Seed and Hormonal Regulation of Gibberellin 20-Oxidase Expression in Pea Pericarp.

To understand further how seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum L.) pericarp at the molecular level, we studied the expression of GA 20-oxidase in this tissue using northern-blot analysis. Pericarp GA 20-oxidase mRNA levels were highest from prepollination (-2 d after anthesis [DAA]) through anthesis (0 DAA), then decreased 3-fold by 2 DAA, and remained at these levels through 6 DAA. The effects of seeds and hormones (4-Cl-IAA and GA3) on the expression of GA 20-oxidase in pea pericarp were investigated over a 36-h treatment period. GA 20-oxidase mRNA levels in 2 DAA pericarp with seeds remained relatively stable throughout the treatment period; however, when the seeds were removed the pericarp transcript levels declined. When 2 DAA deseeded pericarps were treated with 4-Cl-IAA, a significant increase in GA 20-oxidase mRNA levels was detected within 2 h and transcript levels remained elevated for up to 12 h after 4-Cl-IAA application. GA3 significantly decreased GA 20-oxidase mRNA levels in deseeded pericarp within 2 h of application. These data suggest that the previously reported conversion of GA19 to GA20 in pea pericarp is controlled by seeds, 4-Cl-IAA, and GA3 at least in part by regulating GA 20-oxidase mRNA levels in this tissue.

Influence of Auxin and Gibberellin on in Vivo Protein Synthesis during Early Pea Fruit Growth.

Developing pea fruits (Pisum sativum L.) offer a unique opportunity to study growth and development in a tissue that is responsive to both gibberellins (GAs) and auxin (4-chloroindole-3-acetic acid[4-CI-IAA]). To begin a molecular analysis of the interaction of GAs and auxins in pea fruit development, in vivo labeling with [35S]methionine coupled with two-dimensional gel electrophoresis were used to characterize de novo synthesis of proteins during gibberellic acid (GA3)-, 4-CI-indoleacetic acid-, and seed-induced pea pericarp growth. The most significant and reproducible polypeptide changes were observed between molecular weights of 20 and 60. Comparing about 250 de novo synthesized proteins revealed that seed removal changed the pattern substantially. We identified one class of polypeptides that was uniquely seed induced and five classes that were affected by hormone treatment. The latter included 4-CI-IAA-induced, GA3-induced, GA3- and 4-CI-IAA-induced, 4-CI-IAA-repressed, and GA3- and 4-CI-IAA-repressed polypeptides. Similar patterns of protein expression were associated with both hormone treatments; however, changes unique to GA3 or 4-CI-IAA treatment also indicate that the effects of GA3 and 4-CI-IAA on this process are not equivalent. In general, application of 4-CI-IAA plus GA3 replaced the seed effects on pericarp protein synthesis, supporting our hypothesis that both hormones are involved in pea pericarp development.

Seed and 4-chloroindole-3-acetic acid regulation of gibberellin metabolism in pea pericarp.

In this study, we investigated seed and auxin regulation of gibberellin (GA) biosynthesis in pea (Pisum sativum L.) pericarp tissue in situ, specifically the conversion of [14C]GA19 to [14C]GA20. [14C]GA19 metabolism was monitored in pericarp with seeds, deseeded pericarp, and deseeded pericarp treated with 4-chloroindole-3-acetic acid (4-CI-IAA). Pericarp with seeds and deseeded pericarp treated with 4-CI-IAA continued to convert [14C]GA19 to [14C]GA20 throughout the incubation period (2-24 h). However, seed removal resulted in minimal or no accumulation of [14C]GA20 in pericarp tissue. [14C]GA29 was also identified as a product of [14C]GA19 metabolism in pea pericarp. The ratio of [14C]GA29 to [14C]GA20 was significantly higher in deseeded pericarp (with or without exogenous 4-CI-IAA) than in pericarp with seeds. Therefore, conversion of [14C]GA20 to [14C]GA29 may also be seed regulated in pea fruit. These data support the hypothesis that the conversion of GA19 to GA20 in pea pericarp is seed regulated and that the auxin 4-CI-IAA can substitute for the seeds in the stimulation of pericarp growth and the conversion of GA19 to GA20.

Seed effects on gibberellin metabolism in pea pericarp.

Pea fruit (Pisum sativum L.) is a model system for studying the effect of seeds on fruit growth in order to understand coordination of organ development. The metabolism of (14)C-labeled gibberellin A(12) (GA(12)) by pea pericarp was followed using a method that allows access to the seeds while maintaining pericarp growth in situ. Identification and quantitation of GAs in pea pericarp was accomplished by combined gas chromatography-mass spectrometry following extensive purification of the putative GAs. Here we report for the first time that the metabolism of [(14)C]GA(12) to [(14)C]GA(19) and [(14)C]GA(20) occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA(19) to GA(20) and caused the accumulation of radiolabeled and endogenous GA(19). Deseeded pericarp contained no detectable GA(20), GA(1), or GA(8), whereas pericarp with seeds contained endogenous and radiolabeled GA(20) and endogenous GA(1). These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA(19) to GA(20).

Oxidation of indole-3-acetic acid to oxindole-3-acetic acid by an enzyme preparation from Zea mays.

Indole-3-acetic acid is oxidized to oxindole-3-acetic acid by Zea mays tissue extracts. Shoot, root, and endosperm tissues have enzyme activities of 1 to 10 picomoles per hour per milligram protein. The enzyme is heat labile, is soluble, and requires oxygen for activity. Cofactors of mixed function oxygenase, peroxidase, and intermolecular dioxygenase are not stimulatory to enzymic activity. A heat-stable, detergent-extractable component from corn enhances enzyme activity 6- to 10-fold. This is the first demonstration of the in vitro enzymic oxidation of indole-3-acetic acid to oxindole-3-acetic acid in higher plants.

Oxindole-3-acetic Acid, an Indole-3-acetic Acid Catabolite in Zea mays.

A prior study (13) from this laboratory showed that oxidation of exogenously applied indole-3-acetic acid (IAA) to oxindole-3-acetic acid (OxIAA) is the major catabolic pathway for IAA in Zea mays endosperm. In this work, we demonstrate that OxIAA is a naturally occurring compound in shoot and endosperm tissue of Z. mays and that the amount of OxIAA in both shoot and endosperm tissue is approximately the same as the amount of free IAA. Oxindole-3-acetic acid has been reported to be inactive in growth promotion, and thus the rate of oxidation of IAA to OxIAA could be a determinant of IAA levels in Z. mays seedlings and could play a role in the regulation of IAA-mediated growth.