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First published online September 29, 2006; 10.1104/pp.106.086199 Plant Physiology 142:1267-1281 (2006) © 2006 American Society of Plant Biologists
Developmental and Embryo Axis Regulation of Gibberellin Biosynthesis during Germination and Young Seedling Growth of Pea1Plant Physiology and Molecular Biology Research Group, Department of Agricultural, Food, and Nutritional Science, University of Alberta, Edmonton, Alberta, Canada T6G 2P5 (B.T.A., J.A.O., D.M.R.); and Department of Biological Sciences, University of Calgary, Calgary, Alberta, Canada T2N 1N4 (L.V.K.)
The expression patterns of five genes (PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2) encoding five regulatory gibberellin (GA) biosynthesis enzymes (two GA 20-oxidases, a GA 3  -hydroxylase, and two GA 2-hydroxylases) were examined to gain insight into how these genes coordinate GA biosynthesis during germination and early postgermination stages of the large-seeded dicotyledonous plant pea (Pisum sativum). At the time the developing embryo fills the seed coat, high mRNA levels of PsGA20ox2 (primarily responsible for conversion of C20-GAs to GA20), PsGA2ox1 (primarily responsible for conversion of GA20 to GA29), and PsGA2ox2 (primarily responsible for conversion of GA1 to GA8) were detected in the seeds, along with high GA20 and GA29 levels, the enzymatic products of these genes. Embryo maturation was accompanied by a large reduction in PsGA20ox2 and PsGA2ox1 mRNA and lower GA20 and GA29 levels. However, PsGA2ox2 transcripts remained high. Following seed imbibition, GA20 levels in the cotyledons decreased, while PsGA3ox1 mRNA and GA1 levels increased, implying that GA20 was being used for de novo synthesis of GA1. The presence of the embryo axis was required for stimulation of cotyledonary GA1 synthesis at the mRNA and enzyme activity levels. As the embryo axis doubled in size, PsGA20ox1 and PsGA3ox1 transcripts increased, both GA1 and GA8 were detectable, PsGA2ox2 transcripts decreased, and PsGA2ox1 transcripts remained low. Cotyledonary-, root-, and shoot-specific expression of these GA biosynthesis genes and the resultant endogenous GA profiles support a key role for de novo GA biosynthesis in each organ during germination and early seedling growth of pea.
GAs are known to play important roles in germination of a wide range of dicotyledonous plant species (Koornneef and van der Veen, 1980  ; Groot and Karssen, 1987; Hilhorst and Karssen, 1988; Nambara et al., 1991; for review, see Bewley and Black, 1994; Sponsel and Hedden, 2004). In addition to weakening the endosperm and testa, which surrounds the radicle in certain seeds at maturity (Groot and Karssen, 1987; Debeaujon and Koornneef, 2000), GAs appear to control the growth of the embryo axis (Groot and Karssen, 1987), as well as growth of the rapidly developing shoot and root tissues (Reid et al., 2004).
These GA-mediated events in postimbibition germination and seedling growth are thought to be regulated, at least in part, by the modulation of tissue- and cell-specific GA levels and also by altering the ability of the cells to respond to GA (Richards et al., 2001
Our approach in this study was to define the roles of GAs and the regulation of GA biosynthesis during germination and early seedling growth of the large-seeded dicot pea (Pisum sativum). Pea has been used as a model system for explaining the role of GAs in plant growth and development of large-seeded dicots (Reid et al., 2004
In the pea seedling, GA biosynthesis occurs via the early 13-hydroxylation pathway (Sponsel, 1995
GA biosynthesis inhibitor studies using compounds that inhibit several early steps in GA biosynthesis (the cyclization of geranylgeranyl diphosphate to ent-copalyl diphosphate: Sponsel, 1983 ; oxidation of ent-kaurene to ent-kaurenoic acid: Graebe, 1986) as well as later steps (3-hydroxylation of GA20 to GA1; Ross et al., 1993) suggested that de novo GA biosynthesis was not essential for pea seed germination per se, but was, however, essential for the maintenance of normal seedling growth soon after germination (i.e. 4 d after imbibition [DAI]). Furthermore, the na mutation of the pea GA biosynthesis gene NA (PsKAO1, encodes ent-kaurenoic acid oxidase and is mainly expressed in vegetative tissues; Davidson et al., 2003) provides genetic evidence for the role of GAs in early pea vegetative growth. Thus, pea seeds with the na mutation germinate readily (the homolog PsKAO2 is highly expressed in the developing seeds, and this results in normal GA levels in na seeds; Potts and Reid, 1983; Davidson et al., 2003). Subsequently, though, expanding shoot tissues of na show dramatically decreased levels of the growth-active GA1 (Proebsting et al., 1992), and this results in severely dwarfed plants with extremely short internodes (Potts and Reid, 1983) and a markedly reduced taproot length (Yaxley et al., 2001). Additionally, Ross et al. (1993) have shown that GA biosynthesis occurs in shoots of rapidly growing young pea seedlings. The above data, taken together, strongly suggest that, following germination, de novo GA biosynthesis is required for normal pea seedling shoot and root growth.
Although these studies support the importance of GA biosynthesis in pea, the exact timing and tissue-specific nature of de novo GA biosynthesis during these early events in seedling shoot and root growth still are not known (in pea or in other plant species). Since bioactive GA1 in young seedling shoots of pea is low (Ross et al., 1992
The following late GA biosynthesis genes have been identified and reported to be expressed in both vegetative and developing seeds of pea (with one exception): two GA20ox genes, PsGA20ox1 (Martin et al., 1996
This study characterizes the expression pattern of these five key GA biosynthesis genes in developing seeds and mature embryos and in shoots, roots, and cotyledons of pea from 0 to 6 DAI. It also examines the coordination of gene expression among these GA gene family members during the critical stages for germination and seedling establishment in two distinctly different cultivars of pea (‘Alaska,’ a model cultivar for tall [LE] vining pea, and ‘Carneval,’ a model cultivar for semidwarf [le], semileafless field pea). Further, it tests if the embryo axis regulates GA biosynthesis in the cotyledons of germinating seeds. Real-time reverse transcription (RT)-PCR, the current method of choice for obtaining sensitive, specific, and reproducible quantification of mRNA (Bustin, 2000
GA Biosynthesis in Developing Seeds and Mature Embryos
High levels of mRNA were detected for PsGA20ox2, PsGA2ox1, and PsGA2ox2 in developing seeds 20 d after anthesis (DAA; Table I
). These are the genes that encode the enzymes that convert GA53 to GA20 (GA 20-oxidase), and GA20 to GA29 and/or GA1 to GA8 (GA 2
Further maturation of the pea embryo resulted in a large reduction in PsGA20ox2 (665-fold) and PsGA2ox1 (38-fold), but there was no significant change in PsGA2ox2 mRNA levels. This finding is consistent with the decreased levels of GA20 (20-fold) and GA29 (2.3-fold) that we observed in the embryo at maturity (Tables I and II). We would suggest that the high levels of PsGA2ox2 mRNA in 20 DAA and mature embryos, as well as in the 0.5-DAI embryo axis (Table I), and the apparent preference of PsGA2ox2 for GA1 as a substrate (Lester et al., 1999 ) indicate a key role for GA 2-oxidases in the later phase of seed development, thereby assuring minimal levels of growth-active GA1. This would allow the seed to complete normal maturation and possibly aid in prevention of precocious germination. Synthesis of GA1 and GA8 in the embryo during the latter stage of pea seed development was very low or not detectable (Table II; Sponsel, 1983), and these GAs do not accumulate in the mature embryo (Table II; Ross et al., 1993).
During the first 0.5 DAI, mature air-dried seeds of ‘Carneval’ absorbed more water (1.7-fold) than ‘Alaska’ (45.7 and 26.6% relative water content [RWC] at 0.5 DAI, respectively; Fig. 2 ). At 1 DAI, the RWC of cotyledons from both cultivars was similar (50% in ‘Alaska’ and 55% in ‘Carneval’), and the RWC gradually increased from 1 to 6 DAI, reaching 67% to 68% by 6 DAI in both cultivars. The transcript levels of GA20ox, GA3ox, and GA2ox genes in the cotyledons remained relatively constant in ‘Alaska’ (Table I; Figs. 3C , 4C , and 5C ) during the first 0.5 DAI. ‘Carneval’ cotyledons exhibited a 4- to 5-fold decrease in PsGA2ox1 and PsGA2ox2 mRNA levels during their first half-day of imbibition. The much higher levels of these transcripts in the mature seeds of ‘Carneval’ relative to those in ‘Alaska’ are a likely reason for the large decline in their abundance observed during the first half-day of ‘Carneval’ seed imbibition (Table I; Fig. 5F). By 1 DAI, the RWC of ‘Alaska’ cotyledons had increased to 50%, the transcript abundance of PsGA2ox1 had decreased (3.6-fold; Table I), and, coincidentally, the level of GA29 decreased (3.6-fold; Table II).
The decrease in 2 -hydroxylation of GA20 to GA29 in cotyledons at 1 DAI, together with the increase in transcription of PsGA3ox1 (25-fold; Table I) and in the production of GA1 (5-fold; Table II), suggests that the cotyledonary GA20 serves as substrate for in situ 3-hydroxylation into bioactive GA1 (note that GA20 levels also decreased 2.3-fold by 1 DAI; Table II). Although bioactive GAs have a well-defined role in coordinating mobilization of the reserve materials in cereals (Jacobsen et al., 1995), their possible role in mobilization of cotyledonary reserves in dicots remains unclear (Bewley and Black, 1994). The growth-active GA1 in pea cotyledons would not be involved in subsequent growth or photosynthetic processes as the hypogeal cotyledons do not develop into green leaf-like structures that photosynthesize and their cells have a short but normal life span of 2 to 3 weeks (Smith and Flinn, 1967). However, since cell wall loosening enzymes have been shown to be GA inducible during germination of other species (tomato and Arabidopsis; Chen et al., 2001; Ogawa et al., 2003), the bioactive GA1 present in these cotyledons could be necessary for promoting the formation of the large reticulum of intercellular spaces in this tissue during germination (Smith and Flinn, 1967). The increase in cotyledonary intercellular spaces could increase the oxygen diffusion rates through the tissue to support the high respiratory activity of the cotyledonary storage parenchyma cells during storage mobilization (Bain and Mercer, 1966a). Transport of GA20 from the cotyledon to the embryo axis to support synthesis of growth-active GA1 for embryo axis expansion also can occur (Ross et al., 1993).
Radicle protrusion occurred between 1 and 2 DAI (Fig. 2A) and it was accompanied by an increase in the expression of cotyledonary PsGA20ox2 (13-fold in ‘Alaska’; Fig. 3C) and PsGA3ox1 (6-fold in ‘Alaska’; Fig. 4C), together with a decrease in cotyledonary GA20 and GA1 levels (Table II). The cotyledonary PsGA20ox2 mRNA levels that we observed (Fig. 3C) were not consistent with GA feedback regulation, i.e. PsGA20ox2 transcripts increased sharply from 1 to 2 DAI then decreased by 4 DAI, while during this same period GA20 levels decreased from 1 to 4 DAI (Table II). Cotyledonary PsGA3ox1 mRNA levels increased during the first DAI, at the same time endogenous GA1 levels increased (‘Alaska’; Tables I and II). Subsequently, though, from 1 to 2 DAI, PsGA3ox1 transcript abundance in both cultivars continued to increase, while cotyledon GA1 content decreased to undetectable levels (Table II; Fig. 4C). Although feedback up-regulation of PsGA3ox1 transcription by low levels of bioactive GA could be occurring in the cotyledons after 1 DAI (‘Alaska’), the increase in cotyledonary PsGA20ox2 and PsGA3ox1 transcript levels observed during or soon after radicle protrusion in both cultivars (1–2 DAI; Fig. 4C) suggested that a signal from the embryo axis may induce expression of GA biosynthesis genes in the cotyledon. Indeed, Bain and Mercer (1966b)
To examine whether the presence of the embryo axis is required to induce the expression of GA biosynthesis genes and, in turn, the metabolism of GAs in the cotyledons of germinating pea seeds, expression of PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2 and metabolism of [14C]GA20 were compared in the cotyledons of ‘Alaska’ imbibed for 2 d with or without the presence of the embryo axis (axis excised within 2 h after imbibition). Removal of the embryo axis from the cotyledons 2 h after imbibition reduced the transcript abundance of cotyledonary PsGA20ox1 (3-fold), PsGA20ox2 (9-fold), PsGA3ox1 (2-fold), and PsGA2ox1 (7-fold), but had no effect on PsGA2ox2 transcript levels after 2 d of imbibition (Fig. 6
). Embryo axis removal also resulted in more than a 7-fold reduction in the conversion of [14C]GA20 to [14C]GA8 in the 2-DAI cotyledons (Fig. 7
). Since PsGA2ox2 transcript levels were not affected by embryo axis removal, the reduction in conversion of [14C]GA20 to [14C]GA8 is likely due to the lower transcript abundance of cotyledonary PsGA3ox1 (Fig. 6) leading to reduced GA 3
GA Biosynthesis in the Embryo Axis Consistent with a faster initial rate of water imbibition, initiation of radicle protrusion was observed in 53% of the ‘Carneval’ seeds by 1 DAI, and the percentage of germinated seeds increased to 77% by 1.5 DAI. In contrast, only 27% of ‘Alaska’ seeds exhibited radicle emergence at 1.5 DAI. Germination percentages then increased to 90% in ‘Carneval’ and 65% in ‘Alaska’ by 2 DAI. From 0.5 to 1 DAI, embryonic-axis fresh weight (FW) doubled and axis length increased 1.6-fold for both cultivars (Fig. 2, D and E, see inset tables). During the same period, transcript abundance increased markedly for embryo-axis-derived PsGA20ox1 (13-fold in ‘Alaska’) and also for PsGA3ox1 (15-fold in ‘Alaska’; Table I; Figs. 3 and 4). These large increases in gene expression of the GA20ox and GA3ox biosynthesis genes in the embryo axis occur concomitantly with a substantial decrease in PsGA2ox2 transcript abundance (approximately 4-fold) and maintenance of low levels of PsGA2ox1 transcript (Table I; Fig. 5). These data suggest an increased capacity to synthesize and maintain growth-active GA1 in the embryo axis for the rapid expansion that occurs soon after imbibition. The presence of GA20, GA1, and GA8 in the 1-DAI embryo axis is supportive of this hypothesis, especially since GA1 levels were much lower in the mature embryo (0 DAI) than in the 1-DAI embryo axis (Table II).
Increases in GA20ox (AtGA20ox1 and AtGA20ox3), GA3ox (AtGA3ox1 and AtGA3ox2) transcript abundance, and also in the levels of the growth-active GA4 were observed after imbibition of wild-type Arabidopsis seeds (Ogawa et al., 2003
Following seed germination (which occurred approximately 2 DAI; Fig. 2A), the seedling plumules of both cultivars began to appear (4 DAI), and by 5 DAI the shoots of 73% of ‘Alaska’ and 88% of ‘Carneval’ seedlings had emerged. Complete emergence of all germinated seedlings had taken place by 6 DAI for both cultivars. Root FW and length generally exceeded that of the shoot from 2 to 6 DAI for both cultivars (Fig. 2). Finally, both cultivars had similar patterns of shoot and root growth, with a slower growth rate from 2 to 3 DAI followed by a higher rate of growth from 3 to 6 DAI (Fig. 2).
Transcript levels in the shoot for PsGA20ox1 were more abundant (141-fold in ‘Alaska’) than for PsGA20ox2 at 2 DAI, and they increased markedly from 2 to 6 DAI as the shoot elongated rapidly (Figs. 2, D and E, and 3, B and E). Concomitant with these increases in transcript level of PsGA20ox1, endogenous levels of GA20 decreased 5-fold in the embryo axis from 1 to 2 DAI, and GA20 levels continued to decrease from 2 to 4 DAI in the shoot (3.5-fold; Table II). Since the abundance of PsGA20ox1 mRNA was shown to be regulated by the levels of growth-active GA in the shoots of ‘Alaska’ seedlings (application of GA3 reduced and application of GA biosynthesis inhibitor Prohexadione increased PsGA20ox1 mRNA levels; Ayele et al., 2006
Since the expression of shoot-derived PsGA2ox1 remained low (Fig. 5B) and the GA29 concentration (ng g–1 FW) actually decreased in the shoot from 2 to 4 DAI (Table II), we conclude that the reduction in shoot GA20 levels is not a result of increased 2
PsGA3ox1 (LE) transcript levels in the roots were maintained at moderately high levels as the roots of both cultivars grew from 2 to 6 DAI (Fig. 4A; Martin et al., 1997
To further localize expression of GA biosynthesis genes in the root, we analyzed the expression pattern of the GA biosynthesis genes in root tips (4-mm apex, which constitutes about 2.4%–3.3% of the total root FW) and also in the remaining part of the root from 6-DAI seedlings. PsGA20ox2 transcript abundance was similar to that of PsGA20ox1 in the more mature root tissue, but was markedly lower (12- to 24-fold lower) in root tips for both cultivars (Table III
). In ‘Alaska’ pea roots, the first 2 to 3 mm of the root apex consists of the root cap and the root meristem, which contains mostly dividing cells. Behind this zone of cell division is a zone consisting of mainly elongating and differentiating cells (from 2–3 to 11 mm or greater; Rost and Baum, 1988
Cultivar-Specific GA Gene Expression
The relatively similar GA gene expression patterns between the two different genotypes indicate the general nature (and likely importance) of the spatial and temporal regulation of these GA biosynthesis genes in facilitating the establishment of the pea seedling in the first few days after germination. Specific expression pattern differences between the genotypes for the GA3ox and GA2ox genes do occur, and these seem likely to be related to the LE (PsGA3ox1) gene. ‘Carneval’ carries the le-1 mutation (Ayele, 2006
Additionally, we found that from 2 to 6 DAI, PsGA2ox2 transcript abundance is lower in the actively growing shoots and roots of ‘Carneval’ (le-1) than in ‘Alaska’ (LE; Fig. 5, A, B, D, and E). This suggests that one mechanism to compensate for the reduction in enzyme efficiency of the GA 3 In summary, although embryo development is a complex process and the expression patterns of these GA biosynthesis genes and GA1 content are not necessarily the causal factors for specific developmental events, our findings support the emerging hypothesis that endogenous growth-active GA (GA1 in pea) is minimized in the developing embryo to allow for seed maturation processes to proceed. However, GA gene expression and endogenous GA profiles are consistent with previous findings that GA20 (the immediate precursor to the growth-active GA1) is sequestered in the developing pea embryo and significant amounts of GA20 exist in the embryo at maturity. Additionally, high PsGA2ox2 message levels in the mature quiescent embryo and in the embryo axis at 0.5 DAI likely reflect a mechanism where growth-active GA1 is maintained at minimal levels to prevent embryo axis expansion during the later phases of seed maturation and/or under nonoptimal germination conditions.
During seed imbibition, the expression pattern of this suite of GA biosynthesis genes and the concomitant levels of endogenous GAs suggest that pea cotyledons are serving as a reservoir of GA20 (both preexisting and newly synthesized), which is then used as a substrate for GA 3 As the embryo axis initiates growth (by 1 DAI), a dramatic change in the expression patterns of these regulatory GA biosynthesis genes occurs in the axis tissue, providing the embryo axis with a very much increased capacity to produce growth-active GA1 for axis expansion. In the rapidly growing young seedling (2–6 DAI), both shoots and roots display unique expression patterns, which likely provide for coordination of GA biosynthesis within and between these organs. Overall, our results show that coordination of these key GA biosynthesis genes during germination and early seedling growth is highly regulated, and they also suggest that each organ modulates the levels of GA biosynthesis gene transcripts to maintain specific pools of both precursors and growth-active GA1 during seed maturation, germination, and active growth phases (early seedling growth) of the plant.
Plant Material The pea (Pisum sativum) cv ‘Alaska’ (I3) was chosen as a model vining-type pea plant. ‘Alaska’ has normal leaflet morphology (AF), wild-type internode length (LE), white flowers and green cotyledons at maturity, and it begins to flower at approximately the 10th node under long- or short-day conditions. ‘Carneval’ was chosen as a model for semidwarf (semileafless; af) field pea, which is used extensively in crop agriculture. ‘Carneval’ has white flowers and yellow cotyledons at maturity, begins to flower at about the 15th to 17th node under long-day conditions, and was found to contain Mendel's dwarfing gene, le-1 (data not shown). Both cultivars readily germinate upon imbibition with water at 15°C to 25°C.
Mature air-dry seeds of ‘Alaska’ (5.4% RWC) and ‘Carneval’ (5.8% RWC) were planted at a depth of approximately 2.5 cm into moist sterilized sand in 3-L plastic pots (10 seeds per pot), the pots were placed in a growth chamber (Conviron) at 22°C/20°C (day/night) in a 16/8-h photoperiod with cool-white fluorescent and incandescent lights (205 µE m–2 s–1) until harvest. For germination and growth measurements, seeds of each cultivar were harvested at 0.5, 1, 2, 3, 4, and 6 DAI from the sand medium, and separated by dissection into cotyledons and embryo axes (0.5 and 1 DAI) or into cotyledons, roots, and shoots (2–6 DAI; 15–30 seeds or seedlings per time point). Seeds were scored as being germinated when protrusion of the radicle (2–5 mm) through the seed coat was visible. The RWC of the cotyledons was determined by comparing the sample weights before and after drying for 72 h at 60°C, and are expressed on a FW basis. For RNA extraction, seedlings at 0.5, 1, 2, 4, and 6 DAI were separated either into cotyledons and embryo axes (0.5 and 1 DAI), or cotyledons, shoots, and roots (2 and 4 DAI), or cotyledons, shoots, root tips (approximately 4 mm), and remainder of roots (6 DAI), and immediately frozen in liquid N2 and stored at –80°C until extraction. From 2 DAI, only seeds classed as germinated were used. To examine the mRNA levels in the mature embryos (0 DAI), seeds of the two cultivars were immersed in ice:water (1:1, w/v) for 4 h to facilitate seed coat removal, and the embryos (cotyledon plus embryo axis) were immediately frozen in liquid nitrogen and stored at –80°C until RNA extraction. To study whether the presence of the embryo axis was required to induce the expression of GA biosynthesis genes and, in turn, the metabolism of GAs in the cotyledons of germinating pea seeds, mature seeds of ‘Alaska’ were surface sterilized in 1.2% sodium hypochlorite solution for 25 min and rinsed five times with sterile deionized water. Seeds for embryo axis removal were kept immersed in sterile water for 2 h after surface sterilization. After the 2-h imbibition period, the testa and embryo axis were removed without damage to the cotyledon using a scalpel. The cotyledons without an embryo axis or the intact seeds (for cotyledon with embryo axis treatment) were then placed in a 9-cm sterile petri plate (20 seeds per plate) on a sterile Whatman #1 filter paper wetted with 10 mL of sterile deionized water and imbibed in darkness at 22°C/20°C 16/8 h for 2 d. After the 2 d of incubation, a portion of the cotyledons from each treatment (with and without embryo axis) was harvested into liquid nitrogen and stored at –80°C for RNA extraction. The remaining cotyledons or intact seedlings were transferred to petri plates for the [14C]GA20 metabolism study.
Tissues were finely ground in liquid N2, and 200 to 550 mg FW (20-DAA seeds, mature embryos, embryo axes, shoots, roots, or root tips) or 100 to 250 mg FW (cotyledons) subsamples were used for total RNA isolation using a modified TRIzol (Invitrogen) protocol (cotyledon subsamples were taken from two ground cotyledons). After initial extraction with the TRIzol reagent and centrifugation, the supernatant was cleaned by chloroform partitioning (0.2 mL mL–1 TRIzol). The resulting supernatant fraction was then precipitated by first using an isopropanol solution (0.25 mL mL–1 TRIzol) followed by a high salt solution (1.2 M sodium citrate and 0.8 M NaCl) to remove polysaccharides and proteoglycans. The RNA sample was further precipitated with 4 M LiCl and finally followed by a mixture of 3 M sodium acetate (pH 5.2):100% ethanol (1:20, v/v). The precipitate was dissolved in diethylpyrocarbonate-treated water. The integrity of the RNA was verified both electrophoretically and by the average absorption ratio 260 to 280 nm. The total RNA samples of all tissues were then digested with DNase (DNA-free kit; Ambion), and the cotyledonary total RNA samples were further purified with RNeasy columns (Qiagen). Sample RNA concentration was determined in duplicate by A260 measurement, and then the samples were stored at –80°C until quantitation by real-time RT-PCR.
Primers and Probes
All probes were TaqMan MGB and were labeled at the 5' end with fluorescent reporter dye 6-carboxyfluorescein (target gene probes) or VIC (18S-62 reference gene probe), and at the 3' end with nonfluorescent quencher dye (Applied Biosystems). To confirm the PCR product produced by the quantifying primers, RT-PCR amplification products were separated and identified using 1% agarose gel electrophoresis and ethidium bromide staining.
Real-Time RT-PCR Assay
The relative transcript abundance of the target genes in the individual plant samples was determined by the 2–
To study GA metabolism in the cotyledons with the axis attached or the axis removed, [14C]GA20 (specific activity of 34 µCi/µmol) was injected into one cotyledon of 2-DAI ‘Alaska’ seedlings (with axis treatment) or one 2-DAI cotyledon (without axis treatment) at two spots (a total of 2.5 µL of 50% aqueous ethanol; a total of approximately 82,000 dpm). The [14C]GA20-labeled cotyledons were incubated for 12 or 24 h on filter paper moistened with 10 mL of sterile water in 15-cm petri plates (five cotyledons or seedlings per plate) placed at 22°C/20°C (day/night) in a 16/8-h photoperiod as described previously. The incubation period started 4 h into the photoperiod for both treatments. After incubation with the [14C]GA20 substrate, the [14C]GA20-treated cotyledons were harvested onto dry ice and stored at –80°C until extraction. The [14C]GA20-treated cotyledons (five per sample) were homogenized in cold 80% methanol (10 mL per sample) in 30-mL Corex tubes using a polytron homogenizer. 17-[14C]GA7 (approximately 11,000 dpm) was added at homogenization to each sample extract as an external standard for recovery determination of radioactive metabolites at the HPLC step. The extracts were mixed overnight on a shaker (150 rpm) at 4°C in darkness, and then centrifuged for 30 min at 10,000g. The methanolic supernatant was removed, and the residue was resuspended in 5 mL of homogenization solvent and shaken for at least 4 h. The residue extracts were centrifuged for 30 min at 10,000g, and the pooled methanolic extracts were evaporated to the aqueous phase using a SpeedVac concentrator. After adjusting the pH of the aqueous extract to 8.0 with 0.1 N NH4OH, the extract was partitioned four times against n-hexane (5 mL) in 20-mL glass scintillation vials. The aqueous phase was then adjusted to pH 3 with 0.1 N HCl and partitioned five times against ethyl acetate (5 mL). The combined ethyl acetate extract was reduced in volume using the SpeedVac concentrator, and partitioned four times against 5% (w/v) aqueous NaHCO3 (2 mL). The combined NaHCO3 extract was transferred into a 30-mL Pyrex tube placed on ice, pH was adjusted to 3 with 6 N HCl, and then it was partitioned four times against ethyl acetate (5 mL). The ethyl acetate extracts were pooled and evaporated to complete dryness using a SpeedVac concentrator prior to HPLC purification.
For HPLC analysis, the ethyl acetate extracts were dissolved in 400 µL of 20% MeOH, filtered through a 0.45-µm nylon filter (Whatman International), and injected onto a 4.6- x 250-mm C18 column (5 µm; Beckman Instruments). The samples were eluted at 1 mL min–1 flow rate using the following linear gradient of methanol (solvent A) and aqueous 0.01% TFA (solvent B): 20% solvent A for 1 min, gradient to 100% solvent A in 45 min, and isocratic 100% solvent A for 5 min. Radioactivity in the sample effluent was monitored using a flow-through radiochemical detector. Fractions eluting at the retention times of GA8 (8.6 min), GA29 (10.7 min), GA1 (16.1 min), GA29-catabolite (16.4 min), GA20 (24.2 min), and GA7 (27.6 min) were collected and reduced to dryness. The putative GA8, GA29, GA1, and GA29-catabolite fractions were pooled across treatments, methylated using diazomethane, and rechromatographed as their methyl-esters by C18 HPLC, using the same solvent system and radiochemical detection. The [14C]GA methyl-ester fractions that chromatographed at the retention time of the GA methyl-ester standards confirmed the presence of the respective [14C]GA. As the [14C]GA29 and [14C]GA8 methyl-ester fractions were of sufficient quantity, they were converted to their trimethylsilyl ether derivatives (Gaskin and MacMillan, 1991
Developing seeds (20 DAA), mature embryos (cotyledon plus embryo axis), embryo axes, cotyledons, shoots, and roots were freeze-dried and subsequently ground to a fine powder in a mortar and pestle (0.6–11 g dry weight) with liquid N2 and washed sea sand (Fisher Scientific). The tissue powder was homogenized with 80% (v/v) aqueous methanol, and 20 ng of [17,17-2H2]GA1, [17,17-2H2]GA19, [17,17-2H2]GA20, and [17,17-2H2]GA29 and 33 ng of [17,17- 2H2]GA8 (obtained from Prof. L.N. Mander, Research School of Chemistry, Australian National University, Canberra, ACT, Australia) was added to each extract as internal standards for recovery determination at the GC-MS-SIM step. The methanolic extracts were filtered through a 55-mm filter paper (Whatman #2; Whatman International), eluted through a C18 preparative column (3 g of C18 preparative reversed-phase material [Waters] preconditioned with 100% methanol followed by 80% (v/v) aqueous methanol; Koshioka et al., 1983 The extract residue was dissolved in 1 mL of 10% aqueous methanol with 1% acetic acid and injected onto a C18 (µ-Bondapak) Radial-PAK (8-mm x 10-cm) column connected to a Waters HPLC system. The samples were eluted at 2 mL min–1 using the following linear gradient of 10% methanol in 1% acetic acid [water:MeOH:acetic acid, 89:10:1 (v/v); solvent A] and 100% MeOH (solvent B): 0 to 10 min 100% solvent A, 10 to 50 min gradient to 30% solvent A, and 50 to 80 min gradient to 100% solvent B. Fractions eluting at the corresponding retention times of the GAs of interest were collected and dried in vacuo at 35°C.
Fractions containing the putative GAs were dissolved in methanol, methylated using diazomethane, then taken to dryness and trimethylsilylated using BSTFA with 1% TMCS (Gaskin and MacMillan, 1991
The endogenous GAs were identified by SIM of three prominent ions (including the molecular ion M+, except for GA19: i.e. GA8, 596/594, 581/579, 450/448; GA1, 508/506, 493/491, 450/448; GA29, 508/506, 493/491, 449/447; GA20, 420/418, 377/375, 405/403; GA19, 436/434, 376/374, 404/402) characteristic to the corresponding GAs, as well as comparison of the GC retention times of the endogenous GAs with their respective [2H2]GA standards. Endogenous GA concentrations were calculated by reference to the stable isotope-labeled internal standard using equations for isotope dilutions analysis adapted from Gaskin and MacMillan (1991)
We thank Ashley Durec, Bridget McLeod, and Dr. David W. Pearce for technical assistance. Received July 2, 2006; accepted September 20, 2006; published September 29, 2006.
1 This work was supported by the Alberta Agricultural Research Institute and the Natural Sciences and Engineering Research Council of Canada (to J.A.O.).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Jocelyn A. Ozga (jocelyn.ozga{at}ualberta.ca). www.plantphysiol.org/cgi/doi/10.1104/pp.106.086199 * Corresponding author; e-mail jocelyn.ozga{at}ualberta.ca; fax 780–492–4265.
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GA53
Ct method (Livak and Schmittgen, 2001
