Hormone levels and response during de-etiolation in pea

The objective of this study was to increase our understanding of the hormonal regulation of de-etiolation by investigating endogenous hormone levels and response in etiolated pea ( Pisum sativum L.) seedlings after exposure to continuous white light. Recent reports suggest that de-etiolation may result from the down-regulation of an enzyme in the brassinosteroid (BR) biosynthesis pathway in pea. A subsequent review highlighted the need for direct measurements of BR levels to support this hypothesis. We have shown that endogenous castasterone and 6-deoxocastasterone levels are not greatly reduced after exposure to light; indeed, 6-deoxocastasterone levels were actually increased. Similarly, the elongation response to exogenous brassinolide was greater in plants grown in continuous light, or in dark-grown plants that had been transferred into the light, than in plants that were grown in continuous darkness. These results provide further evidence to suggest that BRs do not negatively regulate de-etiolation in pea. However, changes in the levels of several other hormones have also been implicated in light-regulated development. We have simultaneously quantified indole-3-acetic acid (IAA), gibberellin (GA), and abscisic acid levels in whole seedlings, which revealed a complex pattern of changes in the levels of these substances after exposure to light. The first and most dramatic of these changes was a significant reduction in GA(1) levels, which reached a minimum 8 h after exposure to light. Whilst GA(1) levels rapidly decreased, IAA levels remained unchanged in the short term after exposure to light, suggesting that GA(1) levels may be the primary factor regulating the reduction in elongation growth during de-etiolation. In the long term after exposure to light, IAA levels underwent a transitory increase, which peaked at 48 h, and had abated by 96 h. However, abscisic acid levels remained unchanged in the first 1 h after exposure to light before undergoing a steady decline over time. The relative importance of these changes in mediating light-induced changes in plant morphology is discussed.     

The hormonal regulation of de-etiolation

De-etiolation involves a number of phenotypic changes as the plants shift from a dark-grown (etiolated) to a light-grown (de-etiolated) morphology. Whilst these light-induced, morphological changes are thought to be mediated by plant hormones, the precise mechanism/s are not yet fully understood. Here we provide further direct evidence that gibberellins (GAs) may play an important role in de-etiolation, because a similar light-induced reduction in bioactive GA levels was detected in barley (Hordeum vulgare L.), Arabidopsis (Arabidopsis thaliana L.), and pea (Pisum sativum L.). This is indicative of a highly conserved, negative-regulatory role for GAs in de-etiolation, in a range of taxonomically diverse species. In contrast, we found no direct evidence of a reduction in brassinosteroid (BR) levels during de-etiolation in any of these species. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Brassinolide, a growth-promoting steroidal lactone

Brassinolide (BR), a naturally-occurring-steroidal lactone from rape ( Brassica napus L.) pollen, was compared with auxin for activity in a number of bioassay systems. Responses similar to IAA were elicited by BR in bioassays based upon bean hypocotyl hook opening, elongation of maize mesocotyl, pea epicotyl and azuki bean epicotyl sections, and fresh weight increase in Jerusalem artichoke (2,4-D used) and pea epicotyl sections. The azuki bean and dwarf pea epicotyl bioassays were much more responsive to BR than IAA (at 10 μ M ). Responses approximately two-fold greater in magnitude were elicited by IAA in the maize mesocotyl, bean hypocotyl hook and Jerusalem artichoke bioassays. Little or no response was elicited by BR (0.01 to μ M ) in the cress root or decapitated pea-lateral bud bioassays. A powerful synergism between BR and IAA was observed in the azuki bean, pea epicotyl and bean hypocotyl hook bioassays. Although, as previously reported, other steroidal substances are active in some of the bioassay systems tested, none compared with BR in magnitude and diversity of elicited responses.     

UV-B-induced Inhibition of Stem Elongation and Leaf Expansion in Pea Depends on Modulation of Gibberellin Metabolism and Intact Gibberellin Signalling

Information on the involvement of elongation-controlling hormones, particularly gibberellin (GA), in UV-B modulation of stem elongation and leaf growth, is limited. We aimed to study the effect of UV-B on levels of GA and indole-3-acetic acid (IAA) as well as involvement of GA in UV-B inhibition of stem elongation and leaf expansion in pea. Reduced shoot elongation (13%) and leaf area (37%) in pea in response to a 6-h daily UV-B (0.45 W m^?2) exposure in the middle of the light period for 10 days were associated with decreased levels of the bioactive GA₁ in apical stem tissue (59%) and young leaves (69%). UV-B also reduced the content of IAA in young leaves (35%). The importance of modulation of GA metabolism for inhibition of stem elongation in pea by UV-B was confirmed by the lack of effect of UV-B in the le GA biosynthesis mutant. No UV-B effect on stem elongation in the la cry-s (della) pea mutant demonstrates that intact GA signalling is required. In conclusion, UV-B inhibition of shoot elongation and leaf expansion in pea depends on UV-B modulation of GA metabolism in shoot apices and young leaves and GA signalling through DELLA proteins. UV-B also affects the IAA content in pea leaves.     

Changes in gibberellin A(1) levels and response during de-etiolation of pea seedlings

The level of gibberellin A(1) (GA(1)) in shoots of pea (Pisum sativum) dropped rapidly during the first 24 h of de-etiolation. The level then increased between 1 and 5 d after transfer to white light. Comparison of the metabolism of [(13)C(3)H] GA(20) suggested that the initial drop in GA(1) after transfer is mediated by a light-induced increase in the 2beta-hydroxylation of GA(1) to GA(8). A comparison of the elongation response to GA(1) at early and late stages of de-etiolation provided strong evidence for a change in GA(1) response during de-etiolation, coinciding with the return of GA(1) levels to the normal, homeostatic levels found in light- and dark-grown plants. The emerging picture of the control of shoot elongation by light involves an initial inhibition of elongation by a light-induced decrease in GA(1) levels, with continued inhibition mediated by a light-induced change in the plant's response to the endogenous level of GA(1). Hence the plant uses a change in hormone level to respond to a change in the environment, but over time, homeostasis returns the level of the hormone to normal once the ongoing change in environment is accommodated by a change in the response of the plant to the hormone.     

Auxin, ethylene and brassinosteroids: tripartite control of growth in the Arabidopsis hypocotyl

Dark-grown Arabidopsis seedlings develop an apical hook by differential cell elongation and division, a process driven by cross-talk between multiple hormones. Auxins, ethylene and gibberellins interact in the formation of the apical hook. In the light, a similar complexity of hormonal regulation has been revealed at the level of hypocotyl elongation. Here, we describe the involvement of brassinosteroids (BRs) in auxin- and ethylene-controlled processes in the hypocotyls of both light- and dark-grown seedlings. We show that BR biosynthesis is necessary for the formation of an exaggerated apical hook and that either application of BRs or disruption of BR synthesis alters auxin response, presumably by affecting auxin transport, eventually resulting in the disappearance of the apical hook. Furthermore, we demonstrate that ethylene-stimulated hypocotyl elongation in the light is largely controlled by the same mechanisms as those governing formation of the apical hook in darkness. However, in the light, BRs appear to compensate for the insensitivity to ethylene in hls mutants, supporting a downstream action of BRs. Hence, our results indicate that HLS1, SUR1/HLS3/RTY1/ALF1 and AMP1/HPT/COP2/HLS2/PT act on the auxin-ethylene interaction, rather than at the level of BRs. A model for the tripartite hormone interactions is presented.     

Evidence that the mature leaves contribute auxin to the immature tissues of pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

In plants such as the garden pea (Pisum sativum L.), it is widely thought that the auxin indole-3-acetic acid (IAA) is synthesised mainly in the immature tissues of the apical bud and then transported basipetally to other parts of the plant. Consistent with this belief are results showing that removal of the apical bud markedly reduces the IAA content in the stem. However, it has also been suggested that the mature leaves may synthesise substantial amounts of IAA, which enters the basipetal transport stream after being transported to the shoot apex in the phloem (Cambridge and Morris in Planta 99:583–588, 1996). To examine this theory, we defoliated pea plants and measured the effect on IAA content in the remaining shoot tissues. IAA levels were reduced in the internodes, and to a lesser extent in the apical bud, after defoliation, suggesting that mature leaves are indeed an important source of auxin for the shoot. Consistent with this idea, we have demonstrated that mature, fully expanded leaves are capable of de novo IAA synthesis. Furthermore, we report evidence for the presence of IAA in the phloem sap of pea. Together these results support those of Cambridge and Morris, suggesting that mature leaves are a source of the IAA in the basipetal transport stream.     

The characterization of gio, a new pea mutant, shows the role of indoleacetic acid in the control of fruit development by the apical shoot

Fruit-set and fruit growth in pea (Pisum sativum L.) depend on gibberellins (GAs). The authors have isolated a new pea mutant, gio, which appeared spontaneously within the population of the cultivar Alaska, characterized by unpollinated ovaries much less sensitive to applied GAs. The mutant also has elongated peduncles, and is taller than the wild-type (WT) because the upper plant internodes are longer. Contrary to WT, the gio ovaries respond very little to benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid, but become fully sensitive to GA(3) when this hormone is applied together with BAP. The gio phenotype is determined by a mutation at a single mendelian locus. The mutation is recesive, shows incomplete penetrance, and its expression depends on environmental culture conditions. The sensitivity of the ovaries to GA(3) can be recovered by removing the apical shoot (plant decapitation) and by blocking the transport of indoleacetic acid (IAA) from the apical shoot with 2,3,5-triiodobenzoic acid. The content of IAA in methanolic extracts and phloematic exudates of the apical shoot of gio is about double that in the WT. The rate of transport of [(3)H]IAA applied to the apex of the mutant is also twice that in the WT. This indicates that the insensitivity of the gio ovaries to GAs is due to the inhibitory effect of the higher basipetal IAA transport from the shoot. The interaction between the fruit and the apical shoot mediated by IAA probably also involves cytokinins transported from the basal part of the plant.     

Involvement of auxin and CKs in boron deficiency induced changes in apical dominance of pea plants (Pisum sativum L.)

It has previously been shown that boron (B) deficiency inhibits growth of the plant apex, which consequently results in a relatively weak apical dominance, and a subsequent sprouting of lateral buds. Auxin and cytokinins (CKs) are the two most important phytohormones involved in the regulation of apical dominance. In this study, the possible involvement of these two hormones in B-deficiency-induced changes in apical dominance was investigated by applying B or the synthetic CK CPPU to the shoot apex of pea plants grown in nutrient solution without B supply. Export of IAA out of the shoot apex, as well as the level of IAA, Z/ZR and isopentenyl-adenine/isopentenyl-adenosine (i-Ade/i-Ado) in the shoot apex were assayed. In addition, polar IAA transport capacity was measured in two internodes of different ages using 3H-IAA. In B-deficient plants, both the level of auxin and CKs were reduced, and the export of auxin from the shoot apex was considerably decreased relative to plants well supplied with B. Application of B to the shoot apex restored the endogenous Z/ZR and IAA level to control levels and increased the export of IAA from the shoot apex, as well as the 3H-IAA transport capacity in the newly developed internodes. Further, B application to the shoot apex inhibited lateral bud growth and stimulated lateral root formation, presumably by stimulated polar IAA transport. Applying CPPU to the shoot apex, a treatment that stimulates IAA export under adequate B supply, considerably reduced the endogenous Z/ZR concentration in the shoot apex, but had no stimulatory effect on IAA concentration and transport in B-deficient plants. A similar situation appeared to exist in lateral buds of B-deficient plants as, in contrast to plants well supplied with B, application of CKs to these plants did not stimulate lateral bud growth. In contrast to the changes of Z/ZR levels in the shoot apex, which occurred after application of B or CPPU, the levels of i-Ade/i-Ado stayed more or less constant. These results suggest that there is a complex interaction between B supply and plant hormones, with a B-deficiency-induced inhibition of IAA export from the shoot apex as one of the earliest measurable events.     

Brassinosteroids do not undergo long-distance transport in pea. Implications for the regulation of endogenous brassinosteroid levels

It is widely accepted that brassinosteroids (BRs) are important regulators of plant growth and development. However, in comparison to the other classical plant hormones, such as auxin, relatively little is known about BR transport and its potential role in the regulation of endogenous BR levels in plants. Here, we show that end-pathway BRs in pea (Pisum sativum) occur in a wide range of plant tissues, with the greatest accumulation of these substances generally occurring in the young, actively growing tissues, such as the apical bud and young internodes. However, despite the widespread distribution of BRs throughout the plant, we found no evidence of long-distance transport of these substances between different plant tissues. For instance, we show that the maintenance of steady-state BR levels in the stem does not depend on their transport from the apical bud or mature leaves. Similarly, reciprocal grafting between the wild type and the BR-deficient lkb mutants demonstrates that the maintenance of steady-state BR levels in whole shoots and roots does not depend on either basipetal or acropetal transport of BRs between these tissues. Together, with results from (3)H-BR feeding studies, these results demonstrate that BRs do not undergo long-distance transport in pea. The widespread distribution of end-pathway BRs and the absence of long-distance BR transport between different plant tissues provide significant insight into the mechanisms that regulate BR homeostasis in plants.     

Uncoupling brassinosteroid levels and de-etiolation in pea

The suggestion that brassinosteroids (BRs) have a negative regulatory role in de-etiolation is based largely on correlative evidence, which includes the de-etiolated phenotypes of, and increased expression of light-regulated genes in, dark-grown mutants defective in BR biosynthesis or response. However, we have obtained the first direct evidence which shows that endogenous BR levels in light-grown pea seedlings are increased, not decreased, in comparison with those grown in the dark. Similarly, we found no evidence of a decrease in castasterone (CS) levels in seedlings that were transferred from the dark to the light for 24 h. Furthermore, CS levels in the constitutively de-etiolated lip1 mutant are similar to those in wild-type plants, and are not reduced as is the case in the BR-deficient lkb plants. Unlike lip1 , the pea BR-deficient mutants lk and lkb are not de-etiolated at the morphological or molecular level, as they exhibit neither a de-etiolated phenotype or altered expression of light-regulated genes when grown in the dark. Similarly, dark-grown WT plants treated with the BR biosynthesis inhibitor, Brz, do not exhibit a de-etiolated phenotype. In addition, analysis of the lip1lkb double mutant revealed an additive phenotype indicative of the two genes acting in independent pathways. Together these results strongly suggest that BR levels do not play a negative-regulatory role in de-etiolation in pea.     

Effects of boron starvation on boron compartmentation, and possibly hormone-mediated elongation growth and apical dominance of pea (Pisum sativum) plants

Two experiments were carried out to study the effects of boron (B) deficiency on 7-day-old pea plants for 6 or 9 days under controlled growth chamber conditions. Growth and apical dominance (AD) of the plants and their B concentration and compartmentation were followed throughout the starvation period. Additionally, auxin (indoleacetic acid, IAA) concentration in the shoot apex and polar transport from it were measured along with the cytokinin (CK) concentration in the shoot apex and the roots. The results demonstrate that during a 6-day B-deficiency period, B concentration in the water-insoluble residue of the roots was very stable and could not easily be reduced. In contrast, B concentration in the cell sap fraction was very sensitive to external B supply. Twelve hours after transferring the plants from B-sufficient to B-deficient solutions, the B concentration in root cell sap declined to half the concentration of the control plants. In addition, B concentration in the new aerial plant parts, which developed after the onset of the B-deficiency treatment, was extremely low. A decline in elongation growth could be observed as soon as about 4 days after the imposition of B deficiency. This preceded the first measurable growth of lateral buds (release from AD). Before the onset of these morphological changes, there was a considerable decline in CK concentration, accompanied by a dramatic decrease in IAA export out of the shoot apex, a decline in IAA concentration in the shoot apex and the roots and a reduced capacity for polar IAA-transport. These changes are discussed as possible reasons for the observed reduction in elongation growth and AD. These hormonal changes themselves are possibly the result of the decreased symplasmic B concentration, which in turn may be responsible for the reduced concentration in apical CKs. A sequence of events, which may be causally related, is suggested to explain the effects of B deficiency on the growth and AD of pea plants.     

Growth patterns and endogenous indole-3-acetic acid concentrations in current-year coppice shoots and seedlings of two Betula species

Apical dominance, internode elongation, radial growth and xylem cell size in coppice and apical shoots of Betula pubescens B. Pendula were determined and related to endogenous indole-3-acetic acid (IAA) levels, measured by gas chromatography-selected ion monitoring in the apical bud and at three positions along the stem. The effects of defoliation and debudding on morphological and anatomical characters and endogenous IAA levels were also investigated. The coppice shoots displayed superior stem elongation and increased branching during the initial phase of growth, after which their growth pattern was similar to that of the seedlings; however, their radial growth was greater throughout the experiment. Both plant types produced smaller-sized xylem cells at the top of the shoot than at the bottom with coppice shoots tending to form larger tracheids and smaller vessels than the seedlings. There was no consistent difference in IAA concentration between the coppice shoots and the seedlings. Defoliation and debudding reduced the IAA level in the stem within 36 h and it was still low after 25 days. Although the extent of the IAA decrease was similar in both coppice shoots and seedlings, the treatments affected the morphological and anatomical characters differently in the two plant types. The results suggest that the observed differences between seedlings and coppice shoots were not mediated through a drastic change in IAA level.     

Cell division activity during apical hook development

Growth during plant development is predominantly governed by the combined activities of cell division and cell elongation. The relative contribution of both activities controls the growth of a tissue. A fast change in growth is exhibited at the apical hypocotyl of etiolated seedlings where cells grow at different rates to form a hook-like structure, which is traditionally assumed to result from differential cell elongation. Using new tools we show asymmetric distribution of cell division during early stages of hook development. Cell divisions in the apical hook were predominantly found in subepidermal layers during an early step of hook development, but were absent in mutants exhibiting a hookless phenotype. In addition, during exaggeration of hook curvature, which is mediated by ethylene, a rapid change in the combined activities of cell division and cell elongation was detected. Our results indicate a fast change in cell division activity during apical hook development. We suggest that cell division together with cell elongation contributes to apical hook growth. Our results emphasize the change in the relative contribution of cell division and cell elongation in a fast growing structure like the apical hook.     

Hypaphorine from the ectomycorrhizal fungus Pisolithus tinctorius counteracts activities of indole-3-acetic acid and ethylene but not synthetic auxins in eucalypt seedlings

Very little is known about the molecules regulating the interaction between plants and ectomycorrhizal fungi during root colonization. The role of fungal auxin in ectomycorrhiza has repeatedly been suggested and questioned, suggesting that, if fungal auxin controls some steps of colonized root development, its activity might be tightly controlled in time and in space by plant and/or fungal regulatory mechanisms. We demonstrate that fungal hypaphorine, the betaine of tryptophan, counteracts the activity of indole-3-acetic acid (IAA) on eucalypt tap root elongation but does not affect the activity of the IAA analogs 2,4-D ((2,4-dichlorophenoxy)acetic acid) or NAA (1-naphthaleneacetic acid). These data suggest that IAA and hypaphorine interact during the very early steps of the IAA perception or signal transduction pathway. Furthermore, while seedling treatment with 1-amincocyclopropane-1-carboxylic acid (ACC), the precursor of ethylene, results in formation of a hypocotyl apical hook, hypaphorine application as well as root colonization by Pisolithus tinctorius, a hypaphorine-accumulating ectomycorrhizal fungus, stimulated hook opening. Hypaphorine counteraction with ACC is likely a consequence of hypaphorine interaction with IAA. In most plant-microbe interactions studied, the interactions result in increased auxin synthesis or auxin accumulation in plant tissues. The P. tinctorius / eucalypt interaction is intriguing because in this interaction the microbe down-regulates the auxin activity in the host plant. Hypaphorine might be the first specific IAA antagonist identified.     

Characterization of gravitropic inflorescence bending in brassinosteroid biosynthesis and signaling Arabidopsis mutants

The interaction between the plant hormones, brassinosteroids and auxins has been documented in various processes using a variety of plants and plant parts. In this study, detached inflorescences from brassinosteroid biosynthesis and signaling Arabidopsis mutants were evaluated for their gravitropic bending in response to epibrassinolide (EBR) and indole-3-acetic acid (IAA). EBR supplied to the base of detached inflorescences stimulated gravitropic bending in all BR biosynthetic mutants but there was no effect on the BR signaling mutant or wild type plants. When IAA was supplied to the base of BR mutant inflorescences both natural and EBR-induced gravitropic bending was inhibited. Treatment with the auxin inhibitors also decreased both natural and EBR-induced gravitropic bending. No gravitropic bending was observed when the apical tips of BR mutant inflorescences were removed. IAA treatment to the tips of decapitated BR mutant inflorescences restored gravitropic bending to values observed in the inflorescences with an apical tip, however, EBR applied to the tip had no effect. When decapitated inflorescences from BR mutants were treated with IAA to the base and either gel, EBR or IAA was applied to the tip; there was no gravitropic bending. These results show that brassinosteroids have a role in the gravitropic bending response in Arabidopsis and mutants serve to uncover this hidden contributor.     

Effects of a brassinosteroid on growth and electrogenic proton extrusion in Azuki bean epicotyls

Brassinolide, a plant hormone newly isolated from pollen, promotes growth of the stem of a number of plant species. Similar effects are induced by a brassinosteroid (BR), the synthetic 24-epibrassinolide. In this paper the effects of BR on acid secretion and transmembrane electrical potential difference in Azuki bean ( Vigna angularis , Ohwi and Ohashi cv. Takara) epicotyls were determined in short term experiments and compared with the effects on growth. At concentrations between 10^-7 to 10^-5 M , BR stimulates, similarly to indole-3-acetic acid (IAA), growth and H⁺ extrusion and hyperpolarizes the transmembrane electric potential (PD). These effects of BR, as well as those of IAA, are suppressed by inhibitors of RNA and protein synthesis. All these effects of BR and IAA appear roughly additive, even when both hormones are present at their optimal concentrations. The data are interpreted as showing that the action of BR on growth is at least in part mediated by its capability to activate electrogenic proton extrusion. The additivity of the effects of BR and IAA suggests that the primary mechanism of action of the two hormones is different.     

Blue-light-mediated shade avoidance requires combined auxin and brassinosteroid action in Arabidopsis seedlings

Plant growth in dense vegetation can be strongly affected by competition for light between neighbours. These neighbours can not only be detected through phytochrome-mediated perception of a reduced red:far-red ratio, but also through altered blue light fluence rates. A reduction in blue light (low blue) induces a set of phenotypic traits, such as shoot elongation, to consolidate light capture; these are called shade avoidance responses. Here we show that both auxin and brassinosteroids (BR) play an important role in the regulation of enhanced hypocotyl elongation of Arabidopsis seedlings in response to blue light depletion. Only when both hormones are experimentally blocked simultaneously, using mutants and chemical inhibitors, will the response be fully inhibited. Upon exposure to low blue several members of the cell wall modifying XYLOGLUCAN ENDOTRANSGLUCOSYLASE/HYDROLASE (XTH) protein family are regulated as well. Interestingly, auxin and BR each regulate a subset of these XTHs, by which they could regulate cell elongation. We hypothesize that auxin and BR regulate specific XTH genes in a non-redundant and non-synergistic manner during low-blue-induced shade avoidance responses of Arabidopsis seedlings, which explains why both hormones are required for an intact low-blue response.