Auxin-Gibberellin Interactions and Their Role in Plant Growth

J Plant Growth Regul 2002) 20:346±353 DOI: 10.1007/s003440010034

John J. Ross,Ã Damian P. O'Neill, Carla M. Wolbang, Gregory M. Symons, and James B. Reid

School of Plant Science, University of Tasmania, GPO Box 252-55, Hobart, Tasmania 7001, Australia

ABSTRACT Recently it was discovered that auxin promotes auxin as a messenger linking the apical bud with the gibberellin GA) biosynthesis in decapitated stems of biosynthesis of active GAs in the expanding inter- pea Pisum sativum L.) and tobacco Nicotiana tabacum nodes. Finally, new evidence is presented that the

L.), and here we review the evidence for this inter- promotion of growth by GA1 does not depend on action. We also discuss the possible relationship be- GA1-induced increases in auxin content. tween auxin and the mechanisms by which bioactive

GAs such as GA1) regulate their own levels, and the Key words: Auxin; Feed-back regulation; Gibber- implications of the auxin-GA interaction for the ellin; Hormone interactions; Internode elongation; control of plant growth. It is now possible to envisage Pisum sativum

INTRODUCTION Figure 1), reducing the content of bioactive GA1 and thereby resulting in dwar®sm Ingram and Auxin is one of the ®ve ``classical'' plant growth others 1984). More recently, LE was cloned and hormones Kende and Zeevaart 1997) and was the shown to encode the enzyme for the step GA20 to ®rst to be isolated. The discovery of a second growth GA1 Lester and others 1997; Martin and others hormone, gibberellin GA), brought with it much 1997). It was also shown that LE now termed speculation about how auxin and GA might interact PsGA3ox1) is down-regulated by GA1 Martin and Brian and Hemming 1957, 1958; Vlitos and Meudt others 1997; Ross and others 1999), as part of a 1957; Galston and Warburg 1959). This question was, ``feed-back'' mechanism Hedden and Croker 1992). and still is, of paramount importance for under- Auxin research has also made great progress. For standing the hormonal regulation of plant growth. example, there have been major breakthroughs in Nevertheless, much of our knowledge on auxin isolating key elements of the polar auxin transport and GAs has come from studying them indepen- system reviewed by Jones 1998). Furthermore, dently. In GA studies Mendel's le mutant now genes that are up- or down-regulated by auxin have termed le-1) has played a pivotal role. Early research been extensively characterized Abel and Theologis demonstrated the spectacular growth-promoting 1996; Guilfoyle and others 1998). Some of these effects of applying GA3 to intact le-1 plants Brian genes are up-regulated within a few minutes of and Hemming 1955). In the 1980s it was shown that auxin treatment. These ``early'' or ``primary'' genes le-1 blocks the crucial activation step GA20 to GA1 are thought in turn to regulate secondary or ``late'' auxin response genes Abel and Theologis 1996).

Online publication: 18 March 2002 Recently, Ross and O'Neill 2001) proposed a new ÃCorresponding author; e-mail: [email protected] model that brings together the auxin and GA ®elds.

346 Auxin-Gibberellin Interactions 347

the stem, below the application site, while GA29 accumulates Ross 1998). Results from the bushy mutant of pea, which is characterized by profuse branching and short internodes Symons and others 1999), are also consistent with the model. Free IAA levels in intact bushy shoots are markedly reduced compared with wild-type plants G,M. Symons and 14 others unpublished). After feeds of [ C]GA20, the pattern of metabolism in bushy plants was very similar to that observed in decapitated wild-type

plants, with the step GA20 to GA29 predominating over GA20 to GA1 G.M. Symons and others unpublished). As a result, bushy plants contain less

endogenous GA1 than the wild-type. Two other pea mutants, gi and veg1, also contain reduced levels of

both IAA and GA1 Beveridge and others 2001). Therefore there is clear evidence for a link between IAA and GA in pea, but how widespread is the relationship? We chose tobacco as a second species in which to investigate the auxin-GA interaction. Tobacco is not closely related to pea, and any Figure 1. Relevant parts of the GA pathway in pea and similarity in the interaction might indicate that it is tobacco. Solid arrows or bars indicate the steps known to widespread. be affected by IAA, on the basis of metabolism experi- In tobacco, decapitation again inhibited the step ments. The stronger the effect, the broader the arrow or GA20 to GA1, and this was reversed by IAA, but the bar. effects were less dramatic than in pea Wolbang and Ross 2001). The most intriguing outcome was that

The model is based on the discovery that LE the conversion of GA19 to GA20 Figure 1) was PsGA3ox1) is an auxin-regulated gene Ross and strongly inhibited by decapitation. This step, termed others 2000). In pea shoots the main auxin is indole- GA 20-oxidation, is an important, regulated step in 3-acetic acid IAA), which is transported from the a number of species Hedden and Kamiya 1997; apical bud into the elongating internodes Lomax Hedden 1999). Once again, IAA counteracted the and others 1995). Decapitation removing the apical effect of decapitation, increasing GA20 levels to bud) reduces the IAA content of the remaining stem three times that observed in intact plants. Hence, it tissue Beveridge and others 1994; Ross and others was concluded that in tobacco, as in pea, IAA from 2000). In decapitated stems PsGA3ox1 transcript the apical bud is required for GA biosynthesis in levels, and consequently GA1 biosynthesis, are also stems. However, the main steps promoted appear to dramatically reduced Ross and others 2000). In differ between the two species: 20-oxidation in to- contrast, expression of the GA deactivation gene SLN bacco, and 3-oxidation in pea Figure 1).

!PsGA2ox1), which encodes the enzyme for the steps In tobacco, GA19 is known to accumulate to a GA20 to GA29 and at least in vitro)GA1 to GA8 greater extent than other GA intermediates Nilsson Lester and others 1999; Martin and others 1999), is and others 1993), indicating that GA19 to GA20 increased by decapitation Ross and others 2000). might be the rate-limiting step. In contrast, GA19 to The application of IAA in lanolin paste) to the GA20 proceeds rapidly in pea, where the rate-lim- `stump' of decapitated peas restores both PsGA3ox1 iting step is thought to be 3-oxidation Martin and transcript levels and GA1 biosynthesis in the stems. others 1996). Therefore, the GA biosynthetic step Furthermore, IAA down-regulates PsGA2ox1 tran- most affected by IAA in tobacco and pea stems ap- script levels, compared with untreated decapitated pears to correspond with the rate-limiting step. plants Ross and others 2000). In a subsequent In pea stems there was no evidence from endog- study, auxin was shown to exert these effects within enous GA levels for a strong effect of IAA on the

2 h of application O'Neill and Ross unpublished). conversion of GA19 to GA20. However, in pea pods Further evidence for the new model comes from this step does appear to be auxin-regulated, but by a applying auxin transport inhibitors to intact plants different auxin, 4-chloro-IAA van Huizen and Ross 1998). When applied in a ring of lanolin, these others 1997). It was suggested that 4-Cl-IAA might compounds reduce both the IAA and GA1 content of move from young seeds into the pods, promoting the 348 J. J. Ross and others

step GA19 to GA20, and therefore pod elongation esting to note that in Ross and others 2000), de- van Huizen and others 1997). However, while the capitated plants treated with IAA contained 3-fold importance of GA1 for internode elongation is be- more GA1 than did intact plants, indicating that in yond dispute, its role in pod elongation is not so clear some circumstances at least, the stem IAA content

MacKenzie-Hose and others 1998). The main rea- might be limiting for GA1 biosynthesis. son for this is that the pods of certain dwarf mutants such as le-1) elongate to the same extent as wild- type pods, despite being GA1-de®cient Santes and IS THERE ARELATIONSHIP BETWEEN others 1993; MacKenzie-Hose and others 1998). AUXIN AND THE FEED-BACK REGULATION OF GA BIOSYNTHESIS? CRITICAL ASSESSMENT OF THE EVIDENCE: The most widely studied factor in¯uencing GA THE ``PESIGS RULES'' biosynthesis is the ``feed-back'' mechanism, Does the evidence discussed above actually establish whereby bioactive GAs negatively regulate their own production Scott 1990; Croker and others that endogenous IAA is necessary for GA1 biosynthesis in stems? Certainly it seems to meet the re- 1990; Hedden and Kamiya 1997). Recently it has quirements of the ``PESIGS Rules'' Jacobs 1979; been shown that bioactive GAs can also up-regulate Davies 1995), established for determining whether expression of GA deactivation genes Thomas and or not a hormone performs a particular role. The others 1999) and this ``feed-forward'' mechanism, ®rst of these rules ``P'') concerns the presence of the along with feed-back, tends to maintain constant hormone and parallel variation between the level of levels of bioactive GAs such as GA1. How auxin the hormone IAA in this case) and the response in relates to the feed-back and feed-forward mechanisms is not yet known. Because of feed-back reg- question GA1 biosynthesis). IAA is de®nitely present in pea stems, and there were parallel reduc- ulation, wild-type pea plants contain lower levels of tions in levels of the two hormones after treatment PsGA3ox1 transcript than do GA1-de®cient mutants with auxin transport inhibitors Ross 1998), and in Martin and others 1997; Ross and others 1999). In the bushy mutant. The second PESIGS requirement contrast, in decapitated wild-type internodes, a low is also satis®ed, since excision of the apical bud, PsGA3ox1 transcript level is associated with a low thought to be the main source of IAA for stems, GA1 content Ross and others 2000). This indicates that the auxin de®ciency of decapitated internodes reduces stem GA1 content Sherriff and others 1994; Ross and others 2000). The third requirement, sub- might override the feed-back mechanism. stitution, is clearly met by the ability of applied IAA At this stage, however, we cannot be sure that feed-back regulation of PsGA3ox1 occurs in the in- to promote GA1 biosynthesis in decapitated plants Ross and others 2000; Wolbang and Ross 2001). In ternodes themselves, rather than being con®ned to the leaves and/or apical bud Elliott and others isolated stem sections of pea, IAA also promotes GA1 production D.P. O'Neill and J.J. Ross unpublished). 2001). On the other hand, feed-forward regulation The generality of the auxin-GA interaction is indi- of PsGA2ox1 transcript levels is known to occur in cated by the results from tobacco, but we aim to internodes Elliott and others 2001), but is still over- further extend our studies, especially to monocot- ridden by auxin de®ciency: decapitated internodes yledonous species. The ®nal PESIGS requirement contain high, not low, levels of PsGA2ox1 mRNA concerns the speci®city of the response. We have not Ross and others 2000). yet tested a wide range of compounds, but it is clear It might be suggested that the up-regulation of that GA itself does not promote GA biosynthesis in PsGA3ox1 transcript level in GA1-de®cient mutants is 1 1 mediated by an increase in IAA level. However, this is the same way as IAA. Indeed, GA1 is known to in- hibit its own biosynthesis see following section). not supported by evidence that GA1-de®cient mu- Thus, the PESIGS rules seem to be satis®ed with tants contain less, not more, IAA than the wild type regard to demonstrating that IAA is required for Law and Davies 1990; McKay and others 1994). normal GA1 biosynthesis in stems. However, this does not mean that GA1 levels are actually regulated by IAA. It is possible that a threshold level of IAA is IMPLICATIONS OF THE AUXIN-GA required, above which GA1 biosynthesis is normal, RELATIONSHIP FOR GROWTH and below which it is essentially prevented. We are presently studying the quantitative relationship According to the Ross and O'Neill 2001) model, between IAA content and GA1 content. It is inter- IAA is a messenger compound from the apical bud, Auxin-Gibberellin Interactions 349 activating GA biosynthesis in the elongating inter- In contrast to our ®ndings with pea, Collett and nodes. Indeed, the main growth-regulating function others 2000) concluded that there is no interaction of endogenous IAA might be to maintain GA1 levels between auxin and GA in controlling the growth of in the internodes, although our model by no means Arabidopsis hypocotyls. However, it should be borne precludes a direct effect of auxin on growth. Cer- in mind that there are signi®cant differences be- tainly, it is clear that IAA can affect GA1 biosyn- tween pea stems and Arabidopsis hypocotyls. For thesis in very young internodes less than 25% of example, auxin was inhibitory to elongation in their ®nal length) J.J. Ross and C.M. Wolbang Arabidopsis hypocotyls Collett and others 2000) but unpublished), and internodes at this stage of ex- stimulatory in decapitated peas Ross and others pansion are GA-responsive Ross 1998). It is there- 2000). Furthermore, GA affects both cell length and fore highly likely that in these internodes auxin- cell number in pea internodes Reid and others induced GA1 is important for elongation. 1983), but only the former in Arabidopsis hypocotyls It is interesting to reconsider previous studies on Gendreau and others 1997). the interaction between auxin and GA in view of our recent evidence. For example, Davies and Ozbay 1975) and Haga and Iino 1998) decapitated tall pea plants, applied IAA in lanolin paste, measured the EVIDENCE THAT GA1 PROMOTES growth response, and ascribed that response to IAA. ELONGATION PER SE It now appears possible that at least part of the growth response was in fact mediated by an increase in GA1 The suggestion by Ross and O'Neill 2001) that content. Certainly the decapitated plants studied by auxin might promote elongation at least in part) by Davies and Ozbay 1975) were GA-responsive. increasing GA1 levels contrasts with an earlier the- Our results also raise the possibility that if an ory Lantican and Muir 1969; Kuraishi and Muir environmental factor affects auxin content, GA1 1962) that part of the GA response is mediated by levels might also be altered as a consequence. For an increase in auxin level. Further evidence for the example, a conventional wisdom is that pea stems early theory is the apparent correlation between bending towards the light should contain more IAA content and stem elongation across a range of auxin on the shaded side than on their irradiated GA-related mutants Law and Davies 1990). McKay side the Cholodny-Went theory) Kaufman and and others 1994) also found that the internodes of 5839 others 1995). If this is the case, does the shaded side a dwarf mutant with reduced GA1 levels, le also contain more GA1 than the irradiated side? since re-named le-3), contained less IAA than wild- Physico-chemical analyses are required to answer type internodes. Furthermore, GA application was this question. At present it is not even clear that reported to increase IAA levels in intact plants Law auxin itself is asymmetrically distributed across and Hamilton 1984) and in isolated stem sections phototropically bending pea stems. Barratt and Davies 1997). Thus GA1-induced It is clear, however, that there is a gradient of IAA elongation is usually accompanied by an increase in from the tip of the pea shoot to its base Beveridge IAA content, and it has always been dif®cult to and others 1994). Gibberellin A1 levels are also lower exclude the possibility that these increases, al- in mature tissue than in immature tissue Smith and though typically only about 2-fold, actually cause others 1992; Ross 1998), and it might be speculated the growth response. Clearly, it is important to ask, that this is due to the low IAA content of the former. ``Can GA1 promote elongation independently of its However, this appears not to be the case. When de- effect on IAA level?'' capitated mature internodes were treated with IAA, To address that question, we designed experi-

GA20 3-oxidation was certainly enhanced, compared ments to gauge the signi®cance for growth of the with untreated decapitated controls, but the GA1 GA1-induced changes in IAA content, using Men- content was not restored to that of immature tissue del's le-1 dwarf mutant. The le-1 line was 205-, J.J. Ross and others unpublished). The reason for which is isogenic with 205+, the main tall line used this appears to be that in mature pea tissue, GA 2- in previous auxin-GA experiments Ross and others oxidation activity is very strong, and although ma- 2000). In a series of experiments le-1 plants were ture tissue synthesizes GA1, it is rapidly deactivated decapitated and treated with auxin or GA1 and their to GA8. IAA application appears incapable of sub- growth responses were monitored. A previous paper stantially reducing the 2-oxidase activity of mature reported the strong GA response of decapitated le-1 tissue, and it is likely, therefore, that the 2-oxidase internodes Ross 1998). responsible is not PsGA2ox1 which is IAA-respon- The data in Figure 2 show that the GA1 response sive) Ross and others 2000). in decapitated le-1 internodes was 70% of that 350 J. J. Ross and others

Figure 2. Effects of applied GA1 on elongation and hormone content of decapitated le-1 plants. Plants were grown in a heated glasshouse with an 18 h photoperiod, as described previously Ross and others 2000). When the plants had 7 fully expanded leaves node 7 is indicated), uniform plants were either left intact 2 plants on the left) or decapitated immediately below node 8. GA1 was then applied 10 lgin10ll of ethanol) to leaf 6. Internode 7-8 was measured at decapitation initial length indicated by dashed lines) and, 2 days later, measured again and harvested for hormone analysis. Hormone levels were measured using stable isotope-labeled internal standards and GC-MS, as before Ross 1998). The internodes are drawn to scale scale bar = 20 mm). Shown are means and standard errors from 9 Figure 3. Length a) and IAA content b)ofle-1 in- replicates for elongation data and from 2 replicate har- ternodes at several time points after decapitation and GA1 vests each consisting of internodes from 4 or 5 plants) 3treatment, performed as described for Figure 2. Intact, h; for hormone levels. decapitated, n or ); decapitated + GA1, O. Shown are means from 10 replicates for length data and from 2 shown by accompanying intact plants. In both in- replicate harvests each consisting of internodes from 5 to tact and decapitated plants, GA application resulted 7 plants) for IAA levels. Standard errors are shown where 1 they exceed the symbol. in a higher IAA content, con®rming previous results see, for example, Barratt and Davies 1997). How- ever, it is improbable that these small approxi- 28, and 45 h after decapitation/GA1 application. mately 2-fold) changes in IAA mediated the growth There was no evidence for a large early increase response to GA1 because decapitated internodes in IAA content in GA1-treated plants. Decapitation treated with GA1 still contained very little IAA: rapidly reduced IAA content, by approximately 10- about 7% of the level present in the intact inter- fold after 4 h, and this was the case with and without nodes without GA1 measured 2 days after decapi- applied GA1 Figure 3). By 21 h, both the IAA tation). Despite their low IAA content, the content and elongation of decapitated plants were elongation of decapitated GA1-treated internodes increased by GA1, but the level of IAA was still only was more than twice that of intact internodes about 11% of the level in intact internodes. Figure 2). These results clearly break the correla- It was also possible that decapitated plants were so tion between rapid elongation growth and high IAA sensitive to auxin that even the small GA1-induced levels, and indicate that GA1 promotes elongation in increase in IAA content Figures 2 and 3) could ac- its own right, rather than by increasing IAA con- count for the growth response to GA1. To investigate tent. this possibility we applied varying dosages of IAA to

It was possible, however, that GA1 caused a large decapitated plants and monitored the elongation but transient increase in IAA, which had dissipated response compared with decapitated plants treated by 2 days after application. We therefore conducted with GA1. Internodes given the lowest IAA dose a time-course experiment with intact, decapitated, contained a similar level of IAA to GA1-treated and decapitated + GA1 treatments. Internode length plants, but their elongation was not promoted and endogenous IAA levels were measured 4, 9, 21, compared with decapitated controls Table 1). Auxin-Gibberellin Interactions 351

Table 1. Effects of Applied GA1 and IAA on Elongation and IAA Content of Decapitated le-1 Internodes, 48 h after Decapitation

Treatment Elongation mm) IAA level ng.g FW)1) Intact 15.9 0.6 b) 108 7.0 b) Decap. 9.5 0.7 d) 6.0 1.0 e)

Decap. + 10 lgGA1 27.8 2.5 a) 9.4 0.6 d,e) Decap. + IAA/lanolin at 0.006 mg.g)1 9.6 0.8 d) 10.0 0.5 d) Decap. + IAA/lanolin at 0.02 mg.g)1 11.7 0.7 b,c,d) 17.0 0.7 c) Decap. + IAA/lanolin at 0.1 mg.g)1 11.6 1.1 c,d) 72 4 b) Decap. + IAA/lanolin at 1.0 mg.g)1 14.7 0.4 b,c) 312 11 a)

IAA was applied in lanolin paste to the top of the decapitated internode every 12 h, as before !Ross and others 2000). GA1 was applied as described in Figure 2. Data are means s.e. of 8 replicates for elongation and 2 replicates !each comprising 4 plants) for IAA levels. Within a column, means with the same letter, are not signi®cantly different !P < 0.05).

Again, these data indicate that GA1 did not promote surprising that there is such a simple and direct re- elongation by increasing IAA level. Furthermore, lationship between the two classical growth hor- there was no evidence that decapitated plants were mones. Interestingly, as far as stem elongation is more sensitive to IAA than intact plants, consistent concerned, it is now possible to view GA1 as a with a previous ®nding Haga and Iino 1998). component of the auxin signalling pathway. A dif- In summary, it appears that the slight effect of ferent viewpoint is that auxin is important for

GA1 on IAA content, although consistently ob- growth primarily because it increases the level of served, is too small to contribute markedly to GA1- bioactive GA. Regardless of perspective, our results induced elongation. It should be noted also that the provide further evidence that internode elongation effect of IAA on GA1 biosynthesis is much stronger is strongly in¯uenced by the apical bud. When the than the reverse effect. For example, in a previous bud is damaged or removed for example, by graz- study, decapitation reduced IAA content by 6-fold, ing), the levels of both auxin and GA1 decrease in and virtually eliminated the ability to accumulate the internodes, in turn reducing elongation growth. labeled GA1 after feeds of labeled GA20 Ross and Therefore, potentially wasteful extension of the others 2000). In contrast, the data in Figure 2 show decapitated internodes can be avoided, and future that changes in GA1 of more than 1000-fold caused growth concentrated instead in the side shoots that only 2-fold changes in IAA content. grow out after decapitation.

The GA1-induced elongation of decapitated Our present results indicate that in some cir- internodes is also signi®cant because it has been cumstances at least, GA1 can stimulate growth per suggested that, at least in some circumstances, a se, rather than by increasing auxin content. The idea growth response to GA depends on the presence of of GA1 as the ®nal hormonal regulator of stem auxin Brian and Hemming 1958; Barratt and Davies growth is consistent with conclusions from studies 1997). It now appears that under our conditions GA on deep-water rice. Although ethylene, abscisic can in fact strongly promote growth even when acid, and GA are all thought to play a role in reg- auxin levels are low Figures 2 and 3; Table 1). Nev- ulating the elongation of deep-water rice, the actual ertheless, the lower elongation of GA1-treated de- effector is thought to be GA1 Kende and others capitated plants, compared with GA1-treated intact 1998). In the present paper we have referred to the plants Figure 2), might indicate that IAA is required growth-promoting effects of GA1, because the effects for the maximal GA response. The growth difference are dramatic after applying the hormone. However, between GA-treated decapitated and intact plants it should be borne in mind that GA1 is probably an was most likely not due to a difference in GA1 level, inhibitor of a growth inhibitor Brian and Hemming since this level was very high in both cases Figure 2). 1958; Richards and others 2001). Signi®cantly, recent models based on this concept Richards and others 2001) do not appear to involve other hor- CONCLUSIONS mones as mediators of the GA message. Future studies will address the question of In pea and tobacco, and possibly in other species, it whether or not other hormones affect the GA now appears that auxin from the apical bud is re- pathway. It is well known that auxin stimulates the quired for normal GA1 biosynthesis in stems. It is production of ethylene. However, it is unlikely that 352 J. J. Ross and others ethylene mediates the promotion of GA biosynthe- ACKNOWLEDGMENTS sis by IAA, because in terrestrial plants, ethylene is We thank Dean Williams for statistical analyses, actually a growth inhibitor Kende and Zeevaart Emma Bayly-Stark for photography, Noel Davies, 1997; Fuchs and Lieberman 1968). This contrasts Ian Cummings, Tracey Jackson, and Robert Wilt- markedly with its role in submerged plants, where shire for technical assistance, Jane Burrell and An- the increased ethylene level plays a major role in dre Phillips for artwork, and the Australian promoting elongation by enhancing GA levels and/or Research Council for ®nancial support. GA sensitivity Kende and others 1998). In view of our recent results from pea and tobacco, it is interesting to speculate on the auxin-GA REFERENCES relationship in the classical auxin model system, the grass coleoptile. 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