at high irradiance in greenhouse environment (Jacobs et al., 1959), marginal evidence was found in the present study to support Thimann’s contention (Thimann et al., 1971 ; Thimann, 1977) that auxin did inhibit bud growth when the irradiance level was reduced (Fig. 8). The fact that outdoor light control of apical dominance in Ipomoea could be manipulated wholly by the use of shade screens suggested that the response under the present conditions was dependent upon changes in irradiance and not in spectral differences. This result, while not excluding the interaction with indirect light quality effects or with auxin, is also consistent with the nutritional hypothesis of apical dominance via increased photosynthate availability for bud growth at high irradiance. The possibility that the plastic shade screens could affect light quality and, hence, other physiological processes cannot be ruled out. Light is one of many stimuli (e.g. gravity, CO , nutrients, # etc.) which often will promote axillary bud outgrowth (Hillman, 1984). The mechanism by which light induces such outgrowth is unknown. Field and Jackson (1975) point out some of the complexities involved in interpreting light effects on apical dominance. Gregory and Veale (1957) reported that auxin repression of axillary bud outgrowth increased with decreasing light in Linum particularly at low nitrogen levels. Thimann et al. (1971) suggested that high light may promote the synthesis of cytokinins which in turn may reverse the auxin effect. Accumulating evidence in the literature for an indirect role of auxin in apical dominance might also indicate the involvement of certain phytochromemediated processes, interaction with other hormones and second messengers or by decreasing sensitivity to auxin. CONCLUSION The results of this study confirm that the Thimann–Skoog experiment does work for most species and suggests a controlling role for auxin in apical dominance. Most likely the role is indirect, perhaps involving cytokinins (Bangerth, 1994 ; Sandberg et al., 1995). It is hoped that the present accelerating research involving both traditional physiological and molecular approaches with mutants will soon be able to resolve these problems and more fully elucidate this classic developmental phenomenon. A C K N O W L E D G E M E N TS Appreciation is expressed to Daniel Repicz for his diligent efforts with the statistical data and graphs, Annabelle Chern and Liang Shi for their competent assistance with the preliminary dgt tomato experiments, to the Livingston Seed Co. of Columbus, Ohio for their gracious donation of Vicia faba seeds and to the Ohio State University Arabidopsis Biological Resource Center for their generous help in the donation of seeds and propagation of seedlings. LITERATURE CITED Ali A, Fletcher RA. 1970. Hormonal regulation of apical dominance in soybeans. Canadian Journal of Botany 48 : 1989–1994.
266
Cline—Exogenous Auxin Effects in Decapitated Shoots
Anderson AS. 1976. Regulation of apical dominance by ethephon, irradiance and CO . Physiologia Plantarum 37 : 303–308. # Bangerth F. 1994. Response of cytokinin concentration in the xylem exudate of bean plants (Phaseolus ulgaris L.) to decapitation and auxin treatment and relationship of apical dominance. Planta 194 : 439–442. Beveridge CA, Ross JJ, Murfet IC. 1994. Branching mutant rms-2 in Pisum satium. Grafting studies and endogenous indole-3-acetic acid levels. Plant Physiology 104 : 953–959. Brown CL, McAlpine RG, Kormanik PP. 1967. Apical dominance and form in woody plants : a reappraisal. American Journal of Botany 54 : 153–162. Cline MG. 1991. Apical dominance. The Botanical Reiew 57 : 318–358. Cline MG. 1994. The role of hormones and apical dominance. New approaches to an old problem in plant development. Physiologia Plantarum 90 : 230–237. Estruch JJ, Chriqui D, Grossmann K, Spena A. 1991. The plant oncogene rolC is responsible for the release of cytokinins from glucoside conjugates EMBO Journal 10 : 2889–2895. Field RJ, Jackson DI. 1975. Light effects on apical dominance. Annals of Botany 39 : 369–374. Gregory FC, Veale JA. 1957. A reassessment of the problem of apical dominance. Symposia of Society of Experimental Biology 11 : 1–20. Hillman JR. 1984. Apical dominance. In : Wilkins M, ed. Adanced plant physiology. London : Pitman Publishing, 127–148. Hosokawa Z, Shi, L, Prasad TK, Cline MG. 1990. Apical dominance control in Ipomoea nil : the influence of the shoot apex, leaves and stem. Annals of Botany 65 : 547–556. Jacobs WP, Danielson J, Hurst V, Adams P. 1959. What substance normally controls a given biological process ? II. The relation of auxin to apical dominance. Deelopmental Biology 1 : 534–554. Kelly MO, Bradford KJ. 1986. Insensitivity of the diageotropica tomato mutant to auxin. Plant Physiology 82 : 713–717. Klee HJ, Horsch RB, Hinchee MA, Hein MB, Hoffman NL. 1987. The effects of over production of two Agrobacterium tumefaciens TDNA auxin biosynthetic gene products in transgenic petunia plants. Genes and Deelopment 1 : 86–96. Klee HJ, Romano C-P. 1994. The roles of phytohormones in development as studied in transgenic plants. Critical Reiews in Plant Sciences 13 : 311–324. Last RL, Borczak JL, Pruitt KD, Radwanski ER, Rose AB. 1992. The genetics of tryptophan biosynthesis in Arabidopsis. Journal of Experimental Botany Supplement 43 : 18. Lincoln C, Britton J, Estelle M. 1990. Growth and development of the axr1 mutants of Arabidopsis. The Plant Cell 2 : 1071–1080. McIntyre GI. 1977. The role of nutrition in apical dominance. In : Jennings DH, ed. Integration of actiity in the higher plants. Symposium of Society for Experimental Biology, Cambridge : Cambridge University Press, 251–273. Martin GC. 1987. Apical dominance. HortScience 22 : 824–833. Palm K, Hesse T, Moore I, Campos N, Feldwisch J, Garbers C, Hesse F, Snell J. 1991. Hormonal modulation of plant growth : the role of auxin perception. Mechanisms of Deelopment 33 : 97–106. Prasad TK, Cline MG. 1985 a. Mechanical perturbation-induced ethylene releases apical dominance in Pharbitis nil by restricting shoot growth. Plant Science 41 : 217–222.
Prasad TK, Cline MG. 1985 b. Gravistimulus direction, ethylene production and shoot elongation in the release of apical dominance in Pharbitis nil. Journal of Experimental Botany 36 : 1969–1975. Romano C, Cooper ML, Klee HJ. 1993. Uncoupling auxin and ethylene effects in transgenic tobacco and Arabidopsis plants. Plant Cell 5 : 181–189. Russell W, Thimann KV. 1988. The second messenger in apical dominance controlled by auxin. In : Phares RP, Hood SB, eds. Plant growth substances, 1988. New York : Springer-Verlag, 419–427. Sachs T, Thimann KV. 1967. The role of auxins and cytokinins in the release of buds from dominance. American Journal of Botany 54 : 136–144. Sandberg G, Sitbon F, Eklof S, Edlund A, Astot C, Blackwell J, Moritz T, Sundberg B, Olsson O. 1995. Regulation of auxin and cytokinin turnover in plants. 15th International Conference on Plant Growth Substances. Minneapolis. Program-Abstracts 015. Shein T, Jackson DI. 1972. Interaction between hormones, light and nutrition on extension of lateral buds in Phaseolus ulgaris L. Annals of Botany 36 : 791–800. Shen WH, Davioud E, David C, Barbier-Brygoo H, Tempe! J, Guern J. 1990. High sensitivity to auxin is a common feature of hairy root. Plant Physiology 94 : 554–560. Stafstrom J. 1993. Axillary bud development in pea : apical dominance, growth cycles, hormonal regulation and plant architecture. In : Amasino RM, ed. Cellular communication in plants. New York : Plenum Publishing Corp., 75–86. Stafstrom J. 1995. Developmental potential of shoot buds.In : Gartner BL, ed. Plant stems : Physiology and functional morphology. San Diego : Academic Press, 257–279. Tamas IA. 1987. Hormonal regulation of apical dominance. In : Davies PJ, ed. Plant hormones and their role in plant growth and deelopment. Dordrecht : Martinus Nijhoff Publishers, 393–410. Tamas IA. 1995. Hormonal regulation of apical dominance. In : Davies PJ, ed. Plant hormones, physiology, biochemistry and molecular biology 2nd edn. Boston : Kluwer Academic Publishers, 572–597. Tepfer D. 1984. Transformation of several species of higher plants by Agrobacterium rhizogenes : sexual transmission of the transformed genotype and phenotype. Cell 37 : 959–967. Thimann KV. 1937. On the nature of inhibitions caused by auxin. American Journal of Botany 24 : 407–412 Thimann KV. 1977. Hormone action is the whole life of plants. Amherst : University of Massachusetts Press. Thimann KV, Sachs T, Mathur KN. 1971. The mechanism of apical dominance in Coleus. Physiologia Plantarum 24 : 68–72. Thimann KV, Skoog F. 1933. Studies on the growth hormones of plants. III. The inhibition action of growth substance on bud development. Proceedings of the National Academy of Science, USA 19 : 714–716. Wareing PF, Phillips IDJ. 1981. The control of growth and differentiation in plants, 3rd edn. New York : Pergamon Press. White JC. 1976. Correlative inhibition of lateral bud growth in Phaseolus ulgaris L. Effect of application of indol-3yl-acetic acid to decapitated plants. Annals of Botany 40 : 521–529.