INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

In pea (Pisum sativum), normal pericarp growth requires the presence of seeds (Eeuwens and Schwabe, 1975). Removal or destruction of the seeds 2 to 3 d after anthesis (DAA) results in the slowing of pericarp growth and subsequently abscission (Eeuwens and Schwabe, 1975; Ozga et al., 1992). Chemical signals originating from the seeds may be responsible for continued fruit development by maintaining hormone levels in the surrounding tissue (Eeuwens and Schwabe, 1975; Sponsel, 1982). Developing pea seeds and pericarps contain GAs (Garcia-Martinez et al., 1991; Rodrigo et al., 1997) and auxins (4-chloroindole-3-acetic acid [4-Cl-IAA] and indole-3-acetic acid (IAA); Marumo et al., 1968; Magnus et al., 1997). During early pericarp growth (2 DAA), application of the naturally occurring hormones, 4-Cl-IAA (Reinecke et al., 1995) and GA (Eeuwens and Schwabe, 1975; Ozga and Reinecke, 1999), to deseeded pericarp can substitute for seeds and stimulate pericarp growth. However, the other naturally occurring auxin in pea fruit, IAA, was ineffective in promoting growth (Reinecke et al., 1995).

Initial work comparing the growth promoting properties of 4-, 5-, 6-, and 7-Cl-IAAs and the corresponding F-IAA analogs demonstrated that a chlorine at the 4-position of the indole ring (4-Cl-IAA) was required for significant biological activity in pea pericarp growth (Reinecke et al., 1995). Studies comparing the growth-promoting response and the physicochemical properties of 4-Cl-IAA and 4-substituted analogs found that the 4-substituents' size and lipophilicity were associated with auxin's growth-promoting activity on pea pericarp (Reinecke et al., 1999). Pea pericarps respond in a qualitatively different fashion to two naturally occurring auxins that, in a variety of other auxin bioassays, showed only quantitative differences in activity (Reinecke, 1999). These data suggest unique ways of auxin action based on alternative molecular recognition mechanisms in this tissue.

Pea plants metabolize GAs by the early 13-hydroxylation pathway: GA₁₂  GA₅₃  GA₄₄  GA₁₉  GA₂₀  GA₁ (Sponsel, 1995). Previous studies using the pea split-pericarp assay (test compounds are applied to the inner walls of split and deseeded 2-DAA pericarps) have shown that the presence of seeds or the application of 4-Cl-IAA to deseeded pea pericarp stimulated pericarp GA biosynthesis, specifically, the conversion of [¹⁴C]GA₁₉ to [¹⁴C]GA₂₀ (van Huizen et al., 1995), and expression of PsGA20ox1 (codes for enzyme that converts GA₁₉ to GA₂₀; van Huizen et al., 1997). IAA was ineffective in stimulating pericarp PsGA20ox1 expression and growth (Ngo et al., 2002).

Although elongating pollinated pericarps (3 DAA) converted [¹⁴C]GA₁₂ and [¹⁴C]GA₁₉ to [¹⁴C]GA₂₀, conversion to [¹⁴C]GA₁ was not detected after a 24-h incubation period (Ozga et al., 1992; van Huizen et al., 1995). After a 48-h incubation period, Maki and Brenner (1991) reported metabolism of [²H]GA₅₃ to [²H]GA₁ in pollinated pericarp tissue; however, Rodrigo et al. (1997), using 5-DAA pollinated pericarps and [¹⁴C]GA₁₂, obtained results similar to that of Ozga et al. (1992). In addition, Rodrigo et al. (1997) were unable to detect conversion of [¹⁴C]GA₁₂ or [¹⁴C]GA₂₀ to [¹⁴C]GA₁ in 4-DAA pea seeds. A gene that codes for the enzyme that converts GA₂₀ to the biologically active GA₁ (PsGA3ox1; Mendel's LE gene that codes for a GA 3-hydroxylase) has been cloned from pea (Lester et al., 1997; Martin et al., 1997). Using northern-blot analysis, Martin et al. (1997) were unable to detect PsGA3ox1 signal in unopened pea flowers or 1-cm young fruits, but low levels of seed PsGA3ox1 mRNA were detected at 14 DAA. Therefore, either GA 3-hydroxylation of GA₂₀ to GA₁ does not occur in pea pericarp or seeds, or the methods used in the studies were not sensitive enough to detect pericarp PsGA3ox1 message level or GA 3-hydroxylation activity (GA₂₀ to GA₁) in these tissues in two of the three reported studies.

Real-time reverse transcription (RT)-PCR is the method of choice for sensitive, specific, and reproducible quantification of mRNA (Bustin, 2000). The TaqMan (PE-Applied Biosystems, Foster City, CA) chemistry utilizes the 5'  3' nuclease activity of Taq DNA polymerase to cleave a specific probe, labeled with a 5' reporter and a 3' quencher fluorescent dye, during strand elongation of the target sequence to generate a detectable fluorescent signal during amplification (Bustin, 2000). With the use of appropriate standard curves, absolute amounts of mRNA can be calculated. To determine if PsGA3ox1 message occurs in pea pericarp and further understand how seeds and hormones regulate GA biosynthesis in pea fruit, we studied the expression pattern of PsGA3ox1 in pea pericarps and seeds during pollination, development, and auxin- and/or GA-induced pericarp growth using real-time RT-PCR analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Pericarp and Seed Growth

Pericarp growth in length and width was rapid from 2 to 5 DAA and was essentially complete by 8 DAA (Fig. 1, A and C). Subsequently, pericarp diameter rapidly increased from 6 to 12 DAA to accommodate the developing seeds (Fig. 1, B and C). Pericarp fresh weight increased rapidly from 3 to 9 DAA and continued to increase gradually to 20 DAA (Fig. 2B). After pollination, seed fresh weight increased rapidly from 8 to 20 DAA (Fig. 2, A and B). Pericarps from flowers emasculated at 2 DAA and harvested at the equivalent to 1, 0, 1, 2, and 3 DAA (pericarps from non-pollinated ovaries) ranged from 7 to 10 mm in length.

View larger version (22K): [in this window] [in a new window]   Figure 1.   Pea pericarp development. Development of pericarps during the rapid phases of growth in length, width (2, 0, and 1-7 DAA; flower bud and flower at anthesis are at 2 and 0 DAA, respectively; A) and diameter (5-12 DAA; B). Growth of pericarp in length, width, and diameter from 2 to 20 DAA (C). Data are means ± SE, n = 4 to 12.

View larger version (24K): [in this window] [in a new window]   Figure 2.   Pericarp and seed growth. A, Seed development from 6 DAA to cotyledon contact point (20 DAA; from left to right, row 1, 6-9-DAA seeds; row 2, 10-, 12-, 14-, 16-, and 20-DAA seeds; and row 3, embryos dissected from 10-, 12-, 14-, 16-, and 20-DAA seeds). B, Growth in fresh weight of pericarps and seeds from 2 DAA to 20 DAA. Data are means ± SE, n = 4 to 12 for pericarp fresh weight; n = 2 to 4 for seed fresh weight.

PsGA3ox1 mRNA Quantitation

Pea GA3ox87 forward and reverse primers produced a single product of the expected size (87 bp) when completed TaqMan RT-PCR reactions (40 cycles) were analyzed by agarose gel electrophoresis for end-point detection (data not shown). The PsGA3ox1 target sequence (amplicon) was amplified clearly and reproducibly by real-time RT-PCR. Quantification of starting template (plant tissue PsGA3ox1) concentration in real-time RT-PCR assays was achieved by comparison against serially diluted plasmid-generated PsGA3ox1 mRNAs from 5 × 10³ to 5 × 10² femtogram (fg) at one-log unit intervals (Fig. 3). The dilution series had a linear correlation coefficient (r²) of 0.998 that enabled template quantification over at least six orders of magnitude with cycle threshold (C_t) values between 17 and 37 PCR cycles.

View larger version (15K): [in this window] [in a new window]   Figure 3.   Standard curve for PsGA3ox1. In vitro-transcribed PsGA3ox1 mRNAs were serially diluted from 5 × 103 to 5 × 102 fg at one-log unit intervals. To illustrate the data spread over the standard curve dilution series, samples from Fig. 6B were plotted on the standard curve used to derive the starting template concentration of these samples.

Pea 18S small subunit nuclear ribosomal RNA forward and reverse primers produced a single product of the expected size (62 bp) when completed TaqMan RT-PCR reactions (40 cycles) were analyzed by agarose gel electrophoresis for end-point detection (data not shown). The 18S amplicon was used as a loading control to estimate the variation in input total RNA for one complete replication of the pericarp and seed developmental study (2 to 20 DAA) and one complete replication of the hormonal regulation study. The average C_t value (±SD) for the 18S ribosomal amplicon in pericarp samples from 2 to 20 DAA was 19.1 ± 0.25 (n = 28; coefficient of variation [CV] = 1.3%); in seed samples from 2 to 20 DAA, C_t was 19.0 ± 0.25 (n = 26; CV = 1.3%); in pericarps treated with IAA, 4-Cl-IAA, GA₃, or GA₃ + 4-Cl-IAA, and split pod no seeds (SPNS), split pod (SP), and intact pericarp controls, C_t was 20.32 ± 0.21 (n = 56; CV = 1.0%). Because the coefficient of variation of the 18S ribosomal amplicon C_t values was extremely low among all the samples assayed (1%-1.3% CV), PsGA3ox1 mRNA values were not normalized to the 18S signal.

Pea GA3ox126 forward and reverse primers produced a single product of the expected size (126 bp) when completed TaqMan RT-PCR reactions (40) cycles were analyzed by agarose gel electrophoresis for end-point detection (data not shown). The trend in C_t values was similar when 2-, 6-, 10-, and 14-DAA pericarp samples were assayed using GA3ox126 forward and reverse primers (C_t = 29.9, 22.7, 21.6, 22.5, respectively) and GA3ox87 forward and reverse primers (C_t = 32.3, 24.7, 24.0, and 24.2, respectively) with the same fluorescent probe.

A BLAST search of all GenBank, EMBL, DNA Data Bank of Japan, and Protein Data Base database sequences (excluding expressed sequence tag [EST], sequence tagged sites, genome survey sequences, and phase 0, 1, or 2 high-throughput genomic sequences) found significant sequence alignment only between the 86- and 126-bp PCR amplicon sequences and pea GA 3-hydroxylase (PsGA3ox1) sequences (E values 4 × 10⁶⁴ to 4 × 10³⁹). We did not pick up any significant homology to GA 3-hydroxylases from other plant species or other GA 2-oxoglutarate-dependent dioxygenases in pea. In a BLAST search of ESTs in GenBank, EMBL, and DNA Data Bank of Japan, no pea ESTs were found to have significant sequence alignment to the 86- and 126-bp PCR amplicon sequences. However, two EST sequences from Medicago truncatula (E values ranged from 5 × 10²⁶ to 1 × 10¹⁰) and one from Glycine max (E values ranged from 3 × 10⁶ to 2 × 10³) showed significant homology to both amplicons.

Effect of Pollination on PsGA3ox1 mRNA Levels

PsGA3ox1 mRNA levels were minimal in both pericarps (0.04 fg 200 ng of total RNA¹) and ovules (0.13 fg 200 ng of total RNA¹) before pollination (2 DAA; Fig. 4). After pollination (0 DAA), PsGA3ox1 mRNA levels increased dramatically in both tissues (50 times in pericarps and 19 times in ovules), then decreased by 1 DAA to levels approximately 10 and 9 times of that of prepollinated tissues (2 DAA pericarps and ovules, respectively; Fig. 4). Pericarps from non-pollinated ovaries did not exhibit the dramatic increase in PsGA3ox1 mRNA at the equivalent to anthesis (0 DAA) as seen in pericarps from pollinated ovaries. Furthermore, PsGA3ox1 expression in non-pollinated pericarps dramatically increased at the equivalent to 3 DAA, just before pericarp senescence (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Figure 4.   Effect of pollination on PsGA3ox1 mRNA level in pea fruit. PsGA3ox1 mRNA (femtogram 200 ng of total RNA1) in pericarps from pollinated and non-pollinated ovaries, and ovules or seeds at 2 to 3 DAA. Non-pollinated pericarps were green and turgid through the equivalent to 3 DAA, but did not exceed 10 mm in length. Data are means ± SE, n = 3 to 5 for pollinated pericarps; n = 2 for non-pollinated pericarps; n = 3 to 4 for ovules/seeds.

Developmental Profile of PsGA3ox1 mRNA Levels

After pollination, pericarp PsGA3ox1 mRNA levels increased linearly from 6 to 10 DAA (Fig. 5A), coincident with rapid pericarp diameter expansion (6-12 DAA), to accommodate the growing seeds. PsGA3ox1 mRNA levels in seeds (Fig. 5A) increased from 2 to 4 DAA and remained at this level until a further increase from 8 to 12 DAA, when seed development was rapid (Fig. 2A). In general, the seed and pericarp PsGA3ox1 developmental profile was similar when expressed as femtogram 200 ng of total RNA¹ or as femtogram milligram fresh weight¹, with two exceptions (Fig. 5, A and B). The increase in seed PsGA3ox1 transcript level at 4 DAA was magnified when expressed as femtogram milligram fresh weight¹, and pericarp PsGA3ox1 transcript level increased 20 DAA when expressed as femtogram 200 ng of total RNA¹, but was not when expressed as femtogram milligram fresh weight¹. In addition, when comparing the PsGA3ox1 developmental profiles in seeds and pericarps, seeds had lower mRNA levels per total RNA than pericarps, but similar levels when expressed per milligram fresh weight (Fig. 5, A and B). The differences between seeds and pericarps were due to seeds, in general, having higher levels of total RNA per gram of fresh weight than pericarps (data not shown).

View larger version (20K): [in this window] [in a new window]   Figure 5.   Developmental regulation of PsGA3ox1 mRNA levels in pea pericarps and seeds. PsGA3ox1 mRNA levels from 2 to 20 DAA in pericarps and ovules or seeds expressed as femtogram 200 ng of total RNA1 (A) and femtogram milligram fresh weight1 (B). Data are means ±SE, n = 3 to 6 for A; and n = 2 to 6 for B, with one exception, 2 DAA ovule, n = 1 for A and B.

Hormonal Regulation of PsGA3ox1 mRNA Levels

The effect of pericarp splitting and seed removal plus hormone application (at 2 DAA) on pericarp growth 5 d after treatment is shown in Figure 6A. Pericarps continued to grow after splitting of the pericarp 2 DAA without disturbing the seeds (SP; Fig. 6A); however, removal of the seeds at 2 DAA resulted in slowing of pericarp growth and subsequently abscission (not shown). Treatment of deseeded pericarp with GA₃ or 4-Cl-IAA (50 µM) stimulated growth (Fig. 6A), but IAA did not (not shown). Application of GA₃ plus 4-Cl-IAA stimulated pericarp growth to a greater extent than either applied alone (Fig. 6A).

View larger version (28K): [in this window] [in a new window]   Figure 6.   Effect of seeds and hormones on pericarp growth and PsGA3ox1 expression. A, Representative pericarps illustrating the effects of pericarp splitting (SP) and seed removal plus application of GA3 (GA), 4-Cl-IAA (4-Cl), and GA3 plus 4-Cl-IAA (GA + 4-Cl) on pea pericarp growth. Pericarps at 2 DAA were split (SP), or split and deseeded. Tween 80 (0.1% [v/v]) was applied daily to SP, and GA3 and/or 4-Cl-IAA were applied daily to deseeded pericarps (50 µM; 30 µL at 2 and 3 DAA; and 40 µL at 4, 5, and 6 DAA) and harvested for picture at 7 DAA. B, Hormonal regulation of pericarp PsGA3ox1 mRNA levels (fg 200 ng of total RNA1). Two DAA pericarps were split (SP) or split and deseeded (SPNS) and treated with 50 µM (30 µL) of 4-Cl-IAA, GA3, IAA, 4-Cl-IAA plus GA3, or 0.1% (v/v) aqueous Tween 80 (SPNS control) 12 h after deseeding (indicated by arrow; 0 h on x axis). PsGA3ox1 mRNA levels were monitored 2, 4, 8, and 12 h after hormone application. Data are means ± SE, n = 2 to 5.

Two-DAA intact pericarps contained 0.19 ± 0.02 fg 200 ng of total RNA¹ PsGA3ox1 transcript. Pericarp PsGA3ox1 transcript level remained stable 12 h (0 h on Fig. 6B) after splitting of pericarp (SP, 0.15 ± 0.04 fg 200 ng of total RNA¹); however, removal of seeds (SPNS) decreased PsGA3ox1 transcripts to minimally detectable levels 12 h after splitting and deseeding (0.006 ± 0.002 fg 200 ng of total RNA¹, 0 h on Fig. 6B). The effect of seeds and hormones on the expression of PsGA3ox1 in pea pericarp was investigated over a 12-h period (hormones were applied to the pericarps 12 h after deseeding [0 h in Fig. 6B] to allow sufficient time for the pericarps to become depleted of seed-produced factors that might affect pericarp growth). PsGA3ox1 mRNA levels in pericarp with seeds (SP) had increased about 3 times over the 12-h time course (to 0.50 ± 0.16 fg 200 ng of total RNA¹), whereas message from pericarps without seeds (SPNS) remained minimally detectable throughout the time course (Fig. 6B).

When deseeded pericarps were treated with 4-Cl-IAA, a significant increase in PsGA3ox1 mRNA levels was detected within 2 h of hormone application (Fig. 6B). Transcript levels in 4-Cl-IAA-treated deseeded pericarps peaked 4 h after hormone treatment (270-fold increase) and decreased substantially by 12 h after hormone treatment (levels 28-fold above levels at 0 h; Fig. 6B). Application of IAA or GA₃ to deseeded pericarps resulted in further reductions in PsGA3ox1 transcript levels compared with the SPNS treatment (Fig. 6B, insert). Simultaneous treatment of deseeded pericarps with 4-Cl-IAA and GA₃ (4-Cl-IAA + GA₃ treatment) resulted in a much smaller increase in PsGA3ox1 transcript levels 4 h after hormone application than observed in 4-Cl-IAA-induced deseeded pericarps (Fig. 6B).

Comparison of PsGA3ox1 Gene Expression among Pea Tissues

Pericarp tissue at 2 DAA had the lowest expression of PsGA3ox1 among the various tissues assayed (over 1,000 times lower than 3 d after imbibition [DAI] shoots and roots; Table I). By 10 DAA, expression of pericarp PsGA3ox1 expressed as femtogram 200 ng of total RNA¹ was greater than 3-DAI cotyledons and 10-DAA seeds. In 3-DAI seedling tissues, the level of PsGA3ox1 message was: shoot > root tips > cotyledons.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

PsGA3ox1 (Mendel's LE gene) plays an important role in pea plant height (Lester et al., 1997; Martin et al., 1997); however, its role in fruit development has remained unclear. We were able to detect PsGA3ox1 signal in 2-DAA intact pea pericarps on northern blots when 30 µg of total RNA was probed using a [³²P]-labeled riboprobe of the full-length PsGA3ox1 gene; however, the signal was very weak (data not shown). Using northern-blot analysis, Martin et al. (1997) were unable to detect PsGA3ox1 signal in unopened pea flowers or 1-cm young fruits. Therefore, a more sensitive method of mRNA detection was required to study regulation of PsGA3ox1 message level in this tissue. Using real-time RT-PCR analysis, the PsGA3ox1 target sequence was amplified clearly and reproducibly, and quantification of plant tissue PsGA3ox1 mRNA in real-time RT-PCR assays was achieved by comparison against serially diluted plasmid-derived PsGA3ox1 mRNA. In addition, a similar trend in C_t values for 2-, 6-, 10-, and 14-DAA pericarp samples assayed using a second set of forward and reverse primers (GA3ox126; compared with the GA3ox87 primers) is further evidence of the specificity of the assay for PsGA3ox1. Using this assay, we found that PsGA3ox1 mRNA levels were regulated during pollination and subsequent development of pericarps and seeds, as well as during hormone-induced pericarp growth.

Pericarps from unpollinated ovaries contained high levels of PsGA20ox1 mRNA (2 DAA; van Huizen et al., 1997) and GA₂₀ (5 ng g fresh weight¹; emasculated ovaries at the equivalent to 0 and 1 DAA of pea cv Alaska; Garcia-Martinez et al., 1991), but minimally detectable levels of PsGA3ox1 mRNA (Fig. 4) and GA₁ (Garcia-Martinez et al., 1991). After pollination (0 DAA), pericarp PsGA3ox1 mRNA levels increased 50-fold, then rapidly decreased to levels 10-fold above prepollinated levels by 1 DAA and remained at this level through 4 DAA (Figs. 4 and 5). Seed PsGA3ox1 mRNA levels increased 19-fold after pollination (0 DAA), then decreased to levels 9-fold above prepollinated levels by 1 DAA (Fig. 4). Although steady-state GA₁ levels were reported to be the same in pollinated and non-pollinated ovaries at 0 DAA, GA₈ levels were 2 times higher in pollinated than non-pollinated ovaries at this time (Garcia-Martinez et al., 1991). Because GA₈ is the immediate biologically inactive product of GA₁ (as a result of 2-hydroxylation), these data suggest that more GA₁ was synthesized in pollinated than non-pollinated pericarps and/or ovules at 0 DAA. In addition, the large increase in PsGA3ox1 mRNA levels was not observed in non-pollinated pericarps at the equivalent to 0 DAA; instead, a large increase in PsGA3ox1 message was detected at the equivalent to 3 DAA (Fig. 4). This dramatic increase in PsGA3ox1 message at the equivalent to 3 DAA is likely due to feedback regulation of GA biosynthesis, because at this time levels of GA₂₀, GA₁, GA₈, and GA₃ in non-pollinated ovaries are extremely low or not detectable (Garcia-Martinez et al., 1991). However, even if PsGA3ox1 message was converted to active enzyme and substrate (GA₂₀) was available, the ability of non-pollinated pericarps at the equivalent to 3 DAA to respond in growth to GAs (exogenous) is minimal (Garcia-Martinez and Carbonell, 1980). These data show that pollination triggers the synthesis of pericarp PsGA3ox1 mRNA message and suggest that GA₁ is synthesized from the pool of GA₂₀ present in prepollinated pericarps by pericarp GA 3-hydroxylase, and that this pulse of GA₁ stimulates initial fruit set and development.

After pollination, pericarp PsGA3ox1 levels increased linearly from 6 to 10 DAA (Fig. 5A; from 5-8 DAA when expressed as femtogram per milligram fresh weight¹, Fig. 5B). This increase in PsGA3ox1 mRNA levels coincided with the rapid increase in pericarp diameter to accommodate the developing seeds (Fig. 1, B and C). From 4 to 7 DAA, mesocarp cells are continuing to expand and the only increases in cell number occur in cell layers of the endocarp middle zone layer (pericarp wall thickness; Ozga et al., 2002). GA₁ levels in pollinated pericarps (pea cv Alaska) were approximately 1 ng g fresh weight¹ from 4 to 12 DAA, with one exception: 6-DAA pericarps contained about 1.75 ng g fresh weight¹ (Rodrigo et al., 1997). The steady-state GA₁ levels do not increase with increasing PsGA3ox1 message level during this period. It is possible that the turnover rate of GA₁ is substantially higher during this period, resulting in similar steady-state GA₁ levels from 4 to 12 DAA. Alternatively, a substantial amount of the PsGA3ox1 message may be degraded before translation into protein. Monitoring of GA₁ turnover rate and GA 3-hydroxylase protein levels, and sensitive metabolic studies during pericarp development would differentiate between these hypotheses.

A 2-fold increase (femtogram 200 ng of total RNA¹) in seed PsGA3ox1 levels occurs at 4 DAA (16-fold increase when expressed as femtogram milligram fresh weight¹; Fig. 5, A and B), which precedes the peak of GA₁ levels in the seeds at 6 DAA (about 90 ng g fresh weight¹; Rodrigo et al., 1997). At 4 DAA, 86% of the GA₁ observed in the seeds occurred in the testa, with the remainder in the endosperm (Rodrigo et al., 1997). A subsequent increase in seed PsGA3ox1 mRNA levels was observed from 8 to 12 DAA (Fig. 5A), coincident with rapid embryo (Fig. 2, A and B) and endosperm development (Eeuwens and Schwabe, 1975). When data were expressed as femtogram milligram fresh weight¹ (Fig. 5B), seed PsGA3ox1 mRNA levels were maintained between 50 and 75 fg mg fresh weight¹ from 6 to 14 DAA, whereas seed GA₁ levels decreased to 20 ng g fresh weight¹ by 8 DAA and were minimally detectable by 12 DAA (Rodrigo et al., 1997).

Because in vitro metabolism experiments show that [¹⁴C]GA₁₂ is converted to [¹⁴C]GA₁₉ in 4-DAA seeds (Rodrigo et al., 1997), it is possible that GA₁ is synthesized through the early 13-hydroxylation pathway in developing pea seeds; however, using genetic and metabolic studies, an alternative hypothesis for GA₁ synthesis via GA₄ has been proposed. This model proposes that GA₄ is synthesized in the endosperm/embryo (4 DAA) and transported to the testa, where it is 13-hydroxylated to GA₁ (Rodrigo et al., 1997; MacKenzie-Hose et al., 1998). However, conversion of GA₄ or other GA substrates to GA₁ in any seed tissues has not been demonstrated to date. Although it is possible that expression of a second 3-hydroxylase with a different substrate specificity occurs in young seeds, the expression pattern of PsGA3ox1 (LE) in seeds (Figs. 4 and 5) suggests that PsGA3ox1 gene is involved in the production of GAs for seed development. Further metabolism experiments and monitoring of GA₁ turnover rates and GA 3-hydroxylase protein levels during seed development are also required to further understand regulation of GA₁ biosynthesis in seed tissue.

Both PsGA20ox1 and PsGA3ox1 mRNA levels substantially decrease after pollination. This decrease in message levels may be important for fine-tuned regulation of GA biosynthesis by a multitude of factors including hormones (auxins and GAs) during this period. The effects of seeds and hormones (4-Cl-IAA, IAA, and GA₃) on the expression of PsGA3ox1 in pea pericarp were investigated over a 24-h treatment period from 2 to 3 DAA. The presence of seeds was required for PsGA3ox1 gene expression in the pericarp (Fig. 6B). When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1 levels peaked 4 h after hormone treatment (0.006-1.623 fg 200 ng of total RNA¹ or a 270-fold increase). 4-Cl-IAA stimulated the expression of both PsGA20ox1 (van Huizen et al., 1997) and PsGA3ox1 (Fig. 6B) in pea pericarp, suggesting that similar auxin-induced transcription regulatory elements may operate to coordinate regulation of this part of the GA biosynthesis pathway. Also, as with PsGA20ox1, pericarp PsGA3ox1 mRNA levels were stimulated only by biologically active auxin (4-Cl-IAA stimulates pericarp growth and IAA does not; Reinecke et al., 1995; Ngo et al., 2002; Fig. 6B). Similarly, application of IAA to decapitated pea plants restored internode elongation and the level of PsGA3ox1 levels in the elongating internode (Ross et al., 2000). Because PsGA3ox1 mRNA levels were at minimally detectable levels at the time of hormone application (0 h, Fig. 6B), feedback regulation of PsGA3ox1 expression by GA₃ was minimal. However, GA₃ reduced 4-Cl-IAA stimulation of PsGA3ox1 mRNA levels (Fig. 6B), indicating that GA₃ does feedback regulate PsGA3ox1 message levels in the pericarp.

We propose the following working model for hormonal-directed fruit set and seed- and pericarp-coordinated development. Pollination stimulates GA₁ synthesis (via an increase in GA3ox mRNA levels) in both the seeds and pericarp, resulting in initial fruit set and growth of both tissues. Subsequently, seeds maintain growth in the pericarp at least in part by transporting auxin (4-Cl-IAA) to the pericarp, where it stimulates both GA20ox and GA3ox message levels maintaining a critical level of GA₁ for pericarp growth. Biologically active GA (GA₁) also can feedback regulate its synthesis in the pericarp by reducing GA20ox and GA3ox message levels. In addition, 4-Cl-IAA affects fruit growth directly through auxin-mediated responses (van Huizen et al., 1996). Therefore, interaction of both auxin and GAs are required for coordination of fruit and seed development.

PsGA3ox1 mRNA levels were assayed in young pea seedlings to examine relative amounts of PsGA3ox1 transcript in these tissues versus fruit tissues. The levels of PsGA3ox1 mRNA varied considerably among the pea plant organs assayed (Table I), with roots and shoots of 3-DAI seedlings having the greatest levels, followed by 10-DAA pericarps, 10-DAA seeds, 3-DAI cotyledons, 2-DAA seeds, and 2-DAA pericarps in descending order. It is interesting to note that in 3-DAI shoots, PsGA3ox1 message levels were about 1,000 times higher than those of 2-DAA pericarps. It is possible that other GA3ox genes exist in pea (Southern-blot analysis indicated only one gene in the pea genome; Lester et al., 1997; Martin et al., 1997) as in Arabidopsis (four known GA3ox genes; Hedden and Phillips, 2000); however, the low levels of PsGA3ox1 mRNA in 2-DAA pericarp compared with shoots helps explain why conversion of labeled GA₂₀ to GA₁ in pericarps has been difficult to obtain using conventional methods of detection.

It has been proposed that the sensitivity of the fruit to GA may be substantially greater than that of the stem internode. Santes et al. (1993) investigated the effect of the le mutation on the growth and GA content of developing fruits in near-isogenic lines of pea 205⁺ (LELE, PsGA3ox1) and 205 (lele, 1-bp point mutation of LE that greatly increases the K_m of GA 3-hydroxylase; Martin et al., 1997). Stem elongation is reduced in 205 plants (about 65%), but the growth of pods and seeds are less affected by the le mutation. GA₂₀ was less active in stimulating ovary growth on 205 than on 205⁺, whereas GA₁ had similar high activity on both lines. The content of GA₁ in 6-DAA seeds and pericarps of 205 was 2.7 and 7 times (nanograms gram fresh weight¹) or 5 and 10 times lower (nanograms ovary¹) than 205⁺, respectively. The levels of GA₂₀ and GA₂₉ were substantially elevated in 205 compared with 205⁺ in both tissues (Santes et al., 1993). These results suggest that 3-hydroxylation of GA₂₀ to GA₁ is reduced in pericarps and seeds by the le mutation, even though it does not affect final fruit phenotype.

In wild-type (LE) Alaska plants, when the size of the pericarp was plotted against the GA₁ concentration in non-pollinated fruit growing after application of GA₁ or GA₃ to the leaf subtending the fruit, there was a linear relationship from about 0.1 (minimum amount necessary for fruit set and growth) to 2 ng g fresh weight¹ (Rodrigo et al., 1997). Higher concentrations of GA₁ in the pericarp (20 ng g fresh weight¹, obtained by applying 2 µg to the leaf) did not produce substantial further growth. Therefore, the content of GA₁ found in le pods at 6 DAA (0.1 ng g fresh weight¹; Santes et al., 1993) may be sufficient, if not optimal, to stimulate fruit set and development.

In addition, application of a GA biosynthesis inhibitor (LAB 198 999; blocks the later steps in the GA biosynthesis pathway including 3-hydroxylation) to elongating pollinated pericarps inhibited pericarp growth (fresh weight decreased by 26%), and reduced pericarp GA₁ levels 3-fold; and the inhibition was reversed by treatment with GA₁ or GA₃ (Santes and Garcia-Martinez, 1995). Because LAB also produced an enhancement of GA₁₉ and GA₂₀ levels, despite a reduction of fruit growth, these GAs must not be active per se but become so after conversion to an active GA (GA₁). LAB also decreased seed GA₁ content 4-fold and seed growth about 16% in 6-DAA fruits (Santes and Garcia-Martinez, 1995). Our work along with the work of others suggests a role for GA 3-hydroxylase and GA₁ in growth and development of pea pericarps and likely in seeds.

In summary, our work shows the effectiveness of real-time RT-PCR in quantitating mRNA transcripts from genes expressed at very low levels. This work would not have been possible using standard northern-blot analysis. Real-time RT-PCR enabled sensitive, reproducible, and specific detection and quantification of PsGA3ox1 mRNA in pea tissues. We found that PsGA3ox1 mRNA levels were regulated during pollination and subsequent development of the pericarp and seed, as well as during hormone-induced pericarp growth. The presence and developmental pattern of PsGA3ox1 message in pea pericarp suggest that GA₁ is synthesized in the pericarp for fruit growth. 4-Cl-IAA stimulation of pea pericarp PsGA20ox1 and PsGA3ox1 expression (and the lack of stimulation by IAA) suggests biologically active auxin acts in a concerted fashion on the GA biosynthesis pathway to stimulate production of active GAs in the fruit. Further research at the gene, protein, and enzyme level is required to understand fully the role PsGA3ox1 plays in GA biosynthesis during pea fruit development.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plant Material and Treatments

Plants of pea (Pisum sativum L. line I₃ [Alaska-type, LE]) were grown under 16-h-light/8-h-dark photoperiod (19°C/17°C) with an average photon flux density of 402 µE m² s¹ (van Huizen et al., 1995). Pericarps (2, 0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, and 20 DAA) and ovules (2 DAA) or seeds (0, 1, 2, 3, 4, 5, 6, 7, 8, 10, 12, 14, and 20 DAA) were collected from the first to the fifth flowering node for experiments monitoring PsGA3ox1 expression during fruit development. Non-pollinated pericarps (first to fifth flowering node) were emasculated 2 DAA and harvested from the plant at equivalent to 1, 0, 1, 2, and 3 DAA (ovules removed at harvest). For hormone-treated pericarps, one fruit between the third and fifth flowering nodes was used per plant; subsequent flowers and lateral buds were removed as they developed. Terminal apical meristems of plants were intact and pericarps remained attached to the plant during the entire experiment.

Pericarps were treated with hormones using a split-pod technique (Ozga et al., 1992). Fruits at 2 DAA measuring 15 to 20 mm in length were split down the dorsal suture 1 h before the 8-h dark period, and seeds were either left intact (SP) or removed (SPNS). Surgical manipulation of the pea fruit was completed 12 h before all hormone applications. Pericarps were treated with IAA, 4-Cl-IAA, and/or GA₃ (50 µM in 0.1% [v/v] aqueous Tween 80; 30 µL total). All solutions were applied directly to the inside surface of the pericarp wall (endocarp). The SP and SPNS controls were treated with 30 µL of 0.1% (v/v) aqueous Tween 80. Treated pericarps were covered with plastic bags to maintain high humidity. Pericarps were harvested at 2, 4, 8, and 12 h after the hormone treatment. Seeds, if present, were removed from the pericarp at harvest.

For seedling tissues, seeds of pea line I₃ (Alaska-type, LE) were surface sterilized in 80% (v/v) ethanol for 1 min, then gently shaken in 1.5% (v/v) sodium hypochlorite with 0.15% (v/v) Tween 80 at 22°C ± 0.5°C for 20 min. The seeds were rinsed five times with sterile deionized water, and those with intact seed coats were immediately placed 2.5 cm deep into moist sterilized sand in 1-L plastic containers. The containers were placed at 22°C/20°C (16/8 h) in a growth chamber for 3 d. No shoot emergence from the sand had occurred at the time of harvest. All tissues were harvested into liquid N₂ and subsequently stored at 80°C until extraction.

RNA Isolation

Two whole or half pericarps, seeds, and 3-DAI seedling tissues (root tips [first 4 mm], remainder of roots, shoots, and cotyledons) were ground to a fine powder in liquid N₂; and 100 to 500 mg fresh weight pericarp, 10 to 500 mg fresh weight seed, or 60 to 500 mg fresh weight seedling tissue subsamples were used for RNA extraction. Total RNA was extracted using a modified TRIzol (Invitrogen, Carlsbad, CA) procedure. In brief, after initial extraction with the TRIzol reagent and centrifugation, the supernatant was cleaned by chloroform partitioning (0.2 mL per 1 mL of TRIzol reagent). Subsequently, for further purification, the following steps were carried out in order: high salt precipitation (1.2 M NaCitrate and 0.8 M NaCl) to remove polysaccharides and proteoglycans, 4 M LiCl precipitation, followed by precipitation with 1:20 (v/v) solution of 3 M NaAcetate (pH 5):100% ethanol. The total RNA samples were then treated with DNase (DNA-free kit, Ambion, Austin, TX) and stored at 80°C before real-time RT-PCR analysis.

Oligonucleotide Primers and Probe

Oligonucleotide primers for the GA3ox87 amplicon were designed from the GenBank accession number AF001219 sequence to bind to separate exons of PsGA3ox1 to avoid false positive results arising from amplification of contaminating genomic DNA using Primer Express software (PE-Applied Biosystems). For PsGA3ox1 quantitation, the following primers and probe were used to produce an 87-bp amplicon that spans nucleotides 498 to 584 of AF001219: forward primer, 5'-TTCGAGAACTCTGGCCTCAAG-3'; reverse primer, 5'-ATGTTCCTGCTAACTTTTTCATGGTT-3'; and TaqMan MGB fluorescent dye-labeled complement strand probe (PE-Applied Biosystems), 3'-6FAM-ACAATATCACAGAATCTGGT-MGBNFQ-5'. The GA3ox87 amplicon is outside the region proposed as Fe⁺- and 2-oxoglutarate-binding regions for 2-oxoglutarate-dependent dioxygenases of which GA3ox is a member (Martin et al., 1997).

For pea 18S small subunit nuclear ribosomal RNA quantitation, primers were designed from the GenBank accession number U43011 sequence to produce a 62-bp amplicon that spans nucleotides 1,575 to 1,636 of this sequence as follows: forward primer, 5'-ACGTCCCTGCCCTTTGTACA-3'; reverse primer, 5'-CACTTCACCGGACCATTCAAT-3'; and TaqMan fluorescent dye-labeled probe (PE-Applied Biosystems), 5'-VIC-ACCGCCCGTCGCTCCTACCG-TAMRA-3'.

To test the specificity of the GA3ox87 quantitation primers above, a second set of primers (GA3ox126) were constructed to a region that spanned 126 bp from nucleotides 451 to 576 of AF001219 as follows: forward primer, 5'-CATGTGGTATGAGGGATTTACTATCGT-3'; reverse primer, 5'-GCTAACTTTTTCATGGTTTCATCATATT-3'; and TaqMan MGB fluorescent dye-labeled complement strand probe (PE-Applied Biosystems), 3'-6FAM-ACAATATCACAGAATCTGGT-MGBNFQ-5'.

Preparation of RNA Standard Curve

RNA transcripts were generated from a recombinant plasmid carrying the complete cDNA of PsGA3ox1 (GenBank accession no. AF001219). The plasmid was linearized with EcoRI, treated with proteinase K at 37°C for 1 h, extracted with phenol-chloroform, then chloroform, and precipitated with 1:20 (v/v) solution of 3 M NaAcetate (pH 5):100% ethanol at 20°C. RNA transcripts were generated at 37°C for 2 to 4 h using T₃ RNA polymerase with the RiboMax in vitro transcription kit (Promega, Madison, WI). Plasmid DNA was subsequently digested with RNase-free DNase I at 37°C for 15 min, and the transcripts purified by one phenol-chloroform extraction and RNeasy Mini Kit (Qiagen, Mississauga, ON). RNA concentration was determined spectrophotometrically at A₂₆₀.

C_ts for a dilution series of RNA transcripts (0.05, 0.5, 5, 50, and 500 fg) were plotted to yield a standard curve for each 96-well reaction plate. C_t was initially calculated by the TaqMan software to indicate significant fluorescence signals above noise during the early cycles of amplification. When necessary, C_t was adjusted manually to cross an exponential portion of the amplification curves of all samples being compared on the 96-well plate.

TaqMan RT-PCR Assay

The TaqMan One-Step RT-PCR reagent kit (PE-Applied Biosystems) was used throughout as 50-µL reactions in a model 7700 Sequence Detector (PE-Applied Biosystems). The RT-PCR mixture contained 25 µL of 2× Master Mix (containing AmpliTaq Gold DNA polymerase), 1.25 µL of 40× MultiScribe (reverse transcriptase) and RNase Inhibitor Mix, 300 nM forward primer, 300 nM reverse primer, 100 nM probe, and 200 ng of total RNA for PsGA3ox1 or 10 pg of total RNA for 18S rRNA quantitation. Thermal cycling conditions were 48°C for 30 min for RT, 95°C for 10 min for Taq activation, and 40 cycles of 95°C for 15 s and 60°C for 1 min for PCR. Each sample was assayed twice, and the average of the two assays was used to calculate the PsGA3ox1 concentration.