Dennis M. Reinecke

Hormonal Interactions in Fruit Development

Fruit development involves a complex interplay of cell division, differentiation and expansion of sporophytic and gametophytic tissues that is carefully coordinated temporally and spatially. Plant hormones are signal molecules that regulate many processes of plant development, including fruit development leading to mature fruit and viable mature seed. Auxins, gibberellins, cytokinins, abscisic acid, and ethylene have been implicated at various stages of fruit development. In the past, hormone application studies and hormone analysis studies have supported the hypothesis that fruit development is in part regulated by hormonal interaction. More recently, biochemical, genetic, and molecular studies are beginning to unravel the complexities of how hormones affect fruit development. In the current work, we review selected studies that show the interplay between hormones during fruit development, with an emphasis on the interaction between auxin and gibberellin in pea fruit development.

Pollination-, development-, and auxin-specific regulation of gibberellin 3beta-hydroxylase gene expression in pea fruit and seeds.

To understand further how pollination, seeds, auxin (4-chloroindole-3-acetic acid [4-Cl-IAA]), and gibberellins (GAs) regulate GA biosynthesis in pea (Pisum sativum) fruit, we studied expression of the gene PsGA3ox1 that codes for the enzyme that converts GA20 to biologically active GA1 using real-time reverse transcription-polymerase chain reaction analysis. PsGA3ox1 mRNA levels were minimally detectable in prepollinated pericarps and ovules (−2 d after anthesis [DAA]), increased dramatically after pollination (0 DAA), then decreased by 1 DAA. Seed PsGA3ox1 mRNA levels increased at 4 DAA and again 8 to 12 DAA, when seed development was rapid. Pericarp PsGA3ox1 mRNA levels peaked coincidentally with rapid pod diameter expansion (6–10 DAA) to accommodate the growing seeds. The effects of seeds and hormones on the expression of pericarpPsGA3ox1 were investigated over a 24-h treatment period. Pericarp PsGA3ox1 mRNA levels gradually increased from 2 to 3 DAA when seeds were present; however, when the seeds were removed, the pericarp transcript levels dramatically declined. When 2-DAA deseeded pericarps were treated with 4-Cl-IAA, PsGA3ox1mRNA levels peaked 4 h after hormone treatment (270-fold increase), then decreased. PsGA3ox1 mRNA levels in deseeded pericarps treated with indole-3-acetic acid or GA3were the same or lower than deseeded controls. These data show thatPsGA3ox1 is expressed and developmentally regulated in pea pericarps and seeds. These data also show that pericarpPsGA3ox1 expression is hormonally regulated and suggest that the conversion of GA20 to GA1 occurs in the pericarp and is regulated by the presence of seeds and 4-Cl-IAA for fruit growth.

Hormone and Seed-Specific Regulation of Pea Fruit Growth

Growth of young pea (Pisum sativum) fruit (pericarp) requires developing seeds or, in the absence of seeds, treatment with gibberellin (GA) or auxin (4-chloroindole-3-acetic acid). This study examined the role of seeds and hormones in the regulation of cell division and elongation in early pea fruit development. Profiling histone H2A and γ-tonoplast intrinsic protein (TIP) gene expression during early fruit development identified the relative contributions of cell division and elongation to fruit growth, whereas histological studies identified specific zones of cell division and elongation in exocarp, mesocarp, and endocarp tissues. Molecular and histological studies showed that maximal cell division was from −2 to 2 d after anthesis (DAA) and elongation from 2 to 5 DAA in pea pericarp. Maximal increase in pericarp γ-TIP message level preceded the maximal rate of fruit growth and, in general, γ-TIP mRNA level was useful as a qualitative marker for expanding tissue, but not as a quantitative marker for cell expansion. Seed removal resulted in rapid decreases in pericarp growth and in γ-TIP and histone H2A message levels. In general, GA and 4-chloroindole-3-acetic acid maintained these processes in deseeded pericarp similarly to pericarps with seeds, and both hormones were required to obtain mesocarp cell sizes equivalent to intact fruit. However, GA treatment to deseeded pericarps resulted in elevated levels of γ-TIP mRNA (6 and 7 DAA) when pericarp growth and cell enlargement were minimal. Our data support the theory that cell division and elongation are developmentally regulated during early pea fruit growth and are maintained by the hormonal interaction of GA and auxin.

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.

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA20 to bioactive GA1) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA20 to GA29), suggesting a concerted regulation to increase levels of bioactive GA1 following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA1 to GA8) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA1, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [14C]GA12 to [14C]GA1 only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA1 required for initial fruit set and growth.

Effect of halogen substitution of indole-3-acetic acid on biological activity in pea fruit

Abstract Auxins (a class of plant growth hormones naturally present in all plants) have been implicated in fruit growth of pea. Pea ( Pisum sativum L.) fruit contain the auxins indole-3-acetic acid (IAA) and 4-chloroindole-3-acetic acid (4-C1-IAA). Fruits grow poorly and subsequently abscise when seeds are removed two days after anthesis, but 4-C1-IAA can substitute for the seeds in maintaining growth of deseeded fruit (pericarp) in planta . Applications of 4-C1-IAA promoted pericarp growth, the effect increasing with concentration from 1 to 100 μM, but IAA was ineffective in stimulating growth when tested from 0.1 to 100 μM. The effect of the position of the halogen on pericarp growth was examined by assaying the activities of 4-, 5-, 6- and 7-chloro- and fluoro-substituted IAA. The position and type of halogen dramatically affected auxin activity, with the natural product 4-C1-IAA being most effective. Of the other compounds tested, only 5-C1-IAA stimulated pea pericarp elongation, and then only moderately. Fluoro-substituted IAAs did not stimulate pericarp growth, and 4-F-IAA was inhibitory. This study is unique in that it reports the biological activity of 4-C1-IAA and halogen-IAA analogues in tissues of intact plants known to contain 4-C1-IAA. The relative activity of the compounds is discussed in reference to previous reports of auxin activity in other systems, and 4-C1-IAAs possible importance in pea fruit growth.

4-Chloroindole-3-acetic acid and plant growth

Abstract4-Chloroindole-3-acetic acid (4-Cl-IAA) is a potent auxin in various auxin bioassays. Researchers have used 4-Cl-IAA as well as other halogenated auxins in biological assays to understand the structural features of auxins required to induce auxin mediated growth in plants. 4-Cl-IAA is a naturally occurring auxin in plants from the Vicieae tribe of the Fabaceae family; and 4-Cl-IAA has also been identified in one species outside the Vicieae tribe, Pinus sylvestris. The apparent function of the unique auxin 4-Cl-IAA in normal plant growth and development will be discussed with a focus on Pisum sativum and Vicia faba

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-Cl-IAA). Pericarp with seeds and deseeded pericarp treated with 4-Cl-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-Cl-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-Cl-IAA can substitute for the seeds in the stimulation of pericarp growth and the conversion of GA19 to GA20.

4-chloroindole-3-acetic and indole-3-acetic acids in Pisum sativum

Abstract Endogenous IAA and 4-chloroindole-3-acetic acid (4-C1-IAA) were analysed in vegetative and reproductive tissues of the garden pea (Pisum sativum) using GC-MS selected ion monitoring in the presence of stable-isotope labelled internal standards. In fruit collected 3–8 days after anthesis (DAA) conjugates of both auxins were more abundant than the free hormones. Auxin levels (ng g−1 fr. wt) in the seeds were higher by 1–2 orders of magnitude than in the pericarps. However, as seeds are small at this stage, the pericarp nevertheless contains a substantial fraction of the overall quantity of IAA and 4-C1-IAA. While, in young fruit tissues, IAA was more abundant than 4-C1-IAA, the opposite was true for seeds at the ‘table-ready’ stage and for roots of nine-day-old seedlings. Our data suggest that both IAA and 4-C1-IAA are required to coordinate the vegetative and reproductive growth of pea plants.

Interaction of 4-chloroindole-3-acetic acid and gibberellins in early pea fruit development

In pea, normal pod (pericarp) growth requires the presence of seeds; and in the absence of seeds, gibberellins (GAs) and/or auxins can stimulate pericarp growth. To further characterize the function of naturally occurring pea GAs and the auxin, 4-chloroindole-3-acetic acid (4-Cl-IAA), on pea fruit development, profiles of the biological activities of GA3, GA1, and 4-Cl-IAA on pericarp growth were determined separately and in combination on pollinated deseeded ovaries (split-pericarp assay) and nonpollinated ovaries. Nonpollinated ovaries (pericarps) responded differently to exogenous GAs and 4-Cl-IAA than pollinated deseeded pericarps. In nonpollinated pericarps, both GA3 and 4-Cl-IAA stimulated pericarp growth, but GA3 was significantly more active in stimulating all measured parameters of pericarp growth than 4-Cl-IAA. 4-Cl-IAA, GA1, and GA3 were observed to stimulate pericarp growth similarly in pollinated deseeded pericarps. In addition, the synergistic effect of simultaneous application of 4-Cl-IAA and GAs on pollinated deseeded pericarp growth supports the hypothesis that GAs and 4-Cl-IAA are involved in the growth and development of pollinated ovaries.