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.     

Sites of gibberellin biosynthesis in pea seedlings

Potential sites of gibberellin biosynthesis in 10-day-old `Alaska' pea (Pisum sativum L.) seedlings were investigated using a cell-free ezyme system capable of incorporating [¹⁴C]-mevalonic acid into ent-kaurene. In peas, ent-kaurene is assumed to be a committed intermediate in the gibberellin biosynthetic pathway. Comparative results from enzyme assays using extracts from shoot tips, leaf blades, internodes, and root tips indicate that the highest capacity for ent-kaurene (and presumably gibberellin) synthesis is in those tissues with the greatest potential for growth. The highest rates were obtained with extracts prepared from the fifth (youngest) internode, the fourth (youngest) expanded leaf, and the shoot tip itself. This report represents the first direct evidence that the enzymes responsible for early stages in gibberellin biosynthesis occur in internode tissues with potential for rapid elongation.     

CCC-Induced increase of gibberellin levels in pea seedlings

Summary Pea seedlings (cv. Alaska), were treated with two concentrations of (2-chloroethyl)trimethylammonium chloride (CCC) and choline chloride. Treatment with 1 mg/l CCC resulted in as much as a 150fold increase in endogenous gibberellin (GA) levels without there being any parallel stimulation of growth. Plants grown in 1,000 mg/l CCC were severely dwarfed but contained GA levels not significantly different from control plants grown in distilled water. CCC also retarded GA₃-induced growth of pea seedlings. These effects appear to be CCC specific as the CCC analogue choline chloride affected neither the GA content of pea seedlings nor their response to GA₃. The lack of correlation between endogenous GA levels and stem height suggests that in peas the predominant factor in CCC-induced inhibition of stem growth is not related to an effect of CCC on GA biosynthesis.Supported by National Research Council (Canada) grant A-5727.Supported by a Postdoctoral Fellowship from NRC Grant A-2585 to R.P. Pharis.     

Water-deficit effects on carbon and nitrogen metabolism of pea nodules

Two experiments were carried out to investigate the effects of water-deficit stress on carbon and nitrogen metabolism of Pisum sativum nodules. In the first experiment, leaf w was allowed to reach -1.0 MPa over a period of 14 d whilst in the second experiment -1.5 MPa was reached during the same time period. Nodule activities of phosphoenol pyruvate carboxylase, glutamine synthetase, alkaline invertase, pyruvate decarboxylase, alcohol dehdyrogenase, uridine pyro-phosphorylase, and malate dehydrogenase activities were not affected by water-deficit stress. In the first experiment (-1.0 MPa), sucrose synthase (SS), an enzyme which hydrolyses sucrose to support nodule metabolism, declined by 50% in activity and about 25% in content, according to Western immunoblot data. In the second experiment (-1.5 MPa), SS activity decreased by 75% together with glutamate synthase and aspartate aminotransferase which declined by 60% and 40%, respectively. Coincident with the decline of these activities, a dramatic increase in the nodule content of sucrose and a slight increase in the levels of total free amino acids were found. It has been recently suggested that the decline in SS activity and, therefore, a reduced potential to metabolize sucrose may be an important factor contributing to the overall response of soybean nodules to water stress. These results suggest that this observation may be also correct for temperate legumes with indeterminate nodules. However, in this latter case, the activity of some enzymes involved in nitrogen assimilation (glutamate synthase and aspartate aminotransferase) were also affected by water-deficit stress).Key words: Pisum sativum, water stress, nitrogen metabolism, nodule metabolism, pea, sucrose synthase.      

Nodulation phenotypes of gibberellin and brassinosteroid mutants of pea

The initiation and development of legume nodules induced by compatible Rhizobium species requires a complex signal exchange involving both plant and bacterial compounds. Phytohormones have been implicated in this process, although in many cases direct evidence is lacking. Here, we characterize the root and nodulation phenotypes of various mutant lines of pea (Pisum sativum) that display alterations in their phytohormone levels and/or perception. Mutants possessing root systems deficient in gibberellins (GAs) or brassinosteroids (BRs) exhibited a reduction in nodule organogenesis. The question of whether these reductions represent direct or indirect effects of the hormone deficiency is addressed. For example, the application of GA to the roots of a GA-deficient mutant completely restored its number of nodules to that of the wild type. Grafting studies revealed that a wild-type shoot or root also restored the nodule number of a GA-deficient mutant. These findings suggest that GAs are required for nodulation. In contrast, the shoot controlled the number of nodules that formed in graft combinations of a BR-deficient mutant and its wild type. The root levels of auxin and GA were similar among these latter graft combinations. These results suggest that BRs influence a shoot mechanism that controls nodulation and that the root levels of auxin and GA are not part of this process. Interestingly, a strong correlation between nodule and lateral root numbers was observed in all lines assessed, consistent with a possible overlap in the early developmental pathways of the two organs.     

Control of gibberellin levels and gene expression during de-etiolation in pea

Gibberellin A(1) (GA(1)) levels drop significantly in wild-type pea (Pisum sativum) plants within 4 h of exposure to red, blue, or far-red light. This response is controlled by phytochrome A (phyA) (and not phyB) and a blue light receptor. GA(8) levels are increased in response to 4 h of red light, whereas the levels of GA(19), GA(20), and GA(29) do not vary substantially. Red light appears to control GA(1) levels by down-regulating the expression of Mendel's LE (PsGA3ox1) gene that controls the conversion of GA(20) to GA(1), and by up-regulating PsGA2ox2, which codes for a GA 2-oxidase that converts GA(1) to GA(8). This occurs within 0.5 to 1 h of exposure to red light. Similar responses occur in blue light. The major GA 20-oxidase gene expressed in shoots, PsGA20ox1, does not show substantial light regulation, but does show up-regulation after 4 h of red light, probably as a result of feedback regulation. Expression of PsGA3ox1 shows a similar feedback response, whereas PsGA2ox2 shows a feed-forward response. These results add to our understanding of how light reduces shoot elongation during de-etiolation.     

Thermoperiodic stem elongation involves transcriptional regulation of gibberellin deactivation in pea

The physiological basis of thermoperiodic stem elongation is as yet poorly understood. Thermoperiodic control of gibberellin (GA) metabolism has been suggested as an underlying mechanism. We have investigated the influence of different day and night temperature combinations on GA levels, and diurnal steady-state expression of genes involved in GA biosynthesis (LS, LH, NA, PSGA20ox1, and PsGA3ox1) and GA deactivation (PsGA2ox1 and PsGA2ox2), and related this to diurnal stem elongation in pea (Pisum sativum L. cv Torsdag). The plants were grown under a 12-h light period with an average temperature of 17 degrees C. A day temperature/night temperature combination of 13 degrees C/21 degrees C reduced stem elongation after 12 d by 30% as compared to 21 degrees C/13 degrees C. This was correlated with a 55% reduction of GA1. Although plant height correlated with GA1 content, there was no correlation between diurnal growth rhythms and GA1 content. NA, PsGA20ox1, and PsGA2ox2 showed diurnal rhythms of expression. PsGA2ox2 was up-regulated in 13 degrees C/21 degrees C (compared to 21 degrees C/13 degrees C), at certain time points, by up to 19-fold. Relative to PsGA2ox2, the expression of LS, LH, NA, PSGA20ox1, PsGA3ox1, and PsGA2ox1 was not or only slightly affected by the different temperature treatments. The sln mutant having a nonfunctional PsGA2ox1 gene product showed the same relative stem elongation response to temperature as the wild type. This supports the importance of PsGA2ox2 in mediating thermoperiodic stem elongation responses in pea. We present evidence for an important role of GA catabolism in thermoperiodic effect on stem elongation and conclude that PsGA2ox2 is the main mediator of this effect in pea.     

Effects of gibberellin on the cellular dynamics of dwarf pea internode development

This study analysed the dynamics of cell production and extension, and how these were affected by applied gibberellic acid (GA₃), during internode development in dwarf peas (Pisum sativum L. cv. Meteor). Image analysis was used to obtain cell number and length data for entire cell columns along the epidermis, the two outermost cortical layers, and the pith, from internode 7, over a time period covering the whole of the internode's growth phase. For a few days following the inception of an internode at the shoot apex, little further growth occurred, and there was no significant effect of GA₃ on cell division or cell extension. The subsequent growth of the internode was stimulated more than fourfold by GA₃ as a result of the production of more than twice the number of cells, which were twice as long. At least 96.5% of the cells of the mature internode were actually formed within the internode itself during this period of growth, demonstrating that the internode cells themselves represent the morphogenetic site of response to GA₃. Mitoses and cell extension occurred along the full length of the internode throughout its development. The daily changes in cell numbers were modelled by the Richards function, and manipulations of the fitted functions to reveal time trends of absolute and specific cell production rates were performed for each stem tissue. The increase in cell numbers in the +GA₃ plants was brought about by an increase in the rate of cell production, over a shorter time interval; specific cell production rates declined continuously from initial rapid rates in the +GA₃ epidermis and pith, but declined more slowly in the cortex. The control (−GA₃) epidermis and cortex cells exhibited a constant specific cell production rate (i.e. purely exponential) for several days. Cell extension rates were calculated so as to compensate for the size-reduction effects of concurrent cell division. These calculations confirmed that `real' cell extension rates were higher in the +GA₃ internodes. Models of the cellular controls of internode growth, based on the estimated dynamics of cell division and extension, are discussed. Received: 1 July 1997 / Accepted: 30 July 1997     

Phytochrome controlled gibberellin metabolism in etioplast envelopes

Summary Disruption of preparations of intact etioplasts isolated from wheat (Triticum aestivum L.) leaves in the dark leads to an increase in the level of extractable gibberellin-like (GA-like) activity. Irradiation of disrupted preparations with red light causes an alteration in the Rf of part of the GA-like activity. A study of sub-etioplast fractions prepared by gradient centrifugation indicates that the phytochrome and the GA-like substances are associated principally with the etioplast envelope.Abbreviations GA gibberellin - MOPS N-morpholino-3-propanesulphonic acid - EDTA ethylene diamine tetra-acetic acid - ATP adenosine triphosphate - R red light     

Photoperiodic control of gibberellin metabolism in spinach

[(3)H]GA(20) applied to spinach plants (Spinacia oleracea L.) was metabolized to several products. Two of these were identified by combined gasliquid chromatography-radio counting as [(3)H]GA(29) and [(3)H]3-epi-GA(1). Inasmuch as both GA(20) and GA(29) are endogenous gibberellins in spinach (Metzger, Zeevaart 1980 Plant Physiol 65: 623-626), it was concluded that the conversion of GA(20) to GA(29) is a natural process. However, 3-epi-GA(1) was not detected in extracts of spinach shoots analyzed by combined gas chromatography-mass spectrometry. This indicates that the conversion of exogenous [(3)H]GA(20) to [(3)H]3-epi-GA(1) may be an artifact.Long-day pretreatment of spinach shoots caused a 2-fold increase in the rate of [(3)H]GA(20) metabolism over the rate of metabolism in plants maintained under short-day conditions. Furthermore, [(3)H]GA(29) accumulated more rapidly under long than under short days, whereas photoperiodic treatment had no effect on the accumulation of [(3)H]3-epi-GA(1). Thus, the long-day-induced increase in the level of endogenous GA(29) in spinach shoots (Metzger, Zeevaart 1980 Plant Physiol 66: 844-846) appears to be the result of an increased capability to convert GA(20) to GA(29).     

Photocontrol of gibberellin metabolism in situ in maize

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K_m of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K_m of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.