Decapitation Reduces the Metabolism of Gibberellin A20 to A1 in Pisum sativum L., Decreasing the Le/le Difference

Abstract

In the garden pea (Pisum sativum L.), the mutant allele le blocks the conversion of GAZ0 to GAl (Ingram et al., 1984), reducing the endogenous level of GAl (Ross et al., 1992; Smith et al., 1992). This is responsible for the dwarf phenotype of le plants. It has been suggested that le codes for an altered form of GA20 3B-hydroxylase, the enzyme that catalyzes the step GAZo to GAl (Ross et al., 1989; MacMillan, 1990). Recently, however, Smith (1992) questioned this proposa1 in a model addressing severa1 important aspects of GA biosynthesis in pea. According to this model the apical bud itself does not metabolize GAZo to a significant extent. Instead, it is suggested that GAl production is confined largely to young stem tissue, where it is regulated by an inhibitor of GA2o 30-hydroxylation produced by the apical bud (and possibly by young, expanding leaflets). Smith (1992) proposed that in le plants the inhibitor is constitutively produced, thereby blocking GA1 production. Smiths model is based on the metabolism of [13C,3H]GAz0 by excised leaves, stems, and apical buds. The metabolites were chromatographed by HPLC as the free acids (Smith, 1992). In extracts from apical buds and leaves the level of radioactivity co-eluting with GAl (GA1-like) was very low. This level was higher in extracts from stems, but in a11 cases was not affected by the Le/le gene difference.We have investigated the metabolism of [13C,3H]GAz0 in plants either left intact or with their apical buds removed (decapitated). Furthermore, we have reproduced the experimental conditions used by Smith (1992) and have analyzed the resulting extracts by HPLC as the free acids and methyl esters, and by GC-MS. Our results do not support the in- MATERIALS A N D METHODSThe lines used were the isolines 205+ (Le, tall) and 205-(le, dwarf), developed from cross L2 X L77 at Hobart (Tasmania, Australia). The plants were grown, two per pot, in a heated glasshouse under an 18-h photoperiod (Reid and Potts, 1986). Experiments were conducted when the plants had expanded to five to seven leaves (including the scale leaves).The substrate in a11 experiments was [17-13C,3H~]GA~~ (1.27 X 10" Bq mmol-'; Ingram et al., 1984). Two experimental systems were used. First, substrate (2 r g in 10 PL of ethanol) was applied to the second uppermost fully expanded leaf of 12 tall and 12 dwarf plants. Immediately afterward, six tall and six dwarf plants were decapitated by excising just below the node situated three nodes up from the treated leaf. This left three intemodes and two leaves above the treated leaf in decapitated plants. After 2 d, the uppermost two of these intemodes were harvested together (stem portion), as were the stipules, petiole, and leaflets of the uppermost leaf (leaf portion). Intact plants were harvested by first excising the apical portion as before and then harvesting the next two intemodes down and (separately) the associated leaf, as for decapitated plants. A11 excised portions were then immersed in cold (-20°C) methanol.Second, the technique of Smith ...

Auxin Regulation of the Gibberellin Pathway in Pea

“…14 C]GA 20 was added to the liquid Murashige and Skoog without IAA, and the results supported the findings of Sherriff et al (1994): After 6 h, the main metabolite recovered from the segments was […”

“…This is the first time, to our knowledge, that isolated stem tissue of pea, or any isolated section of pea, has been unequivocally shown to exhibit PsGA3ox1 activity. Past studies have attempted to culture excised pea stem segments to investigate the effect of the le mutation on the metabolism of applied radiolabeled GA 20 (Smith, 1992;Sherriff et al, 1994). In light of current results, the lack of GA 1 peaks observed by Sherriff et al (1994) was attributable to the absence of auxin in the incubation medium, which resulted in the majority of applied GA 20 being converted to GA 29 (Fig.…”

“…The system has the advantage of allowing the addition and quick uptake of IAA and other compounds as well as radiolabeled GA substrates for use in GA metabolism studies. Sherriff et al (1994) reported no evidence of GA 1 -or GA 8 -like peaks when excised LE stem segments, cultured in liquid Murashige and Skoog, were fed [ 13 C…”

“…Earlier, Smith (1992) proposed that GA 1 was a product of GA 20 in Murashige and Skoogcultured stem tissue, but Sherriff et al (1994) suggested that the product concerned was in fact GA 29 -catabolite. The subsequent work of Ross et al (2000) suggests that the lack of GA 1 -like peaks (and therefore a lack of GA 8 -like peaks) reported by Sheriff et al might be attributable to the absence of IAA in the culture medium.…”

Effects of Auxin Transport Inhibitors on Gibberellins in Pea

“…These metabolites were [ 13 C, 3 H]GA 1 , [ 13 C, 3 H]GA 29 , [ 13 C, 3 H]GA 8 , [ 13 C, 3 H]GA 81 , as found before (Ross et al 1995, Sherriff et al 1994. Metabolite levels were determined by HPLC radiocounting (Table 3 and Fig.…”