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First published online June 24, 2005; 10.1104/pp.105.062190 Plant Physiology 138:1794-1806 (2005) © 2005 American Society of Plant Biologists Flowering of the Grass Lolium perenne. Effects of Vernalization and Long Days on Gibberellin Biosynthesis and Signaling1Commonwealth Scientific and Industrial Research Organization Plant Industry, Canberra, Australian Capital Territory 2601, Australia
Almost 50 years ago, it was shown that gibberellin (GA) applications caused flowering in species normally responding to cold (vernalization) and long day (LD). The implication that GAs are involved with vernalization and LD responses is examined here with the grass Lolium perenne. This species has an obligatory requirement for exposure to both vernalization and LD for its flowering (inflorescence initiation). Specific effects of vernalization or LD on GA synthesis, content, and action have been documented using four treatment pairs: nonvernalized or vernalized plants exposed to short days (SDs) or LDs. Irrespective of vernalization status, exposure to two LDs increased expression of L. perenne GA 20-oxidase-1 (LpGA20ox1), a critical GA biosynthetic gene, with endogenous GAs increasing by up to 5-fold in leaf and shoot. In parallel, LD led to degradation of a DELLA protein, SLENDER (within 48 h of LD or within 2 h of GA application). There was no effect on GA catabolism or abscisic acid content. Loss of SLENDER, which is a repressor of GA signaling, confirms the physiological relevance of increased GA content in LD. For flowering, applied GA replaced the need for LD but not that for vernalization. Thus, GAs may be an LD, leaf-sourced hormonal signal for flowering of L. perenne. By contrast, vernalization had little impact on GA or SLENDER levels or on SLENDER degradation following GA application. Thus, although vernalization and GA are both required for flowering of L. perenne, GA signaling is independent of vernalization that apparently impacts on unrelated processes.
Flowering of many vernalization-responsive plants often requires long days (LDs) and this restricts their reproduction to spring and summer. For simplicity, many studies have focused either on vernalization or daylength response. Thus, with the LD- and cold-responsive dicot Arabidopsis (Arabidopsis thaliana), cultivars and lines have been chosen that respond strongly to vernalization, and they have generally been grown in LD to avoid any affect of daylength (for review, see Mouradov et al., 2002  ). Conversely, for defining the nature of daylength response, ecotypes have been used that show no vernalization requirement for their flowering in LD (Gocal et al., 2001b; Mouradov et al., 2002). Likewise, with monocots, most understanding has come from studies with species showing such single-process response as with the LD grass Lolium temulentum, which has no requirement for vernalization (for review, see Evans and King, 1985). Such experimental systems do not address potential similarities and synergies between vernalization and daylength response and this is the focus of this study with Lolium perenne, a perennial near-relative of L. temulentum and which, for flowering, requires both LD and vernalization (Cooper, 1957).
The vernalization response, at least for Arabidopsis, involves FLC, FRI, and a number of other flowering time genes (for review, see Sung and Amasino, 2004
LD response involves enhanced synthesis of the GA class of plant growth regulator with the grass L. temulentum (King and Evans, 2003
Given such information about vernalization and daylength response, the question of their interaction could be an academic one. However, early studies with dicots raised the likelihood of vernalization acting on GA biosynthesis (for review, see Lang, 1965
Species such as L. perenne that require both vernalization and LD for their flowering provide ideal material for examining both responses together. Furthermore, because the changes in GA biosynthesis in LD are well characterized for the annual L. temulentum (King and Evans, 2003
A Clone of L. perenne That Requires Vernalization and Long Days for Inflorescence Formation
Generally, L. perenne requires vernalization for flowering but, since it is an obligate out-crosser, the level of genetic variation within a population can be significant. For example, in some varieties, up to 20% of the plants may flower without cold treatment (Cooper, 1957
As shown in Figure 1A, for clone CPM-1, vernalization for 8 weeks followed by a 14-LD exposure strongly induced flowering assessed on day 15 as either apex length or floral score. All plants flowered and their score of 8 to 10 indicates floret and anther primordia initiation; a score of 2 is the minimum for inflorescence formation (Evans et al., 1990
Stock plants of CPM-1 held in noninductive warm conditions (22°C) in LD have remained vegetative for many (>4) years during this study while, after vernalization, plants held in SD had not flowered after 4 months (data not shown). Thus, this clone of L. perenne shows a clear and absolute requirement for both LD and vernalization for flowering. In addition, flowering increased quantitatively with increases in the duration of vernalization or LD (Fig. 1). Stem length increased in association with flowering, but, as we discuss later, there is no obligatory link with flowering. To examine interactions between vernalization, LD, and GA biosynthesis, we developed a standard experimental protocol involving all four combinations of vernalizationxdaylength treatments. As shown in Figure 2, flowering occurred only when the plants were first vernalized for 8 weeks in SD and then exposed to 2 LD and these conditions were used routinely in subsequent experiments.
It is clear from the evidence in Figure 2 that vernalization and LD do not substitute for each other and so are likely to act in different ways. In fact, for L. perenne, vernalization leads to a more or less permanent change of state. For example, if the exposure to 2 LD was delayed following vernalization, flowering was as good as, or better than, that of plants transferred immediately to 2 LD (Fig. 3). Thus, the vernalization experience is "remembered" and probably occurs in the shoot apex, as is known for a number of plant species (Lang, 1965 ; Metzger, 1988). By contrast, photoperiod perception is likely to be localized in the leaf, as has been shown for L. temulentum (see ref. in Evans and King, 1985), and to involve GA synthesis (King and Evans, 2003). Below, in examining these concepts with L. perenne, we consider firstly whether GAs are part of the LD and vernalization response and, secondly, how vernalization may function to remove a restriction on gibberellin action.
Applied GA Can Replace the LD, But Not the Vernalization Requirement for Flowering
A single drop of GA3 applied to the recently expanded leaf caused flowering in SD for vernalized plants of L. perenne (Fig. 4). Nonvernalized plants did not flower with GA3 treatment whether or not they had been exposed to LD, while control plants treated with ethanol only flowered if vernalized and exposed to LD. Clearly, for flowering, applied GA3 only replaces the need for LD exposure and only in vernalized plants. While control plants exposed to 1 LD did not flower, GA3 acted additively with this or the 2-LD exposure to give substantial responses (Fig. 4). With L. temulentum there is similar additivity between LD and GA (Evans et al., 1990
Of the other bioactive GAs we tested, both GA1 and GA5 replaced the LD requirement for flowering in vernalized plants grown in SD (Fig. 5A). By contrast, GA4 was not florally active but did stimulate stem elongation, while a synthetic derivative of GA4, 2,2-dimethyl GA4, was extremely active both for flowering and stem elongation (Fig. 5B), as we previously reported for L. temulentum (Evans et al., 1990 ).
Based on the data in Figures 4 and 5, over these three different experiments with vernalized plants in SD, the order of potency of applied GAs for floral induction was: 2,2-dimethyl GA4  GA3 GA1 > GA5 > GA4. Although application of GA1, GA3, and 2,2-dimethyl GA4 caused large increases in stem elongation (about 6-fold), GA5 had a small effect (Fig. 5). Thus, GA5 behaves like LD in stimulating flowering more than stem elongation, a distinction we have extensively documented for L. temulentum (King and Evans, 2003). On the one hand, because applied GA replaced only the LD requirement for flowering of L. perenne, then the response to vernalization must be distinct from that to LD. On the other hand, for flowering and stem elongation there was no response to applied GA unless the plants had been vernalized and, later, we examine the possibility that vernalization acts on GA signaling.
It was likely that LD would increase the endogenous GA content in shoots of L. perenne given the ability of leaf-applied GA to replace its LD requirement for flowering (above). Such an increase is confirmed by the data in Figure 6. Relative to plants in SD, exposure to 2 LD led to significant increases in GA content for both the leaf and young shoot tissues. Increases after 2 LD were 5-fold or greater for the precursor, GA20, and up to 4-fold in the shoot for bioactive GA1. There were smaller increases in the GA catabolic products (GA8 and GA29). Similar trends were also found in two further biologically independent experiments (data not shown).
GA53, the initial substrate for GA 20-oxidases, was difficult to detect, and, although LD consistently reduced its content, replication was insufficient for assessment of statistical significance. There were, however, significant decreases in GA19, an intermediate substrate of GA 20-oxidases, along with an increase in GA20, the final product of this biosynthetic enzyme (Fig. 6). Moreover, the LD decrease in leaf GA19 of about 8 to 10 ng g–1 was matched by the increases in GA20 plus GA1, the latter being a product of GA20.
Since GA contents changed in parallel in both leaf and shoot tissues, light during the LD may well be sensed directly in both the leaf and the sheath. Daylength sensing in the flowering of the related species, L. temulentum, is predominantly in the leaf tissue (Evans and King, 1985 In contrast to LD, vernalization had little effect on GA levels; in a few instances there were small but significant differences, but they were less than 2-fold. For example, vernalized shoots contained approximately 1.3- to 1.5-fold more GA19 than nonvernalized shoots (Fig. 6). A two-way ANOVA also showed a small but significant interaction between LD and vernalization. Overall, however, GA content was most affected by LD whether or not the plants had been vernalized. Given the indication above that LD regulates the activity of a GA 20-oxidase, we applied its substrate, 14[C]-GA53, to the leaf or injected it into the air cavities of the shoot. Using HPLC to separate the products, various expected radiolabeled intermediates were detected including GA44, GA19, GA20, and GA1. Although there was a significant increase (40%) in the metabolism of GA53 in LD, less than 5% of the total applied radioactivity was metabolized in the leaf. Such response allowed insufficient precision for analysis of the effect of environment on GA metabolism. As an alternative approach, therefore, we analyzed expression of a GA 20-oxidase gene.
Increased GA biosynthesis in LD was associated with a decrease in GA19 and an increase in GA20 (Fig. 6), which suggests an increase in the activity of a GA 20-oxidase biosynthetic gene. We therefore cloned a GA 20-oxidase, LpGA20ox1, from a vegetative L. perenne cDNA library using mixed cDNA probes produced from barley (Hordeum vulgare) GA 20-oxidases of vegetative tissue and young grain. Although Xu et al. (2002)
Based on Southern-blot hybridization, Xu et al. (2002)
Catalytic activity of the putative GA 20-oxidase we cloned from vegetative tissue was tested by heterologous expression in Escherichia coli. The coding region of the cDNA was ligated in-frame into pET11d, and the construct (named pET20ox) was transformed into a host E. coli strain. Examination of the protein profile of a total cell lysate by SDS-PAGE confirmed the presence of an extra band of the expected size (approximately 46 kD) and this was present only after isopropylthio-
The LpGA20ox1 protein was functional since, in vitro, it converted two substrates, GA53 and GA19, to their expected products, which were characterized here from their gas chromatography (GC) retention times and mass spectrometry (MS) ion spectra. As shown in Figure 7A, [14C]GA53 was converted to [14C]GA44, [14C]GA19, and [14C]GA20. In separate experiments [14C]GA19, the last substrate of this multifunctional enzyme, was converted to [14C]GA20 (Fig. 7B). Evidence for a small amount of conversion of [14C]GA19 to [14C]GA17 was also found but the ion spectrum was not conclusive. The empty vector (pET11d) transformant was inactive (data not shown). Thus, the cloned LpGA20ox1 was indeed a multifunctional GA 20-oxidase capable of converting GA53 through GA44 and GA19 to GA20. These results for LpGA20ox1 extend the report of Xu et al. (2002)
GA Biosynthesis: LpGA20ox1 Expression Is Up-Regulated by LD Exposure Apparently, LpGA20ox1 expresses at a relatively low level in vegetative tissues as it was only detectable using reverse transcription (RT)-PCR. As seen in Figure 8A, the gene is expressed in all vegetative tissue types of L. perenne, including fully expanded leaf blades, the corresponding leaf sheaths, elongating shoot tissues (that had been enveloped in the sheaths), tiller bases (including the shoot apex and primordial leaf blades), and roots.
To establish up-regulation by LD or vernalization of LpGA20ox1 expression in shoots and leaves, a semiquantitative assay was used. In this assay, transcript detection increased in parallel with input RNA amount or when the samples were spiked with different amounts of plasmid-derived RNA. To ensure the assays were quantitative, they were run for 30 cycles that proved to be nonsaturating and, hence, were sensitive to differences in source RNA amount. Input RNA, assessed by RT-PCR using rRNA primers, was relatively constant and was used to normalize for RNA loading across plant samples. In the elongating shoot tissues, exposure to LD caused large and consistent increases across experiments of 5- to 30-fold in LpGA20ox1 expression for harvests at 2 AM toward the end of the second LD. A less dramatic increase was evident in LD leaves (Fig. 8B). What is interesting is that, as for GA content, LD increased LpGA20ox1 expression both in vernalized plants that flowered and in nonvernalized plants that never flowered.
Our evidence above links environment, GAs, and flowering on the basis that: (1) GAs replace the need for LD; (2) LD increases endogenous GA content; and (3) activity of a critical GA biosynthetic gene increases in LD. As a logical next step, we focused on GA signaling to confirm that GA is part of the LD flowering response. We were also interested in establishing whether the failure of applied GA to cause flowering of nonvernalized plants (Fig. 4) was due to a block in GA signaling. Therefore, we examined both the levels of SLN, a DELLA protein that restricts GA signal transduction (Olszewski et al., 2002 We used immunoblots to examine effects of environment on the level of SLN and, as shown in Figure 9A, LD led to substantial loss of SLN within 2 d. Protein loading may have differed slightly across samples, but this fails to account for the large differences in SLN signal. The loss of SLN after 2 LD reflects the increase in GA1 by this time (Fig. 6), while GA3 application to plants in SD led to disappearance of SLN protein (Fig. 9B), albeit more rapidly (within 2 h) than occurred in LD. Thus, the level of SLN is a direct indication of GA content whether applied or endogenous. In contrast to the response to LD, vernalization did not cause any reduction in SLN levels (Fig. 9), which fits with the fact that vernalization had no significant effect on endogenous GA1 content (Fig. 6).
Interestingly, the effect of GA on SLN degradation was the same in both nonvernalized and vernalized samples (Fig. 9B), indicating that the inability of nonvernalized plants to respond to GAs is not related to this component of the GA-signaling cascade. Overall, therefore, SLN protein content of shoot pieces of L. perenne provides independent confirmation of increased GA biosynthesis in LD but also excludes any possibility of vernalization directly interacting with GA signaling to enhance the response to LD.
We cannot explain why SLN increased 1 and 2 d after vernalization in one experiment (Fig. 9A). There might be a transient effect of moving plants to a warmer temperature but we did not examine this further. As for the two SLN bands detected on the immunoblots, these have also been found with SLN from barley (Gubler et al., 2002
It is conceivable that ABA could block GA response, so we measured ABA content in leaf and shoot tissue. As shown in Table I, neither vernalization nor daylength reduced ABA content as determined by GC-MS/selected ion monitoring measurements, which included an appropriate deutero ABA internal standard. We could not obtain a sufficiently large tissue sample to determine ABA content of the shoot apex, but in an earlier study with L. temulentum, although response to cold was not examined, LD had no effect on shoot apex ABA content (see ref. in Evans and King, 1985
Almost 50 years ago, Lang (1956) reported that applied GA3 caused flowering of plants in noninductive conditions. However, for perennial plant species that require both LD and vernalization for their flowering, it has remained unclear whether changes in endogenous GA content are important. Here, with L. perenne, a perennial grass, we have focused on the role of GAs in a vegetatively propagated clone (CPM-1), which shows an obligate requirement for both vernalization and LD exposure for its flowering (inflorescence formation). LD increases GA content, whereas vernalization is required for the action of that GA. How vernalization might affect GA action is one consideration of this discussion.
For flowering, plants of clone CPM-1 of L. perenne cv Yatsyn responded after vernalization for 8 weeks in SD followed by 2 LD in warm conditions (Figs. 1–3
Because L. perenne clone CPM-1 has an obligate requirement for vernalization and LD for its flowering, it has proven invaluable for determining the role of environment in GA synthesis and signaling. Most important has been the comparison of four treatment combinations: nonvernalized SD, nonvernalized LD, vernalized SD, and vernalized LD. Together these features have allowed us to distinguish LD-specific effects on GA biosynthesis from effects of vernalization on GA signaling. However, as summarized in Thomas and Vince-Prue (1997)
Applied GAs including GA1, GA3, and GA5 induced flowering of vegetative, vernalized plants of L. perenne so replacing the requirement for LD (Figs. 4 and 5). By contrast, applied GAs never caused flowering or any associated stem elongation if the plants were not vernalized (Fig. 4). Because endogenous GA content of leaves and shoot pieces increased on exposure to 2 LD (Fig. 6), the induction of flowering by GAs applied in SD implies that they are part of the LD signal. This conclusion is strongly supported by the parallel and more extensive evidence for the annual grass, L. temulentum. Its leaf GA content increases in LD (Gocal et al., 1999
In shoots of dicots, LD also increases GA contents in Arabidopsis (Xu et al., 1997
Specificity of these GA increases in Lolium for LD and not for vernalization is supported by a number of our findings. Firstly, whether or not the plants were vernalized, the content of various GAs (precursor, bioactive and catabolic forms) changed in LD (Fig. 6). This LD response reflected increased GA biosynthesis since GA19, a GA 20-oxidase substrate, decreased and GA20, its product, increased (Fig. 6). Secondly, again irrespective of vernalization status, exposure to LD increased expression of LpGA20ox1, a GA 20-oxidase biosynthetic gene (Fig. 8). Lastly, both GA application and LD exposure led to degradation of the GA signaling protein SLN (Fig. 9), but vernalization had no such effect. Thus, LD and GA regulate flowering responses of L. perenne in a way that is distinct from any effect of vernalization, a finding dependent on our use of all four combinations of vernalization and daylength treatments. This conclusion also fits with genetic studies with Arabidopsis, which indicate distinct roles for GAs and vernalization in its flowering (Michaels and Amasino, 1999
As an aside, our prior studies with L. temulentum (for review, see King and Evans, 2003
In the presence of GA, vernalized plants of L. perenne flower and their stems elongate dramatically, whereas nonvernalized plants remain vegetative with little stem elongation (Figs. 2, 4, and 5). Here, in examining how vernalization might alter the response to GA, we focus on processes closely linked to GA action, namely: (1) reduced GA catabolism at, or near, the shoot apex; (2) reduced activity of SLN, a negative regulator GA of signaling; (3) reduction in ABA content; and (4) activation of expression of "floral" promotory genes.
Were vernalization to reduce GA catabolism by GA 2-oxidases, then it would enhance the effectiveness of applied or endogenous GAs. These enzymes inactivate both active GAs and precursors such as GA20 (Hedden and Phillips, 2000
Considering the second process (listed above), some members of the DELLA family of proteins are negative regulators of GA signaling (for review, see Olszewski et al., 2002
The possibility that ABA might counteract GA response (for review, see Davies, 1995
We have not examined the fourth explanation involving vernalization activating a particular GA-promoted, floral pathway but consider below two vernalization-responsive genes that show LD and/or GA regulation. One obvious candidate gene is SOC1 (Suppressor of Overexpression of Constans), whose expression in dicots is regulated by GA (Bonhomme et al., 2000
Plant Materials Plants of Lolium perenne L. cv Yatsyn were vegetatively propagated using rooted tillers, individual clones being derived from single seeds. For each experiment, the rooted tillers were grown for 2 weeks in 8-h SDs in sunlit, temperature-controlled cabinets with daily watering with a Hoagland Number 2 nutrient solution. Then the plants were either exposed to cold (8°C vernalization) or held at an average temperature of 22°C (nonvernalizing conditions) for 8 weeks. Vernalized and nonvernalized plants were then grown at 22°C either remaining in SD or exposed to LD (8 h sunlight plus a 16-h overnight exposure to low intensity incandescent lamps at 10 µmol m–2 s–1). Thus, the four treatments compared in this paper are nonvernalized SD, nonvernalized LD, vernalized SD, and vernalized LD with only the last treatment being florally inductive. This precisely regulated flowering response was evident in all our studies (see "Results").
For measuring flowering, plants were dissected, the shoot apex floral stage was scored according to Evans et al. (1990)
Various GAs, GA3 (Sigma Chemicals, St. Louis) and GA1, GA4, 2,2-dimethyl GA4, GA5 (from Professor L.N. Mander, Research School of Chemistry, Australian National University, Canberra, Australia) were applied to the center of the most recently expanded leaf blade at a dose of 25 µg in 10 µL of 95% (v/v) ethanol. Control plants received 10 µL of 95% (v/v) ethanol.
Endogenous GAs were quantified by GC-MS in leaf blades and shoots from nonvernalized and vernalized plants exposed to SD or to 2 LD. The harvest was at 10 AM on the morning following the second of 2 LD that began 1 week after the plants had been returned to warm conditions following 8 weeks of vernalization. In two repeat experiments (data not shown), the 2-LD exposure began immediately after vernalization. This earlier exposure to 2 LD was almost as florally effective as exposure to 2 LD after a 1-week delay (compare with Fig. 2) and LD had a comparable effect on GA content but the extractions were not replicated so statistical significance could not be evaluated.
The fully expanded leaf blades, or shoots excluding leaf blades, of 16 plants were frozen in liquid nitrogen, ground to a fine powder, and lyophilized. Samples of up to 160 mg dry weight were extracted in 150 mL of 80% methanol/water (v/v) at 4°C overnight. Dideuterated and tritiated standards (from Professor L.N. Mander, Research School of Chemistry, Australian National University, Canberra, Australia) were added and the extract was evaporated to the water phase. After freeze-thawing, any precipitate was removed by centrifugation and the supernatant was adjusted to pH 2.5, filtered through a 0.45 micron Millipore filter and partitioned three times against equal volumes of ethyl acetate. The ethyl acetate-soluble phase was dried, redissolved in water, adjusted to pH 8, and loaded onto 2 mL QAE Sephadex columns that were washed twice with water, pH 8, and then eluted in 5 mL of 0.2 M formic acid onto preconditioned C-18 Sep-pak cartridges (Waters, Milford, MA). GAs and ABA were eluted with 5 mL of 80% (v/v) methanol and further purified by HPLC on a 25 cmx4.6 mmx5 µm particle size C18 column (Allsphere ODS-2, Alltech, Deerfield, MI) with a linear gradient over 40 min from 19% (v/v) methanol in 2 mM acetic acid to 100% methanol at a flow rate of 1 mL min–1. After methylation using diazomethane, GA derivatives were trimethylsilylated with pyridine and BSTFA (N,O-bis-trimethylsilyltrifluoracetamide) for at least 30 min at 90°C. GA and ABA derivatives were injected onto a 25 m x 0.2 mm x 0.25 µm film thickness fused silica column (BPX-5, SGE, Austin, TX). GC conditions were as in Green et al. (1997) The ions monitored appear in Table II.
Cloning and Characterization of an L. perenne GA 20-Oxidase cDNA
A GA 20-oxidase gene sequence was isolated from a cDNA library of above-ground vegetative tissue of L. perenne cv Yatsyn screened with barley (Hordeum vulgare) GA 20-oxidase cDNAs. Construction of this library and screening and characterization of gene sequences was as described in McAlister et al. (1998) To test enzymatic activity of LpGA20ox1, a PCR fragment from the cDNA was generated using the primers pLp-GA20ox08 5'-CATGCCATGGTGCAGCCTGTGTTC-3' (+strand; start codon underlined), and pLp-GA20ox09 5'-GAAGATCTTATGTCGTCGTCGTCGTG-3' (–strand). The PCR fragment was ligated into the NcoI and BglII sites of the pET11d expression vector and expressed in Escherichia coli strain BL21. After protein expression was induced with IPTG, the cells were lysed in 200 mM Tris-HCl, pH 7.4, 1 mg lysozyme mL–1, and 10 mM dithiothreitol. After freeze-thawing, the lysate was centrifuged at 4°C and the supernatant was stored at –80°C. Expression of the protein was verified by SDS-PAGE against noninduced controls. Protein activity was assayed based on metabolism of the precursor [14C]GA53 to the radiolabeled products GA44, GA19, GA17, and GA20, or of [14C]GA19 conversion to GA20. In each assay, 75 µL of lysate (pET20ox or pET) were incubated with 0.3 nmol of precursor in a 200-µL (final volume) reaction solution containing 95 mM Tris, pH 8.0, 4.5 mM dithiothreitol, and a cofactor mix (2 mM L-ascorbic acid, 2 mM 2-oxoglutarate, 0.1 mM iron sulfate, 0.1% bovine serum albumin, and 0.05% catalase). Metabolism was allowed to proceed at 30°C overnight before the reaction was stopped by adding 0.25 volumes of acetic acid. GAs in each sample were semipurified using a Sep-pak C18 column and then methylated and trimethylsilylated before GC-MS identification. The ions monitored included those shown in the preceding section as ion 2+ but now for [14C].
Total RNA was isolated from leaf blades and shoots using Qiagen RNeasy Plant Mini kits (Qiagen, Clifton Hills, Victoria, Australia) according to the manufacturer's instructions. cDNA synthesis and PCR-amplification were performed in the same reaction using a SuperScript One-Step RT-PCR kit (Invitrogen, Mt. Waverley, Victoria, Australia) according to the manufacturer's instructions. The LpGA20ox1-specific primers used were pLp-RGL (5'-GCCTCGGG*GAGGTGTACG-3') and pLp-LHQ (5'-TCCTGGTGGAGGATGGTGAG-3'). The expected product (232 bp) corresponded to the LpGA20ox1 transcript and not to genomic DNA since pLp-RGL crosses the predicted intron site as indicated by the asterisk. The RT-PCR components included: SuperScript One-Step RT-PCR Reaction buffer with extra MgSO4 to 1.5 mM, left and right primers (1.2 µM each), 0.2 mM dNTPs; SuperScript II H– Reverse Transcriptase/Platinum Taq HiFi mix (2%), and total RNA (100 ng). cDNA synthesis was performed at 50°C for 30 min. This was followed by PCR that included an initial denaturing step at 94°C for 2 min and then successive cycles of amplification involving a denaturing step at 94°C for 15 s, an annealing step at 63°C for 30 s, and an extension step at 68°C for 1 min. To detect relative differences in transcript levels, amplification was performed when PCR product was accumulating exponentially with respect to cycle number (between 30 and 34 cycles). Equivalence of total input RNA was assessed from 25S rRNA content determined in separate amplifications with the primers: pLp-25S-L (5'-GAAGGGTTCGAGTTGGAGCA-3') and pLp-25S-R (5'-CCCCCGATGCCTCTAATCAT-3') at a low concentration of 0.1 µM but using the same RT-PCR conditions as for LpGA20ox1. The primers were based on unpublished sequence of a Lolium temulentum cDNA for 25S rRNA. The lower rRNA primer concentration relative to LpGA20ox1 primers ensured that product accumulation reached the exponential phase after 30 cycles. Semiquantitation of RT-PCR products was based on the intensity of each ethidium-stained band using ImageQuant version 3.3 software (Amersham Biosciences, Uppsala), and LpGA20ox1 transcript levels were normalized against the 25S rRNA products.
Shoot pieces (i.e. shoots excluding leaf blades and the two oldest leaf sheaths) from six plants were harvested, frozen in liquid nitrogen, and total protein was extracted by grinding in 500 µL of sample buffer (62.5 mM Tris-HCl, pH 6.8; 70 mM SDS; 10% glycerol [v/v]; 0.015 mM bromphenol blue; and 1% [v/v] Sequence data from this article have been deposited with the EMBL/GenBank data libraries under accession number DQ071620 for LpGA20ox1.
The L. perenne cDNA library was a kind gift from John Watson, (CSIRO Plant Industry, Canberra). The barley GA 20-oxidase cDNA probes were kindly provided by Peter Chandler (CSIRO Plant Industry). The purified primary barley SLN antibody used in western-blot analysis was generously provided by Frank Gubler (CSIRO Plant Industry). Andrew Poole (CSIRO Plant Industry) is thanked for support with GA and ABA extraction and analysis. Lloyd Evans (CSIRO Plant Industry) and Ed Newbigin (The University of Melbourne, Melbourne, Australia) are thanked for helpful discussions. Received March 3, 2005; returned for revision April 14, 2005; accepted April 19, 2005.
1 This work was supported by the Dairy Research and Development Corporation, Australia (Ph.D. scholarship to C.P.M.). 
2 Present address: ensis, PO Box E, 4008 Kingston, ACT 2604, Australia. Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.105.062190. * Corresponding author; e-mail rod.king{at}csiro.au; fax 616262465000.
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), and a nonresponsive clone (
), which has not been included in subsequent studies. Values are means ± SE shown as bars where larger than the symbols.
-galactoside induction of cells transformed with pET20ox (data not shown). 
-Amylase production during grain maturation and its relation to endogenous gibberellic acid content. Plant Physiol 114: 203–212
