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First published online January 15, 2004; 10.1104/pp.103.030460 Plant Physiology 134:769-776 (2004) © 2004 American Society of Plant Biologists Auxin from the Developing Inflorescence Is Required for the Biosynthesis of Active Gibberellins in Barley Stems1School of Plant Science, University of Tasmania, G.P.O. Box 252–55, Hobart, Tasmania 7001, Australia (C.M.W., J.J.S., J.J.R.); and Commonwealth Scientific and Industrial Research Organization Plant Industry, G.P.O. Box 1600, Canberra, Australian Capital Territory 2601, Australia (P.M.C.)
Multiple gibberellins (GAs) were quantified in the stems of intact, decapitated, and decapitated auxin-treated barley (Hordeum vulgare) plants. Removal of the developing inflorescence reduced the endogenous levels of indole-3-acetic acid (IAA), GA1, and GA3 and increased the level of GA29 in internodal and nodal tissues below the site of excision. Application of IAA to the excised stump restored GA levels to normal in almost all cases. The conversion of [14C]GA20 to bioactive [14C]GA1 and of [14C]GA5 to bioactive [14C]GA3 was reduced by decapitation, and IAA application was able to restore conversion rates back to the levels found in intact plants. The amount of mRNA for the principal vegetative 3-oxidase (converting GA20 to GA1, and GA5 to GA3) was decreased in decapitated plants and restored by IAA application. The results indicate that the inflorescence of barley is a source of IAA that is transported basipetally into the internodes and nodes where bioactive GA1 and GA3 are biosynthesized. Thus, IAA is required for normal GA biosynthesis in stems, acting at multiple steps in the latter part of the pathway.
The classical plant hormones auxin and GA, which are responsible for elongation and various other processes, have been investigated extensively both individually and simultaneously, the latter in an effort to elucidate the relationship between them. In the past, many conflicting theories have been put forward concerning their interactions (Brian and Hemming, 1958  ; Kurashi and Muir, 1962; Law and Davies, 1990; van Huizen et al., 1997), but recently, it was discovered that in pea (Pisum sativum) stems the auxin indole-3-acetic acid (IAA) promotes GA biosynthesis. Auxin was found to increase the transcript level of a GA 3-oxidase, PsGA3ox1, encoded by Mendel's LE gene, and to reduce that of a GA 2-oxidase, PsGA2ox1, which encodes an enzyme for GA deactivation (Ross et al., 2000). Later, this model was extended into another dicot species, tobacco (Nicotiana tabacum), where IAA predominantly promotes a different biosynthetic step, GA 20-oxidation (Wolbang and Ross, 2001), highlighting the comparable yet distinct nature of the auxin-GA relationship in that species.
The aim of the present research was to determine whether auxin promotes GA biosynthesis in monocotyledons, in particular the grasses. Previous studies on GAs at the seedling and successive stages of grass development are abundant (Fujioka et al., 1988a
Koning et al. (1977 We focus on the maturing reproductive plant, specifically the p-1 internode and the nodes above and below it. We employ a number of techniques to investigate the auxin-GA relationship including decapitation and IAA application, GA quantification and metabolism studies, and determination of mRNA levels by quantitative PCR. In addition, analyses of specific areas of growth within the p-1 internode of barley (Hordeum vulgare) are undertaken. Evidence is presented that the auxin IAA promotes GA biosynthesis in barley, and the role of the developing inflorescence is discussed in relation to this auxin/GA interaction.
Effects of Decapitation and IAA Application on Endogenous GA Levels
Experiments were conducted with oat and barley. Oat plants were included to enable comparison with previous reports (Kaufman et al., 1976
Decapitation of barley plants also reduced IAA, GA1, and GA3 levels in the p-1 internode, in this case harvested with the intercalary meristem at the base of the internode (Table II). Similar results were obtained in a separate experiment (Fig. 2), in which the p-1 internode and the p node above it (including the intercalary meristem of the p internode) were harvested separately. Application of IAA again restored IAA, GA1, and GA3 levels to at least those found in intact internodes. The GA3, GA1, and IAA data in Table II were obtained after purification of the samples by HPLC as methyl esters. This was necessary to remove an impurity that co-eluted with GA3 as free acids on HPLC and that produced a 504 mass-to-charge ratio ion that eluted very close to GA3 on gas chromatography-mass spectrometry (GC-MS) with the nonpolar column used in this work. This compound may correspond to that previously reported by Croker et al. (1990
The level of GA29 was increased by decapitation, whereas those of GA20 and GA19 were largely unaffected or reduced slightly (Fig. 2). GA5 was not successfully quantified. GA levels were generally greater in the nodal zone than in the p-1 internode (Fig. 2). This is consistent with the localization of growth in the basal portion of the internode (Table III). In intact internodes, the level of GA1 was approximately 2-fold greater than that of GA3 (Table II). Metabolic relationships between the GAs are shown in Figure 3.
The inflorescence of barley was found to contain approximately 3-fold more IAA than, and comparable levels of GA1 and GA3 to, the p-1 internode (data not shown). Other auxins (4Cl-IAA and indolebutyric acid [IBA]) were not detectable in the p-1 internodal tissue, although internal standards for both were recovered by GC-MS (data not shown). Decapitation generally reduced the elongation of the p-1 internode in both barley and oat (data not shown). Application of IAA did not consistently restore growth to that of intact plants. However, in the experiment reported in Table I, IAA significantly stimulated elongation of oat internodes (mean elongation values for decapitated and auxin-treated decapitated internodes were 6.0 ± 1.1 and 15.3 ± 2.5 mm, respectively).
Quantification of GA1 levels and measuring internode lengths of the grd2-463 3-oxidase mutant (Chandler and Robertson, 1999
[14C]GA20 and [14C]GA5 were fed to barley stems to clarify which steps leading to bioactive GA1 and GA3 were affected by decapitation and subsequent IAA application. After feeds of [14C]GA20 to the p-1 internode of intact, decapitated, and decapitated plants treated with IAA, HPLC chromatograms contained peaks corresponding to GA29, as shown by GC-MS, and the un-metabolized GA20. Further large peaks eluted in fractions 12 to 14 and 41 to 42 (data not shown). There was a peak eluting near, but slightly earlier than, GA1 (data not shown), but GC-MS analysis of the fractions from the authentic GA1 zone confirmed that this peak was not GA1. Figure 4 shows GC-MS traces confirming the presence of GA1 in both the intact and IAA-treated cases and its virtual absence in the decapitated case. The other peak in the GC-MS traces (mass-to-charge ratio 508) corresponds to 1 ng of [2H2] GA29 added as an "internal standard" after HPLC. The other unknown peaks were also analyzed by GC-MS. The substance eluting close to GA1 was not epi-GA1. Furthermore, GA29-catabolite, GA5, and GA6, were eliminated as candidates for the unidentified peak eluting in fractions 41 and 42. Similarly, the peak in fraction 12 to 14 was found not to correspond to either GA8 or epi-GA8.
[14C]GA20 was also fed to pseudostem segments from 2-week-old seedlings (two leaves expanded). Excised pseudostem segments consisted of blade and sheath and were floated in Murashige and Skoog medium with or without IAA. Harvests were made after incubation periods of 6, 12, and 24 h. Metabolites were separated by HPLC as methyl esters. Figure 5 shows HPLC chromatograms and corresponding GC-MS traces for tissue harvested at 6 h. Aliquots from the GA29 and GA1 zones, containing radioactive peaks, were combined and subjected to selected ion monitoring GC-MS. At all time points, the ratio of GA1 to GA29 was greater in the IAA-treated case.
The HPLC chromatograms after feeds of [14C]GA5 showed four major radioactive peaks corresponding to GA3, GA5, and two unknown peaks (Fig. 6). [14C]GA5 was converted to [14C]GA3 in the intact and IAA-treated case but to a lesser extent in the decapitated case (Fig. 6). Analysis by GC-MS confirmed that decapitation reduced the conversion of [14C]GA5 to [14C]GA3.
To examine the molecular basis of auxin effects on the GA biosynthetic pathway, we monitored the mRNA levels of the Hv3ox2 gene. This gene encodes the main enzyme for the steps GA20 to GA1 and GA5 to GA3 (data not shown). Barley 3ox2 forward and reverse primers produced a single product of the expected size of 139 bp when completed SYBR Green PCR reactions were analyzed by agarose gel electrophoresis. Sequencing of the PCR product further confirmed that the correct target sequence was being amplified (data not shown). The results demonstrated a decrease in the amount of 3ox2 mRNA in decapitated plants and a restoration by IAA (Fig. 7).
Removal of the developing inflorescence of barley and oat reduced the IAA content of the stem. This appears to be the first direct evidence, to our knowledge, that the inflorescence acts as a source of IAA for the stems of these agronomically important species. The high level of IAA in the inflorescence is consistent with that evidence.
In barley and oat, the levels of the bioactive GAs, GA1 and GA3, in stems were also reduced by inflorescence excision. However, exogenous IAA was able to restore the content of these GAs to at least the levels found in intact plants. Obtaining this result in oats allows a direct comparison with the findings of Koning et al. (1977
The importance of GA1 and GA3 for stem growth in barley is demonstrated by the effects of the mutation to the Grd2 locus, which specifically reduces the content of these GAs and, as a consequence, stem elongation (Chandler and Robertson, 1999 A range of other GAs was also quantified in barley stems, including GA19, GA20, and GA8 (Fig. 2). There was no evidence from the levels of these GAs that the effect of decapitation on GA1 and/or GA3 content was primarily because of decreased synthesis of GA20 or increased deactivation of GA1. Therefore, we investigated the effects of decapitation and auxin application on the steps immediately after GA20.
Metabolism studies with [14C]GA20 revealed that IAA promotes the 3-oxidation of GA20 to GA1, as in pea (Ross et al., 2000 Quantitative PCR demonstrated the relationship between IAA content and GA 3-oxidase (Hv3ox2) mRNA levels in barley stems. mRNA levels of Hv3ox2 were reduced in decapitated plants, compared with intact plants, whereas IAA application reversed this effect (Fig. 7).
The present results extend the effects of auxin to the pathway GA20 to GA5 to GA3, an important branch commonly found in grasses. The metabolism of GA5 to GA3 has been observed in rice (Oryza sativa; Kobayashi et al., 1991 It appears, therefore, that IAA is required for the normal biosynthesis of active GAs in stems of barley and oats. The inflorescence is a major contributor to the auxin pool of elongating stems, in which the bioactive GAs GA1 and GA3 are produced. In this way, the inflorescence potentially plays a critical role in controlling the levels of key GAs in the stem and, therefore, stem elongation. Furthermore, IAA appears to be needed for GA biosynthesis in barley leaves. Our results provide the first evidence, to our knowledge, that the auxin-GA relationship recently discovered in pea and tobacco also exists in monocotyledonous plants, despite their radically different growth habit. This finding indicates the evolutionary antiquity of the auxin-GA interaction.
Plant Material and Growing Conditions
Barley (Hordeum vulgare var. Himalaya), a GA 3oxidase mutant of this line (grd2-463), and oat (Avena sativa) seeds were sown in 10-cm-diameter pots, and plants were grown at a density of one plant per pot. The growing medium was a 6:4 (w/v) mixture of composted pine bark:sand (pH 6). Plants were watered daily, and a complete nutrient solution containing N:P:K at 23:4:13 (v/v) was provided weekly. Plants were grown in a heated greenhouse with a photoperiod of 18 h obtained by extending the natural light as described previously (Beveridge and Murfet, 1996
The rapidly expanding p-1 internode of barley was divided into three segments. A small window in the sheath was removed, and a correction pen was used to mark the lowermost and the uppermost 20 mm of the internode. Sections were measured until fully expanded. The final lengths of the p-1 and p-2 internodes were recorded for both the wild type and the grd2-463 mutant in a separate experiment.
Plants were either left intact or decapitated. Decapitation involved excising the developing inflorescence (which at this stage was enclosed within the sheath of the flag leaf) at the top of the peduncular internode using a razor blade (Fig. 1). One-half of the decapitated plants were treated with IAA in hydrous lanolin (3 mg g–1); the remainder received lanolin alone. The auxin/lanolin or lanolin paste was re-applied 8, 20, 32, and 44 h after decapitation; on each occasion, the previous lanolin was removed. Plant portions harvested for hormone metabolism and quantification studies included the p-1 internode and, in some cases, the nodes on either side (p and p-1 nodes). Sheath material was removed from these portions. Nodal segments included the node itself and approximately 10 mm of internodal tissue on either side. The p-1 internode was young and still actively elongating. At the time of experimentation, plants were approximately 5 to 6 weeks old, and the p-1 internode was 70 to 100 mm in length. The potential final length of this internode was greater than 200 mm in our conditions. Two replicates were harvested for each type of segment and treatment. Number of plants per replicate varied from three to 10. For metabolism studies, [14C]GA5 or [14C]GA20 (Prof. Lewis N. Mander, Australian National University, Canberra) was injected into the p-1 internode in 10 µL of 1:1 (v/v) distilled water:methanol solution at a rate of 10,000 dpm plant–1. In [14C]GA5 experiments, substrate was also injected into the p-1 node. Substrate was injected concurrently with decapitation and initial auxin treatment. Tissue was harvested 48 h after injection and immersed in cold (–20°C) 80% (v/v) methanol containing butylated hydroxyltoluene. We also studied the metabolism of [14C]GA20 by pseudostem segments from 2-week-old seedlings (two leaves expanded). The expanded leaf blades were excised at the ligules, and the pseudostem consisted of sheath and immature leaves encased within. Five pseudostem segments were placed in each petri dish, and segments were approximately 6 to 7 cm long. Segments were incubated at 24°C in 6-cm petri dishes containing 10 mL of sterile liquid Murashige and Skoog medium, with or without added IAA (5 µg mL–1). Substrate was added at a rate of 500,000 dpm per petri dish. Segments were harvested at three time points: 6, 12, and 24 h after the beginning of the incubation period. Tissue was removed from the incubation medium, washed with distilled water, and placed in cold (–20°C) 80% (v/v) methanol containing butylated hydroxyltoluene.
For the extraction of GAs and auxins, tissue was homogenized, and the extracts were held at 4°C for 24 h, before filtering (Whatman no. 1, Whatman, Clifton, NJ). Endogenous levels of GA1, GA19, GA20, GA3, GA5, GA29, and GA8 were quantified using deuterated internal standards as described previously (Lawrence et al., 1992
For quantification of endogenous GAs samples were subjected to HPLC as free acids (Ross, 1998
[14C]GA5 and [14C]GA20 metabolites were subjected to HPLC radiocounting as methyl esters. The solvent program ran from 30% to 60% (v/v) methanol in distilled water during 35 min, using an exponential program, followed by an isocratic (60% [v/v]) elution. The flow rate was 1.6 mL min–1, and 1-min fractions were collected and assayed for radioactivity as before (Ross et al., 1995
GA and auxin samples were derivatized for GC-MS by first adding 10 µL of pyridine and 40 µL of bis-trimethylsilyltrifluoroacetamide with 1% (v/v) trimethylchlorosilane, followed by heating at 80°C for 20 min. Samples were then dried, and a further 15 µL of bis-trimethylsilyltrifluoroacetamide with 1% (v/v) trimethylchlorosilane was added, followed by heating at 80°C for 15 min. GC-MS was performed as described previously (Ross, 1998
The nodal portion was harvested from intact, decapitated, and decapitated IAA-treated plants. Samples were ground into a powder with a mortar and pestle in liquid N2 and approximately 100 mg (fresh weight) of tissue was used for RNA extraction. Total RNA was extracted using the Qiagen RNA easy kit (Qiagen, Hilden, Germany) with an on column DNase digestion. The RNA was eluted in 50 µL of RNase-free water. The RNA concentration was determined by measuring the absorption at 260 nm (A260). Reverse transcription was carried out on 5 µg of RNA, before quantitative PCR, using first strand cDNA synthesis (Invitrogen, Carlsbad, CA) using SuperScript III and 250 ng of random hexamers.
For Hv3ox2 quantification, primers were designed by Primer3 (http://www-genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi), to flank an intron. The following primers were used to produce a 139-bp amplicon: forward primer, 5'-TCCTCCTTCTTCTCCAAGTG-3'; and reverse primer, 5'-TGTGGAACTCCTCCATCAC-3'. Primers and probe for the 18S amplicon were as designed by Ozga et al. (2003 For quantification of Hv3ox2, the QuantiTect SYBR Green PCR kit (Qiagen) was used to make up a 20-µL reaction for a Rotorgene 2000 (Corbett Research, Mortlake, NSW, Australia). The PCR mixture consisted of 10 µL of 2x Qiagen QuantiTect SYBR Green PCR Master Mix and 400 nM of both the forward and reverse primer. The template was cDNA generated from 50 ng of total RNA as the starting material. Thermal cycling conditions for SYBR Green PCR were 95°C for 15 min and 60 cycles of 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s. The SYBR Green PCR product was then sequenced to confirm that the correct fragment was being amplified. 18S was selected to correct for variations in input template quantity. For the quantification of 18S, the QuantiTect Probe PCR Kit (Qiagen) was used to make up a 20-µL reaction. The PCR mixture consisted of 10 µL of Taq-Man Probe PCR Mastermix with HotStarTaq DNA polymerase, 400 nM of both the forward and the reverse primer, and 0.1 to 0.2 µM probe. The template was cDNA generated from 50 ng of total RNA as the starting material. Thermal cycling conditions were 95°C for 15 min followed by 45 cycles of 94°C for 15 s and 60°C for 60 s. Standard curves were generated for both Hv3ox2 and 18S. A standard curve was generated for Hv3ox2 from a plasmid carrying the complete cDNA of Hv3ox2 of known quantity. A standard curve for 18S was generated from one of the samples (from intact nodal tissue).
We thank Dr. Noel Davies (Central Science Laboratory, University of Tasmania, Australia) and Tracey Jackson, Ian Cummings, and Professor Lewis Mander (Australian National University, Canberra) for labeled GAs. Received July 17, 2003; returned for revision August 18, 2003; accepted October 29, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.030460.
1 This work was supported by the University of Tasmania Institutional Research Grants Scheme. * Corresponding author; e-mail John.Ross{at}utas.edu.au; fax 03–62262698.
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