Gibberellin Metabolism in Cell-Free Extracts from Spinach Leaves in Relation to Photoperiod

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 14C-labeled gibberellin A12 (GA12) 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 [14CJGA12 to [14C1GA1g and [14C1GA20 occurs in pericarp of seeded pea fruit. Removal of seeds from the pericarp inhibited the conversion of radiolabeled GA19 to GA20 and caused the accumulation of radiolabeled and endogenous GA19. Deseeded pericarp contained no detectable GA20, GA1, or GA8, whereas pericarp with seeds contained endogenous and radiolabeled GA20 and endogenous GA1. These data strongly suggest that seeds are required for normal GA biosynthesis in the pericarp, specifically the conversion of GA1, to GA20.In pea (Pisum sativum L.), normal pod (pericarp) growth requires the presence of seeds (4); developing seeds contain high levels of GAs3 (11,12); and the requirement for seeds can be replaced by application of the plant hormone, GA (4, 17). Based on these observations, it has been assumed that the GAs biosynthesized by seeds are transported to the pericarp and regulate pericarp growth (6). However, Sponsel (17) proposed an alternative hypothesis that seeds may promote pericarp growth by maintaining GA biosynthesis in the pericarp. To test these hypotheses, our group initiated studies on GA biosynthesis in pea pericarp. The early 13-hydroxylation pathway is known to occur in pea (9): GA12 --+GA53 -* GA44 -* GA19 -, GA20 -. GA1 -+ GA8. We GA12 occurs in the pericarp and is regulated remotely by the presence of seeds. MATERIALS AND METHODS Plant Material and TreatmentsSeeds of pea (Pisum sativum L.) line I3 (Alaska-type) were grown as described by Maki and Brenner (13). Flowering began approximately 30 d after planting. Day 0 of fruit development was defined as the time when the petals were fully reflexed.The following surgical procedure was used to split the pericarp and remove fertilized ovules (seeds). The pericarp of ovaries (pericarp + seeds) were split down the dorsal suture, either without disturbing the seeds (SP treatment), or alternatively, the seeds were removed from the SP (deseeded) using forceps (SPNS treatment). High humidity was maintained by enclosing the pericarp in clear plastic bags. Terminal apical meristems of plants were intact and ovaries were fertilized in all treatments.To measure the effect of seed number on pericarp elongation, 2 DAA pericarp of ovaries were split as described above or left intact. Seeds were removed to obtain a final seed number of 0, 2, 4, 6, or 8 (all). The seeds removed were most distal to the midpoint of the pericarp, the remaining seeds being attached to the longitudinal midportion of the pe...

Section: Resultsmentioning

confidence: 99%

Section: Plant Materials and Treatmentsmentioning

confidence: 99%

Seed Effects on Gibberellin Metabolism in Pea Pericarp

Ozga¹,

Brenner²,

Reinecke³

1992

“…Large increases in GA content preceding stem elongation have also been observed in Agrostemma githago (Jones and Zeevaart, 1980). In spinach, the photoperiod is known to regulate at least two steps in GA metabolism, namely, the conversion of GAS3 to GA44 and of GAI9 to GAZ0 (Gilmour et al, 1986;Zeevaart et al, 1990;Talon et al, 1991b). Furthermore, L3H]GAZo was metabolized more rapidly in spinach plants in LD than in SD conditions (Metzger and Zeevaart, 1982), and treatment with a growth retardant led to a more rapid decline of the GA content of spinach in LD than in SD conditions (Zeevaart, 1971(Zeevaart, , 1974.…”

mentioning

confidence: 84%

“…In the case of spinach, no ent-kaurene synthetase activity was detectable in cell-free extracts from leaves (R.J. Bensen and J.A.D. Zeevaart, unpublished data), although active preparations for GA conversions late in the pathway can be obtained from such leaves (Gilmour et al, 1986). Consequently, it has not been possible until now to compare the rates of ent-kaurene biosynthesis in plants grown under SD and LD conditions.…”

mentioning

confidence: 99%

ent-Kaurene Biosynthesis Is Enhanced by Long Photoperiods in the Long-Day Plants Spinacia oleracea L. and Agrostemma githago L

Zeevaart

Gage

1993

The effect of photoperiod on ent-kaurene biosynthesis was determined in the long-day (LD) plants spinach (Spinacia oleracea 1.) and Agrostemma githago L . Further metabolism of ent-kaurene was blocked by application of the growth retardant tetcyclacis, and ent-kaurene accumulation was measured by isotopic dilution using gas chromatography-selected ion monitoring (CC-SIM) (E.Physiol 96: 1099-1104). In spinach, the rate of ent-kaurene accumulation in shoots grown under LD conditions was 3 times higher than in shoots grown under short-day (SD) conditions. ent-Kaurene also accumulated in fully expanded leaves, but at a lower rate than in shoots (15 and 55 pmol g-' dry weight h-', respectively). In Agrostemma, ent-kaurene accumulated at a rate 2.5 times higher in plants grown under LD conditions than in those grown under SD conditions. In spinach, enhanced ent-kaurene accumulation was detectable after 1 long day, and with exposure to additional long days, the rate of ent-kaurene accumulation increased further.Conversely, when plants were exposed to LD conditions and then returned to SD conditions, the rate of ent-kaurene accumulation decreased. Following tetcyclacis application, ent-kaurene accumulation was observed in all parts of spinach that were analyzed, but there were large quantitative differences between organs of different ages. As the leaves matured, enf-kaurene biosynthesis declined. Petioles accumulated more ent-kaurene than the corresponding leaf blades. It is concluded that stimulation of entkaurene biosynthesis by LD conditions leads to a higher rate of gibberellin biosynthesis, which is essential for stem elongation in rosette plants.There is considerable evidence that the rate of GA biosynthesis in LD rosette plants is higher under LD conditions, when stem elongation and flower formation take place, than during vegetative growth in the rosette stage under SD conditions. For example, the GA content increased severalfold after the transfer of Silene plants from SD to LD conditions

“…Light affects kaurene biosynthesis in pea seedling shoots (1 1), kaurenoic acid oxidation in G. fujikuroi (16), and GA20 (23). Furthermore, photoperiod has been shown to influence the metabolism of GA19 (14), GA20 (20), and GA53 (13,14) (Fig. 5).…”

Section: Light-induced [14c]kaurene Metabolismmentioning

confidence: 99%

Separation of Light-Induced [¹⁴C]ent-Kaurene Metabolism and Light-Induced Germination in Grand Rapids Lettuce Seeds

Hazebroek

Coolbaugh²

1991

ABSTRACTidentified in seeds incubated in constant white light, while no ethyl acetate-soluble metabolites were found in seeds incubated in the dark. In time course experiments using acid scarified seeds, metabolism began after 18 hours of incubation and greatly increased after 24 hours of incubation in 20% PEG. By 48 hours, several unidentified, more polar metabolites were found. Germination was induced in seeds imbibed in 20% PEG by 4 hours of red or 4 hours of white light following 20 hours in the dark, and was fully reversed by 2 hours of far red light. However, in metabolism experiments, [14Clent-kaurene oxidation was observed only with constant white light. These results indicate that although ent-kaurene oxidation is a light sensitive step in the biosynthesis of gibberellins in Grand Rapids lettuce seeds, ent-kaurene metabolism is not required for light-induced germination.