Evidence that auxin promotes gibberellin A1 biosynthesis in pea

Authors


*For correspondence (fax +61 3 62 262698; e-mail John.Ross@utas.edu.au).

Summary

In shoots of the garden pea, the bioactive gibberellin (GA1) is synthesised from GA20, and the enzyme which catalyses this step (a GA 3-oxidase –- PsGA3ox1) is encoded by Mendel's LE gene. It has been reported previously that decapitation of the shoot (excision of the apical bud) dramatically reduces the conversion of [3H]GA20 to [3H]GA1 in stems, and here we show that endogenous GA1 and PsGA3ox1 transcript levels are similarly reduced. We show also that these effects of decapitation are completely reversed by application of the auxin indole-3-acetic acid (IAA) to the ‘stump’ of decapitated plants. Gibberellin A20 is also converted to an inactive product, GA29, and this step is catalysed by a GA 2-oxidase, PsGA2ox1. In contrast to PsGA3ox1, PsGA2ox1 transcript levels were increased by decapitation and reduced by IAA application. Decapitation and IAA treatment did not markedly affect the level of GA1 precursors. It is suggested that in intact pea plants, auxin from the apical bud moves into the elongating internodes where it (directly or indirectly) maintains PsGA3ox1 transcript levels and, consequently, GA1 biosynthesis.

Introduction

Auxin and gibberellin (GA) are ‘classical’ plant hormones that are thought to play crucial roles in regulating plant growth ( Kende & Zeevaart 1997). In the garden pea (Pisum sativum L.), the importance of GAs was first highlighted by research on Mendel's le-1 mutant ( Brian & Hemming 1955). Subsequently, Ingram et al. (1984) showed that the LE gene controls the 3β-hydroxylation of GA20 to GA1 (the bioactive GA in pea). The le-1 mutant is dwarf because it synthesises less GA1 than the wild-type, and the same is true for several other GA synthesis mutants ( Reid 1993; Ross et al. 1997 ). Demonstrating a role for auxin in elongation, however, has relied more on the effects of applying the hormone ( Haga & Iino 1998; Yang et al. 1993 ) or inhibitors of its transport ( McKay et al. 1994 ). For example, auxin promotes the elongation of decapitated pea stems ( Davies & Ozbay 1975; Haga & Iino 1998).

Despite this progress it is still not known how auxin and gibberellin interact, if at all, in exerting their effects ( Kende & Zeevaart 1997). It has been suggested that GA1 deficiency results in IAA deficiency, although only small reductions in IAA content were found in the le-1 mutant ( Law & Davies 1990). Recent evidence for an auxin/GA relationship of some type comes from studies involving auxin transport inhibitors. These compounds, applied in a ring of lanolin around the stem, inhibit the downward movement of IAA ( Lomax et al. 1995 ), reducing endogenous IAA levels below the ring ( McKay et al. 1994 ; Ross 1998). Interestingly, auxin transport inhibitors also dramatically reduced the GA1 content of the stem ( Ross 1998), and decapitation was earlier reported to cause similar effects ( Sherriff et al. 1994 ). Two hypotheses can be advanced to account for these observations ( Ross 1998). The first postulates that GA1, like IAA, is synthesised mainly in the apical bud and transported downwards, and that this transport is inhibited by auxin transport inhibitors. The second is that while GA1 is synthesised in the expanding internodes (and/or their subtending leaves), auxin is required for this to occur. According to the second hypothesis, when decapitation reduces the auxin level in stems, a decrease in GA1 content would follow as a consequence.

We have tested these hypotheses by decapitating tall (wild-type) pea plants and applying IAA to the ‘stump’ at frequent intervals, in order to maintain a high IAA level in the internodes concerned. The aim was to determine whether or not exogenous IAA can substitute for the apical bud in maintaining stem GA1 levels. We report here on the effects of decapitation and IAA treatment on GA metabolism and endogenous GA levels. In addition, we have monitored the transcript abundance of Mendel's LE gene (PsGA3ox1) and gene PsGA2ox1 ( Lester et al. 1999 ). PsGA2ox1 encodes the enzyme (a GA 2-oxidase) for the step GA20 to GA29 ( Fig. 1).

Figure 1.

The late steps in GA biosynthesis in pea shoots, showing the genes and metabolites monitored.

Results

When [13C3H]GA20 was applied to leaf 6 of intact plants, the main metabolites in internode 6 (after 48 h) were [13C3H]GA1, [13C3H]GA29 and [13C3H]GA8 ( Fig. 2). Decapitation virtually eliminated the [13C3H]GA1 peak (as described previously by Sherriff et al. 1994 ), and it was completely restored by applying IAA ( Fig. 2). The identity of this peak was confirmed by gas chromatography-MS (GC-MS), exploiting the 13C label of the substrate and of its metabolites. The mass spectrum corresponding to [13C3H]GA1 in the internodes from IAA-treated plants was: 507 (M+), 100; 492, 11; 449, 23; 448, 17; 378, 13; 377, 19; 376, 15; 314, 12; 236, 7; 208, 25. The spectrum also contained some endogenous GA1 (15%). The identities of the other metabolites, [13C3H]GA8, [13C3H]GA29 and [13C3H]GA29-catabolite were also confirmed by GC-MS. In the experiment represented in Fig. 2, internode 6 of intact plants was 88% of its final length at the beginning of the experiment, and 100% expanded at harvest. In additional experiments, we analysed metabolites in internodes which were only 20% expanded at harvest, and again found that decapitation reduced, and IAA treatment increased, radiolabelled GA1 levels (data not shown).

Figure 2.

The effects of decapitation and IAA application on [13C3H]GA20 metabolites in internode 6 (the internode between nodes 6 and 7).

Plants were either left intact (top), decapitated above node 7 (middle), or decapitated above node 7 and then treated with IAA (bottom). [13C3H]GA20 was applied to leaf 6 (indicated by arrows). Metabolites are shown as peaks of radioactivity (separated by HPLC). The total amounts of radioactivity (dpm × 10−3) recovered from internode 6 were intact, 8.3 ± 0.4; decapitated, 29.9 ± 2.7; and decapitated + IAA, 35.0 ± 0.6. The IAA levels in internode 6 (ng.gFW−1) were intact, 136.6 ± 8.4; decapitated, 22.7 ± 3.8; and decapitated + IAA, 618.0 ± 49.1 (n = 3).

The effects of IAA application on [13C3H]GA20 metabolites in other plant portions are shown in Table 1. The dramatic effect of IAA on [13C3H]GA1 levels was largely confined to the expanding internodes. In extracts from the leaves to which the substrate was applied, there were no peaks coeluting with GA1. The data in Table 1 show that the overall production of [13C3H]GA1 in decapitated plants was stimulated markedly by IAA.

Table 1. . Effects of IAA on [13C3H]GA20 metabolites throughout the plant. Plants were decapitated above node 6 and treated with lanolin or IAA; [13C3H]GA20 was then applied. The expanding internodes (internode 5 plus the stump of internode 6), the leaf treated with [13C 3H]GA20 (leaf 5), the remainder of the shoot system and the roots were harvested separately
Treatment
Portion[13C3H]GA20
Fractions 44, 45
[13C3H]GA1
Fractions 31–33
[13C3H]GA29
Fractions 24–26
[13C3H]GA8
Fractions 15–18
Total
in portion
  1. Data are total radioactivity (dpm × 10−3) for each GA recovered from the portion; HPLC fractions are indicated.

  2. a Roots contained large amounts of [13C3H]GA29-catabolite and [13C 3H]GA8-catabolite.

Expanding internodes1.7038.27.872.0
Leaf 5219.7056.27.6357.5
LanolinRemainder of shoot8.66.249.723.0130.4
Roots a002.62.639.7
Total230.06.2146.741.0599.6
Expanding internodes9.948.823.414.6108.5
Leaf 5213.0078.27.2376.0
IAARemainder of shoot5.46.832.115.390.4
Roots a002.01.729.8
Total228.355.6135.738.8604.7

To investigate whether other GA biosynthesis steps were affected by decapitation and IAA treatment, we quantified the endogenous levels of GA19, GA20, GA1, GA29 and GA8 in internode 6 from intact, decapitated and IAA-treated plants. Decapitation dramatically reduced GA1 levels (10-fold) and, to a lesser extent, GA8 levels (50%), and caused GA29 to accumulate (threefold, Fig. 3). In decapitated plants, the concentration of GA19 increased slightly whilst that of GA20 decreased slightly. These effects were completely reversed by IAA application. Indeed, IAA treatment resulted in a GA1 level three times greater than in intact plants, in keeping with the IAA level, which was nearly four times greater ( Table 2). It appears that the main effects of decapitation and IAA application are on GA20 metabolism, although the altered ratio of GA19 to GA20 indicates that there might be a small effect on this step. Importantly, the IAA levels in IAA-treated decapitated plants, while elevated, varied little between replicates and, indeed, between experiments ( Table 2 and Fig. 2). In contrast, IAA application to stems treated with an auxin transport inhibitor did not increase the IAA level in the stem (data not shown).

Figure 3.

Effects of decapitation and IAA application on endogenous GA levels in internode 6.

Plants were either left intact (white), decapitated above node 7 (stippled), or decapitated above node 7 and then treated with IAA (black). Data are shown as means of three replicates, with standard errors. Arrows indicate metabolic relationships. IAA levels and elongation data are shown in Table 2.

Table 2. . Effects of decapitation and IAA treatment on growth and IAA level in internode 6
Treatment
Fresh weight (g)Elongation
(mm)
IAA
(ng gFW−1)
  1. Elongation is shown as the mean ± standard error of the difference between internode length at decapitation (approximately 98 mm in all cases) and the length 48 h later (n = 15). For the other means there were three replicates, each replicate consisting of five plants. GA levels from these plants are shown in Fig. 3.

Intact1.56 ± 0.0321.2 ± 1.8140.3 ± 8.4
Decapitated1.43 ± 0.059.8 ± 1.612.9 ± 0.8
Decapitated + IAA1.74 ± 0.0420.7 ± 2.3546.7 ± 9.4

To examine the molecular basis of the effects of IAA on GAs, we monitored the expression of the PsGA3ox1 and PsGA2ox1 genes, which encode enzymes for the steps GA20 to GA1 and GA20 to GA29, respectively ( Fig. 1; Lester et al. 1997 ; Lester et al. 1999 ; Martin et al. 1997 ; Martin et al. 1999 ). The level of PsGA3ox1 transcript in internode 6 was dramatically reduced by decapitation, but was restored by IAA application to the stump ( Fig. 4). In complete contrast, decapitation increased PsGA2ox1 transcript levels, an effect opposed by IAA application ( Fig. 4).

Figure 4.

Effects of decapitation and IAA application on PsGA3ox1 and PsGA2ox1 transcript levels in internode 6, monitored by Northern analysis.

Plants were either left intact, decapitated above node 7, or decapitated above node 7 and then treated with IAA. Ten μg of total RNA was loaded in each lane. Corresponding gels stained with ethidium bromide are shown: ribosomal RNAs are visible, indicating the loading of lanes. Conditions differed for the two genes; for example, autoradiograph exposure times for PsGA3ox1 and PsGA2ox1 were 48 h and 6 h, respectively.

Discussion

The results indicate that applied IAA can completely substitute for the apical bud in maintaining stem GA1 content. This clearly supports the second of the two hypotheses mentioned in the Introduction, namely that GA1 biosynthesis can occur elsewhere than the apical bud but only when the level of IAA is adequate. We suggest that decapitation affects GA levels by firstly removing the source of auxin (the apical bud) and thereby reducing the auxin content of the stem. This in turn leads (directly or indirectly) to a dramatic reduction in the transcript abundance of PsGA3ox1 and to an increase in that of PsGA2ox1 ( Fig. 4). The stem is then essentially unable to convert GA20 to GA1 ( Figs 2 and 3), while the step GA20 to GA29 is up-regulated ( Table 1 and Fig. 3). Application of IAA to decapitated plants increases the IAA content, restoring PsGA3ox1 and PSGA2ox1 transcript levels to normal ( Fig. 4) and thereby increasing the GA1 level. The corollary is that in the intact pea plant a supply of auxin from the apical bud is necessary to maintain normal GA1 levels in the stem.

It is clear that IAA promoted overall GA1 biosynthesis in decapitated pea shoots, as indicated by analysis of all remaining portions after treatment with [13C3H]GA20 ( Table 1). However, it is formally possible that, in addition, IAA influenced internode GA1 content by promoting GA1 transport from leaves to stems. The fact that this was not the main effect of IAA is indicated by two lines of evidence. First, there were no peaks attributable to [13C3H]GA1 in extracts from the leaves treated with [13C3H]GA20 ( Table 1), and secondly, there was a strong effect of IAA on PsGA3ox1 transcript level in the stem itself ( Fig. 4).

The mechanism by which IAA affects PsGA3ox1 transcript levels is presently unknown. There is no evidence as yet that the LE promoter contains sequences associated with the better-known auxin response elements ( Guilfoyle et al. 1998 ). However, there is evidence that another auxin, 4-chloroindole-3-acetic acid, affects the mRNA abundance of a different GA biosynthesis gene, a GA 20-oxidase, in pea pericarp ( van Huizen et al. 1997 ).

The association between low PsGA3ox1 transcript level and low GA1 level, observed here in decapitated internodes, is somewhat unusual, since reduced GA1 content (for example, in GA synthesis mutants) is often associated with high PsGA3ox1 transcript abundance ( Martin et al. 1997 ; Ross et al. 1999 ). This is because GA1 negatively regulates the PsGA3ox1 transcript level ( Martin et al. 1997 ). It appears that in decapitated internodes, auxin deficiency completely overrides the relief of negative feedback which might be expected to result from GA1 deficiency. The present results raise the question of whether the up-regulation of PsGA3ox1 transcript levels in GA1-deficient mutants is mediated by an increase in IAA level. However, this appears not to be the case because the IAA level in GA1-deficient mutants is actually reduced (albeit slightly) rather than elevated, compared with the WT ( Law & Davies 1990; McKay et al. 1994 ).

The present results have other significant implications for understanding the hormonal regulation of stem growth. In view of the importance of GA1 for elongation ( Ingram et al. 1984 ; Kende & Zeevaart 1997), and the present evidence that auxin affects GA1 levels (in stems, at least), it is possible that in intact plants part of the growth response to auxin is mediated by GA1. Certainly, the large changes in GA1 level observed here ( Fig. 3) would be expected to affect elongation, given that le mutations, which reduce GA1 content by six- to 10-fold, produce plants about 70% shorter than the WT ( Ingram et al. 1984 ; Ross et al. 1995 ). However, it is probable that auxin also directly affects elongation, given the rapid effects of applied IAA ( Haga & Iino 1998; Yang et al. 1993 ). In our experiments, decapitation reduced whilst IAA restored internode elongation growth ( Table 2), but it is not clear if this was a direct effect of IAA, or mediated by GA1. It does seem clear, however, that the observed effect of IAA on GA1 levels was not a result of enhanced elongation, because IAA-treated plants contained more GA1 than intact plants ( Fig. 3), but their elongation was not greater ( Table 2).

In summary, the results indicate that in pea stems the biosynthesis of GA1 from GA20 depends in some way on the presence of auxin. This new link between these two classical hormones provides valuable insight into both the regulation of GA levels and the mechanism of auxin action.

Experimental procedures

Plant material

The pea (Pisum sativum L.) line used was the tall (wild-type) line 205+. Plants were grown, two per pot, in a heated glasshouse as described previously ( Beveridge & Murfet 1996). The photoperiod was 18 h, provided by extending the natural photoperiod at its beginning and end with a mixture of white fluorescent (40 W) and incandescent (100 W) lights (intensity 25 μmol m−2 s−1 at pot top). All node counts began from the cotyledons as zero. Internode 6 was the internode between nodes 6 and 7, and leaf 6 was the leaf at node 6 ( Fig. 2).

Treatments and harvesting

For each experiment (apart from that represented in Table 1), we chose plants with seven expanded leaves (including scale leaves), and with the internode between nodes 7 and 8 (internode 7) 20–40 mm long (15–30% of its potential final length). Plants were then either decapitated approximately 15 mm above node 7, or left intact (see Fig. 2). Half of the decapitated plants were treated immediately with IAA (Sigma) in hydrous lanolin (3000 parts per million; approximately 15 mg lanolin per plant); the other half received lanolin only. IAA/lanolin or pure lanolin were re-applied after a further 8, 20, 32 and 44 h, on each occasion removing the previous lanolin. It was reasoned that this application frequency should maintain a high level of IAA in internode 6 (approximately 100 mm long at the beginning of the experiments), since a typical rate of IAA movement is 10 mm h−1 ( Johnson & Morris 1989). Where required, immediately after decapitation and the first IAA treatment, plants were treated (on the leaf at node 6, termed leaf 6) with 90 000 dpm (per plant) of [17,17 13C3H]GA20 (88% 13C, 15 mCi mmol−1; from Dr C.L. Willis, University of Bristol, Bristol, UK) in 10 μl of ethanol. In separate experiments, after 48 h (from the time of decapitation) internode 6 was harvested for analysis of [13C3H]GA20 metabolites ( Fig. 2), endogenous GAs ( Fig. 3), or transcript levels ( Fig. 4). IAA levels were also analysed in the first two cases. Typically, there were 15 plants per treatment, which were harvested in three replicate groups of five. The experiment represented in Table 1 was conducted similarly, except that plants were decapitated above node 6, [13C3H]GA20 was applied to leaf 5, and (after 2 days) leaf 5 (excluding stipules), expanding internodes (internode 5 plus the ‘stump’ of internode 6, excluding the top 3 mm), the remainder of the shoot (including the stiplules at node 5) and the roots were harvested and analysed separately. There were nine plants per treatment.

Material for GA analyses was immediately immersed in cold (−20°C) 80% methanol, containing butylated hydroxytoluene (Sigma, 250 mgl−1), and stored in a freezer (at −20°C). Material for Northern analysis was immersed in liquid nitrogen and extracted immediately.

Extraction and analyses

For GA and IAA analyses tissue was homogenised, hormones were extracted at 3°C for 24 h, and extracts were then filtered (Whatman no. 1). In [13C3H]GA20 metabolism experiments, separate aliquots of the filtrate were taken for radiocounting (to measure the total radioactivity), for IAA determinations, and for analysis by HPLC-radiocounting. For the latter purpose, extracts were purified using Sep-Pak C18 cartridges and chromatographed as methyl esters as previously ( Ross 1998). One minute fractions were collected for radiocounting. In some cases 50% aliquots of HPLC fractions were counted, with the remainder stored for confirmation of metabolite identity by full-scan GC-MS, performed as described previously ( Ross et al. 1995 ). Endogenous GAs and IAA were analysed by GC-MS-selected ion monitoring with internal standards, as described previously ( Ross 1998), but using 13C6 IAA (Cambridge Isotope Laboratories, Woburn, MA, USA) as an internal standard.

Northern analyses, using total RNA extracted by a phenol/SDS method ( Ausubel et al. 1994 ), were performed basically as described previously ( Lester et al. 1999 ). DNA probes were labelled with 32P by random priming (Bresatec, Adelaide, South Australia). After hybridization, filters were washed with 2 × SSC at 42°C and with 0.2 × SSC at 65°C.

Acknowledgements

We thank Professor Peter Davies for first suggesting to us that auxin might affect GA biosynthesis in stems; Dr Diane Lester for helpful advice; Dr Noel Davies (Central Science Laboratory, University of Tasmania, Australia), Ian Cummings and Tracey Jackson for technical assistance; Dr Christine Willis and Professor Lewis Mander for labelled GAs; Jane Burrell for artwork; Professor Jim Reid for pea seed; and the Australian Research Council for financial assistance.

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