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The synthesis and deactivation of bioactive gibberellins (GA) are regulated by auxin and by GA signalling. The effect of GA on its own pathway is mediated by DELLA proteins. Like auxin, the DELLAs promote GA synthesis and inhibit its deactivation. Here, we investigate the relationships between auxin and DELLA regulation of the GA pathway in stems, using a pea double mutant that is deficient in DELLA proteins. In general terms our results demonstrate that auxin and DELLAs independently regulate the GA pathway, contrary to some previous suggestions. The extent to which DELLA regulation was able to counteract the effects of auxin regulation varied from gene to gene. For Mendel's LE gene (PsGA3ox1) no counteraction was observed. However, for another synthesis gene, a GA 20-oxidase, the effect of auxin was weak and in WT plants appeared to be completely over-ridden by DELLA regulation. For a key GA deactivation (2-oxidase) gene, PsGA2ox1, the up-regulation induced by auxin deficiency was reduced to some extent by DELLA regulation. A second pea 2-oxidase gene, PsGA2ox2, was up-regulated by auxin, in a DELLA-independent manner. In Arabidopsis also, one 2-oxidase gene was down-regulated by auxin while another was up-regulated. Monitoring the metabolism pattern of GA(20) showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.

Gibberellin (GA) production by soil fungi has received little attention, although substantial work has been carried out on other aspects of plant growth promoting fungi (PGPF). In our studies we investigated GA production and growth-promoting capacity of a novel fungal strain isolated from the roots of soil-grown cucumber. Pure cultures of 19 endophytic fungi were tested for shoot length promotion of Waito-C rice to identify the GA production capacity of these fungal isolates. Isolate MH-6 significantly increased shoot length (12.9 cm) of Waito-C, in comparison to control treatments. Bioassay with culture filtrate (CF) of MH-6 also significantly promoted growth attributes of cucumber plants. Analysis of MH-6 CF showed the presence of physiologically active (GA1, 1.97 ng/mL; GA3, 5.18 ng/mL; GA4, 13.35 ng/mL and GA7, 2.4 ng/ mL) in conjunction with physiologically inactive (GA9 [0.69 ng/mL], GA12 [0.24 ng/mL], GA15 [0.68 ng/ mL, GA19 [1.94 ng/mL and GA20 [0.78 ng/mL]) gibberellins. The CF of MH-6 produced greater amounts of GA3, GA4, GA7 and GA19 than wild type Fusarium fujikuroi, a fungus known for high production of GA. The fungal isolate MH-6 was identified as a new strain of Cladosporium sp. on the basis of sequence homology (99%) and phylogenetic analysis of 18S rDNA sequence.

The plant hormone gibberellin (GA) is crucial for multiple aspects of plant growth and development. To study the relevant regulatory mechanisms, we isolated a rice mutant earlier flowering1, el1, which is deficient in a casein kinase I that has critical roles in both plants and animals. el1 had an enhanced GA response, consistent with the suppression of EL1 expression by exogenous GA(3). Biochemical characterization showed that EL1 specifically phosphorylates the rice DELLA protein SLR1, proving a direct evidence for SLR1 phosphorylation. Overexpression of SLR1 in wild-type plants caused a severe dwarf phenotype, which was significantly suppressed by EL1 deficiency, indicating the negative effect of SLR1 on GA signalling requires the EL1 function. Further studies showed that the phosphorylation of SLR1 is important for maintaining its activity and stability, and mutation of the candidate phosphorylation site of SLR1 results in the altered GA signalling. This study shows EL1 a novel and key regulator of the GA response and provided important clues on casein kinase I activities in GA signalling and plant development.

1-Deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) is an important enzyme involved in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway which provides the basic five-carbon units for isoprenoid biosynthesis. To investigate the role of the MEP pathway in plant development and metabolism, we carried out detailed analyses on a dxr mutant (GK_215C01) and two DXR transgenic co-suppression lines, OX-DXR-L2 and OX-DXR-L7. We found that the dxr mutant was albino and dwarf. It never bolted, had significantly reduced number of trichomes and most of the stomata could not close normally in the leaves. The two co-suppression lines produced more yellow inflorescences and albino sepals with no trichomes. The transcription levels of genes involved in trichome initiation were found to be strongly affected, including GLABRA1, TRANSPARENT TESTA GLABROUS 1, TRIPTYCHON and SPINDLY, expression of which is regulated by gibberellic acids (GAs). Exogenous application of GA(3) could partially rescue the dwarf phenotype and the trichome initiation of dxr, whereas exogenous application of abscisic acid (ABA) could rescue the stomata closure defect, suggesting that lower levels of both GA and ABA contribute to the phenotype in the dxr mutants. We further found that genes involved in the biosynthetic pathways of GA and ABA were coordinately regulated. These results indicate that disruption of the plastidial MEP pathway leads to biosynthetic deficiency of photosynthetic pigments, GAs and ABA, and thus the developmental abnormalities, and that the flux from the cytoplasmic mevalonate pathway is not sufficient to rescue the deficiency caused by the blockage of the plastidial MEP pathway. These results reveal a critical role for the MEP biosynthetic pathway in controlling the biosynthesis of isoprenoids.

Plant hormones are small molecules that play important roles throughout the life span of a plant, known as auxin, gibberellin, cytokinin, abscisic acid, ethylene, jasmonic acid, salicylic acid, and brassinosteroid. Genetic and molecular studies in the model organism Arabidopsis thaliana have revealed the individual pathways of various plant hormone responses. In this study, we selected 479 genes that were convincingly associated with various hormone actions based on genetic evidence. By using these 479 genes as queries, a genome-wide search for their orthologues in several species (microorganisms, plants and animals) was performed. Meanwhile, a comparative analysis was conducted to evaluate their evolutionary relationship. Our analysis revealed that the metabolisms and functions of plant hormones are generally more sophisticated and diversified in higher plant species. In particular, we found that several phytohormone receptors and key signaling components were not present in lower plants or animals. Meanwhile, as the genome complexity increases, the orthologue genes tend to have more copies and probably gain more diverse functions. Our study attempts to introduce the classification and phylogenic analysis of phytohormone related genes, from metabolism enzymes to receptors and signaling components, in different species., (Copyright 2010 Institute of Genetics and Developmental Biology and the Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

African and Asian populations of Fusarium spp. (Gibberella fujikuroi species complex) associated with Bakanae of rice (Oryzae sativa L.) were isolated from seeds and characterized with respect to ecology, phylogenetics, pathogenicity and mycotoxin production. Independent of the origin, Fusarium spp. were detected in the different rice seed samples with infection rate ranges that varied from 0.25% to 9%. Four Fusaria (F. andiyazi, F. fujikuroi, F. proliferatum and F. verticillioides) were found associated with Bakanae of rice. While three of the Fusaria were found in both African and Asian seed samples, F. fujikuroi was only detected in seed samples from Asia. Phylogenetic studies showed a broad genetic variation among the strains that were distributed into four different genetic clades. Pathogenicity tests showed that all strains reduced seed germination and possessed varying ability to cause symptoms of Bakanae on rice, some species (i.e. F. fujikuroi) being more pathogenic than others. The ability to produce fumonisins (FB(1) and FB(2)) and gibberellin A3 in vitro also differed according to the Fusarium species. While fumonisins were produced by most of the strains of F. verticillioides and F. proliferatum, gibberellin A3 was only produced by F. fujikuroi. Neither fumonisin nor gibberellin was synthesized by most of the strains of F. andiyazi. These findings provide new information on the variation within the G. fujikuroi species complex associated with rice seed and Bakanae disease.

We have examined the role of gibberellins (GAs) in plant development by expression of the pea GA 2-oxidase2 (PsGA2ox2) cDNA, which encodes a GA inactivating enzyme, under the control of the MEDEA (MEA) promoter. Expression of MEA:PsGA2ox2 in Arabidopsis caused seed abortion, demonstrating that active GAs in the endosperm are essential for normal seed development. MEA:PsGA2ox2 plants had reduced ovule number per ovary and exhibited defects in phyllotaxy and leaf morphology which were partly suppressed by GA treatment. The leaf architecture and phyllotaxy defects of MEA:PsGA2ox2 plants were also restored by sly1-d which reduces DELLA protein stability to increase GA response. MEA:PsGA2ox2 seedlings had increased expression of the KNOTTED1-like homeobox (KNOX) genes, BP, KNAT2 and KNAT6, which are known to control plant architecture. The expression of KNOX genes is also altered in wild-type plants treated with GA. These results support the conclusion that GAs can suppress the effects of elevated KNOX gene expression, and raise the possibility that localized changes in GA levels caused by PsGA2ox2 alter the expression of KNOX genes to modify plant architecture.

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. GAs were first isolated as metabolites of a fungal rice pathogen, Gibberella fujikuroi, since renamed Fusarium fujikuroi. Although higher plants and the fungus produce structurally identical GAs, significant differences in their GA pathways, enzymes involved and gene regulation became apparent with the identification of GA biosynthetic genes in Arabidopsis thaliana and F. fujikuroi. Recent identifications of GA biosynthetic gene clusters in two other fungi, Phaeosphaeria spp. and Sphaceloma manihoticola, and the high conservation of GA cluster organization in these distantly related fungal species indicate that fungi evolved GA and other diterpene biosynthetic pathways independently from plants. Furthermore, the occurrence of GAs and recent identification of the first GA biosynthetic genes in the bacterium Bradyrhizobium japonicum make it possible to study evolution of GA pathways in general. In this review, we summarize our current understanding of the GA biosynthesis pathway, specifically the genes and enzymes involved as well as gene regulation and localization in the genomes of different fungi and compare it with that in higher and lower plants and bacteria.

Background: Analysis of molecular evolutionary patterns of different genes within metabolic pathways allows us to determine whether these genes are subject to equivalent evolutionary forces and how natural selection shapes the evolution of proteins in an interacting system. Although previous studies found that upstream genes in the pathway evolved more slowly than downstream genes, the correlation between evolutionary rate and position of the genes in metabolic pathways as well as its implications in molecular evolution are still less understood., Results: We sequenced and characterized 7 core structural genes of the gibberellin biosynthetic pathway from 8 representative species of the rice tribe (Oryzeae) to address alternative hypotheses regarding evolutionary rates and patterns of metabolic pathway genes. We have detected significant rate heterogeneity among 7 GA pathway genes for both synonymous and nonsynonymous sites. Such rate variation is mostly likely attributed to differences of selection intensity rather than differential mutation pressures on the genes. Unlike previous argument that downstream genes in metabolic pathways would evolve more slowly than upstream genes, the downstream genes in the GA pathway did not exhibited the elevated substitution rate and instead, the genes that encode either the enzyme at the branch point (GA20ox) or enzymes catalyzing multiple steps (KO, KAO and GA3ox) in the pathway had the lowest evolutionary rates due to strong purifying selection. Our branch and codon models failed to detect signature of positive selection for any lineage and codon of the GA pathway genes., Conclusion: This study suggests that significant heterogeneity of evolutionary rate of the GA pathway genes is mainly ascribed to differential constraint relaxation rather than the positive selection and supports the pathway flux theory that predicts that natural selection primarily targets enzymes that have the greatest control on fluxes.

Precise coordination between stamen and pistil development is essential to make a fertile flower. Mutations impairing stamen filament elongation, pollen maturation, or anther dehiscence will cause male sterility. Deficiency in plant hormone gibberellin (GA) causes male sterility due to accumulation of DELLA proteins, and GA triggers DELLA degradation to promote stamen development. Deficiency in plant hormone jasmonate (JA) also causes male sterility. However, little is known about the relationship between GA and JA in controlling stamen development. Here, we show that MYB21, MYB24, and MYB57 are GA-dependent stamen-enriched genes. Loss-of-function of two DELLAs RGA and RGL2 restores the expression of these three MYB genes together with restoration of stamen filament growth in GA-deficient plants. Genetic analysis showed that the myb21-t1 myb24-t1 myb57-t1 triple mutant confers a short stamen phenotype leading to male sterility. Further genetic and molecular studies demonstrate that GA suppresses DELLAs to mobilize the expression of the key JA biosynthesis gene DAD1, and this is consistent with the observation that the JA content in the young flower buds of the GA-deficient quadruple mutant ga1-3 gai-t6 rga-t2 rgl1-1 is much lower than that in the WT. We conclude that GA promotes JA biosynthesis to control the expression of MYB21, MYB24, and MYB57. Therefore, we have established a hierarchical relationship between GA and JA in that modulation of JA pathway by GA is one of the prerequisites for GA to regulate the normal stamen development in Arabidopsis., Competing Interests: The authors have declared that no competing interests exist.

As sessile organisms, plants modulate their growth rate and development according to the continuous variation in the conditions of their surrounding environment, an ability referred to as plasticity. This ability relies on a web of interactions between signaling pathways triggered by endogenous and environmental cues. How changes in environmental factors are interpreted by the plant in terms of developmental or growth cues or, in other words, how they contribute to plant plasticity is a current, major question in plant biology. Light stands out among the environmental factors that shape plant development. Plants have evolved systems that allow them to monitor both quantitative and qualitative differences in the light that they perceive, that render important changes in their growth habit. In this review we focus on recent findings about how information from this environmental cue is integrated during de-etiolation and in the shade-avoidance syndrome, and modulated by several hormone pathways-the endogenous cues. In some cases the interaction between a hormone and the light signaling pathways is reciprocal, as is the case of the gibberellin pathway, whereas in other cases hormone pathways act downstream of the environmental cue to regulate growth. Moreover, the circadian clock adds an additional layer of regulation, which has been proposed to integrate the information provided by light with that provided by hormone pathways, to regulate daily growth.

How vernalization - exposure to a period of cold - induces flowering in Arabidopsis has been intensively investigated at the genetic and moleular levels. Recent papers, including one in BMC Plant Biology, shed light on changes in gene regulation that occur on vernalization in cereals.

Fusarium verticillioides (Gibberella fujikuroi mating population A [MP-A]) is a widespread pathogen on maize and is well-known for producing fumonisins, mycotoxins that cause severe disease in animals and humans. The species is a member of the Gibberella fujikuroi species complex, which consists of at least 11 different biological species, termed MP-A to -K. All members of this species complex are known to produce a variety of secondary metabolites. The production of gibberellins (GAs), a group of diterpenoid plant hormones, is mainly restricted to Fusarium fujikuroi (G. fujikuroi MP-C) and Fusarium konzum (MP-I), although most members of the G. fujikuroi species complex contain the GA biosynthesis gene cluster or parts of it. In this work, we show that the inability to produce GAs in F. verticillioides (MP-A) is due to the loss of a majority of the GA gene cluster as found in F. fujikuroi. The remaining part of the cluster consists of the full-length F. verticillioides des gene (Fvdes), encoding the GA(4) desaturase, and the coding region of FvP450-4, encoding the ent-kaurene oxidase. Both genes share a high degree of sequence identity with the corresponding genes of F. fujikuroi. The GA production capacity of F. verticillioides was restored by transforming a cosmid with the entire GA gene cluster from F. fujikuroi, indicating the existence of an active regulation system in F. verticillioides. Furthermore, the GA(4) desaturase gene des from F. verticillioides encodes an active enzyme which was able to restore the GA production in a corresponding des deletion mutant of F. fujikuroi.

The rice pathogen Fusarium fujikuroi is well known for its ability to produce the plant hormones gibberellins (GAs). However, the majority of closely related Fusarium species is unable to produce GAs although the GA gene cluster is present in their genomes. In this study, we analyzed five orchid-associated Fusarium isolates for their capacity to produce GAs. Four of them did not produce any GAs and were shown not to contain any GA biosynthetic genes. However, the fifth isolate, which has been identified as F. proliferatum based on five molecular markers, produced significant amounts of GAs in contrast to previously characterized F. proliferatum strains. We focused on the molecular characterization of two GA-specific genes, ggs2 and cps/ks, both inactive in F. proliferatum strain D-02945. Complementation of a F. fujikuroi Deltaggs2 mutant with the ET1 ggs2 gene fully restored GA biosynthesis, confirming that the orchid-associated isolate contains an active gene copy. A possible correlation between GA production and their role in plant-fungal interactions is discussed.

Multiple genetic pathways act in response to developmental cues and environmental signals to promote the floral transition, by regulating several floral pathway integrators. These include FLOWERING LOCUS T (FT) and SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1). We show that the flowering repressor SHORT VEGETATIVE PHASE (SVP) is controlled by the autonomous, thermosensory, and gibberellin pathways, and directly represses SOC1 transcription in the shoot apex and leaf. Moreover, FT expression in the leaf is also modulated by SVP. SVP protein associates with the promoter regions of SOC1 and FT, where another potent repressor FLOWERING LOCUS C (FLC) binds. SVP consistently interacts with FLC in vivo during vegetative growth and their function is mutually dependent. Our findings suggest that SVP is another central regulator of the flowering regulatory network, and that the interaction between SVP and FLC mediated by various flowering genetic pathways governs the integration of flowering signals.

The sequence of Gid1 (a gene for a gibberellin (GA) receptor from rice) was used to identify a putative orthologue from barley. This was expressed in E. coli, and produced a protein that was able to bind GA in vitro with both structural specificity and saturability. Its potential role in GA responses was investigated using barley mutants with reduced GA sensitivity (gse1 mutants). Sixteen different gse1 mutants each carried a unique nucleotide substitution in this sequence. In all but one case, these changes resulted in single amino acid substitutions, and, for the remaining mutant, a substitution in the 5' untranslated region of the mRNA is proposed to interfere with translation initiation. There was perfect linkage in segregating populations between new mutant alleles and the gse1 phenotype, leading to the conclusion that the putative GID1 GA receptor sequence in barley corresponds to the Gse1 locus. Determination of endogenous GA contents in one of the mutants revealed enhanced accumulation of bioactive GA(1), and a deficit of C(20) GA precursors. All of the gse1 mutants had reduced sensitivity to exogenous GA(3), and to AC94377 (a GA analogue) at concentrations that are normally 'saturating', but, at much higher concentrations, there was often a considerable response. The comparison between barley and rice mutants reveals interesting differences between these two cereal species in GA hormonal physiology.

Seed dormancy provides a mechanism for plants to delay germination until conditions are optimal for survival of the next generation. Dormancy release is regulated by a combination of environmental and endogenous signals with both synergistic and competing effects. Molecular studies of dormancy have correlated changes in transcriptomes, proteomes, and hormone levels with dormancy states ranging from deep primary or secondary dormancy to varying degrees of release. The balance of abscisic acid (ABA):gibberellin (GA) levels and sensitivity is a major, but not the sole, regulator of dormancy status. ABA promotes dormancy induction and maintenance, whereas GA promotes progression from release through germination; environmental signals regulate this balance by modifying the expression of biosynthetic and catabolic enzymes. Mediators of environmental and hormonal response include both positive and negative regulators, many of which are feedback-regulated to enhance or attenuate the response. The net result is a slightly heterogeneous response, thereby providing more temporal options for successful germination.

Phytochrome regulates lettuce (Lactuca sativa L. cv. Grand Rapids) seed germination via the control of the endogenous level of bioactive gibberellin (GA). In addition to the previously identified LsGA20ox1, LsGA20ox2, LsGA3ox1, LsGA3ox2, LsGA2ox1, and LsGA2ox2, five cDNAs were isolated from lettuce seeds: LsCPS, LsKS, LsKO1, LsKO2, and LsKAO. Using an Escherichia coli expression system and functional assays, it is shown that LsCPS and LsKS encode ent-copalyl diphosphate synthase and ent-kaurene synthase, respectively. Using a Pichia pastoris system, it was found that LsKO1 and LsKO2 encode ent-kaurene oxidases and LsKAO encodes ent-kaurenoic acid oxidase. A comprehensive expression analysis of GA metabolism genes using the quantitative reverse transcription polymerase chain reaction suggested that transcripts of LsGA3ox1 and LsGA3ox2, both of which encode GA 3-oxidase for GA activation, were primarily expressed in the hypocotyl end of lettuce seeds, were expressed at much lower levels than the other genes tested, and were potently up-regulated by phytochrome. Furthermore, LsDELLA1 and LsDELLA2 cDNAs that encode DELLA proteins, which act as negative regulators in the GA signalling pathway, were isolated from lettuce seeds. The transcript levels of these two genes were little affected by light. Lettuce seeds in which de novo GA biosynthesis was suppressed responded almost identically to exogenously applied GA, irrespective of the light conditions, suggesting that GA responsiveness is not significantly affected by light in lettuce seeds. It is proposed that lettuce seed germination is regulated mainly via the control of the endogenous content of bioactive GA, rather than the control of GA responsiveness.

Bioactive gibberellins (GAs) are diterpene plant hormones that are biosynthesized through complex pathways and control diverse aspects of growth and development. Biochemical, genetic, and genomic approaches have led to the identification of the majority of the genes that encode GA biosynthesis and deactivation enzymes. Recent studies have highlighted the occurrence of previously unrecognized deactivation mechanisms. It is now clear that both GA biosynthesis and deactivation pathways are tightly regulated by developmental, hormonal, and environmental signals, consistent with the role of GAs as key growth regulators. In some cases, the molecular mechanisms for fine-tuning the hormone levels are beginning to be uncovered. In this review, I summarize our current understanding of the GA biosynthesis and deactivation pathways in plants and fungi, and discuss how GA concentrations in plant tissues are regulated during development and in response to environmental stimuli.

A higher concentration of H2O2 was detected in the sense transgenic potato plant (SS4) with the lily chCu,ZnSOD sequence, whereas higher levels of O2(-) was detected in the antisense transgenic plant (SA1) than the WT plant. The elongation growth in SA1 was significantly inhibited by treatment with diphenyleneiodonium, an inhibitor of O2(-) generation, and promoted in the SS4 on treatment with herbicide methyl viologen, a generator of apoplastic O2(-) . Higher concentrations of GAs were detected during plant growth and the early stage of tuberization in SA1. Complete recovery of the above elongation growth and microtuberization pattern in transgenic plants following treatment of GA(3) or an inhibitor of gibberellin synthesis, paclobutrazol, indicate that these changes were mainly caused by active GA levels. In conclusion, a specific ROS (O2(-) ) acts as a signal transducer via GA biosynthetic pathways for the regulation of plant growth and tuber development of potato.

Background: Heterosis in internode elongation and plant height are commonly observed in hybrid plants, and higher GAs contents were found to be correlated with the heterosis in plant height. However, the molecular basis for the increased internode elongation in hybrids is unknown., Results: In this study, heterosis in plant height was determined in two wheat hybrids, and it was found that the increased elongation of the uppermost internode contributed mostly to the heterosis in plant height. Higher GA4 level was also observed in a wheat hybrid. By using the uppermost internode tissues of wheat, we examined expression patterns of genes participating in both GA biosynthesis and GA response pathways between a hybrid and its parental inbreds. Our results indicated that among the 18 genes analyzed, genes encoding enzymes that promote synthesis of bioactive GAs, and genes that act as positive components in the GA response pathways were up-regulated in hybrid, whereas genes encoding enzymes that deactivate bioactive GAs, and genes that act as negative components of GA response pathways were down-regulated in hybrid. Moreover, the putative wheat GA receptor gene TaGID1, and two GA responsive genes participating in internode elongation, GIP and XET, were also up-regulated in hybrid. A model for GA and heterosis in wheat plant height was proposed., Conclusion: Our results provided molecular evidences not only for the higher GA levels and more active GA biosynthesis in hybrid, but also for the heterosis in plant height of wheat and possibly other cereal crops.

Carrizo citrange (Citrus sinensisxPoncirus trifoliata) is a citrus hybrid widely used as a rootstock, whose genetic manipulation to improve different growth characteristics is of high agronomic interest. In this work, transgenic Carrizo citrange plants have been produced overexpressing sense and antisense CcGA20ox1 (a key enzyme of GA biosynthesis) under control of the 35S promoter to modify plant architecture. As expected, taller (sense) and shorter (antisense) phenotypes correlated with higher and lower levels, respectively, of active GA1 in growing shoots. In contrast, other phenotypic characteristics seemed to be specific to citrus, or different from those described for similar transgenics in other species. For instance, thorns, typical organs of citrus at juvenile stages, were much longer in sense and shorter in antisense plants, and xylem tissue was reduced in leaf and internode of sense plants. Antisense plants presented a bushy phenotype, suggesting a possible effect of GAs on auxin biosynthesis and/or transport. The main foliole of sense plants was longer, although total leaf area was reduced. Leaf thickness was smaller in sense and larger in antisense plants due to changes in the spongy parenchyma. Internode cell length was not altered in transgenic plants, indicating that, in citrus, GAs regulate cell division rather than cell elongation. Interestingly, the phenotypes described were not apparent when transgenic plants were grafted on non-transgenic rootstock. This suggests that roots contribute to the GA economy of aerial parts in citrus and opens the possibility of using the antisense plants as dwarfing rootstocks.

The expression patterns of five genes (PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2) encoding five regulatory gibberellin (GA) biosynthesis enzymes (two GA 20-oxidases, a GA 3beta-hydroxylase, and two GA 2beta-hydroxylases) were examined to gain insight into how these genes coordinate GA biosynthesis during germination and early postgermination stages of the large-seeded dicotyledonous plant pea (Pisum sativum). At the time the developing embryo fills the seed coat, high mRNA levels of PsGA20ox2 (primarily responsible for conversion of C20-GAs to GA(20)), PsGA2ox1 (primarily responsible for conversion of GA(20) to GA(29)), and PsGA2ox2 (primarily responsible for conversion of GA(1) to GA(8)) were detected in the seeds, along with high GA(20) and GA(29) levels, the enzymatic products of these genes. Embryo maturation was accompanied by a large reduction in PsGA20ox2 and PsGA2ox1 mRNA and lower GA(20) and GA(29) levels. However, PsGA2ox2 transcripts remained high. Following seed imbibition, GA(20) levels in the cotyledons decreased, while PsGA3ox1 mRNA and GA(1) levels increased, implying that GA(20) was being used for de novo synthesis of GA(1). The presence of the embryo axis was required for stimulation of cotyledonary GA(1) synthesis at the mRNA and enzyme activity levels. As the embryo axis doubled in size, PsGA20ox1 and PsGA3ox1 transcripts increased, both GA(1) and GA(8) were detectable, PsGA2ox2 transcripts decreased, and PsGA2ox1 transcripts remained low. Cotyledonary-, root-, and shoot-specific expression of these GA biosynthesis genes and the resultant endogenous GA profiles support a key role for de novo GA biosynthesis in each organ during germination and early seedling growth of pea.

The Unifoliata (Uni) gene plays a major role in development of the compound leaf in pea, but its regulation is unknown. In this study, we examined the effects of plant hormones on the expression of Uni, PsPK2 (the gene for a pea homolog of Arabidopsis PID, a regulator of PIN1 targeting), PsPIN1 (the major gene for a putative auxin efflux carrier) and LE (a gibberellin biosynthesis gene, GA3ox), and also examined mutual hormonal regulation of these genes, in pea shoot tips, including a number of mutants. The Uni promoter possessed putative auxin and gibberellin response elements. The PsPIN1 mRNA levels were increased in afila, which replaces leaflets with branched tendrils; and reduced in tendrilless, which replaces tendrils with leaflets, compared with the wild type (WT). In contrast, mRNA levels of LE were increased in uni and tendrilless and decreased in afila compared with the WT. Uni, PsPK2 and PsPIN1 are positively regulated by gibberellin and auxin, and were induced to higher levels by simultaneous application of auxin and gibberellin. Auxin induction of Uni, PsPK2 and PsPIN1 did not require de novo protein synthesis. LE was positively regulated by auxin and cytokinin. In conclusion, these results support the hypothesis that auxin and gibberellin positively regulate Uni, which controls pea compound leaf development. Also, Uni, PsPIN1, PsPK2 and LE are expressed differentially in the leaf mutants, suggesting that mutual regulation by auxin and gibberellin promotes compound leaf development.

Gibberellins control various aspects of growth and development. Here, we identified a gene, designated paclobutrazol resistance1 (PRE1), by screening Arabidopsis activation-tagged lines. PRE1 encodes a helix-loop-helix protein and belongs to a small gene family. Physiological and genetic analysis indicated that overexpression of PRE1 altered various aspects of gibberellin-dependent responses such as germination, elongation of hypocotyl/petiole, floral induction and fruit development, and suppressed gibberellin-deficient phenotypes of the ga2 mutant. Expression of some gibberellin-responsive genes was also affected by PRE1. Expression of PRE1 was shown to be early gibberellin inducible in the wild-type plants and under control of SPY and GAI, upstream negative regulators of gibberellin signaling. The shortened hypocotyl length phenotype of the gai-1 mutant was suppressed by PRE1 overexpression. Ectopic overexpression of each of the four PRE1-related genes conferred pleiotropic phenotypes similar to PRE1 overexpression, indicative of overlapping functions among the PRE gene family. Our results of gain-of-function studies suggest that PRE genes may have a regulatory role in gibberellin-dependent development in Arabidopsis thaliana.

A recessive gibberellin (GA)-insensitive dwarf mutant of rice, gibberellin-insensitive dwarf1 (gid1), has been identified, which shows a severe dwarf phenotype and contains high concentrations of endogenous GA. To elucidate the function of gid1, proteins regulated downstream of gid1 were analysed using a proteomic approach. Proteins extracted from suspension-cultured cells of gid1 and its wild type were separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). Of a total of 962 proteins identified from the suspension-cultured cells, 16 were increased and 14 were decreased in gid1 compared with its wild type. Among the proteins hyper-accumulated in gid1 were osmotin, triosephosphate isomerase, probenazole inducible protein (PBZ1) and pathogenesis-related protein 10. Of these four genes, only the expression of PBZ1 was increased by exogenous GA3 application. Expression of this gene was also enhanced in shoots of the wild type by cold stress or by rice blast fungus infection. Under normal growth conditions, there was more PBZ1 protein in gid1 than in the wild type. In addition, gid1 showed increased tolerance to cold stress and resistance to blast fungus infection. The entcopalyl diphosphate synthase (OsCPS) genes, which encode enzymes at the branch point between GA and phytoalexin biosynthesis, were expressed differentially in gid1 relative to the wild type. Specifically, OsCPS1, which encodes an enzyme in the GA biosynthesis pathway, was down-regulated and OsCPS2 and OsCPS4, which encode enzymes in phytoalexin biosynthesis, were up-regulated in gid1. These results suggest that the expression of PBZ1 is regulated by GA signalling and stress stimuli, and that gid1 is involved in tolerance to cold stress and resistance to blast fungus.

The plant hormone, gibberellin (GA), regulates plant growth and development. It was first isolated as a superelongation-promoting diterpenoid from the fungus, Gibberella fujikuroi. G. fujikuroi uses different GA biosynthetic intermediates from those in plants to produce GA3. Another class of GA-producing fungus, Phaeosphaeria sp. L487, synthesizes GA1 by using the same intermediates as those in plants. A molecular analysis of GA biosynthesis in Phaeosphaeria sp. has revealed that diterpene cyclase and cytochrome P450 monooxygenases were involved in the plant-like biosynthesis of GA1. Fungal ent-kaurene synthase is a bifunctional cyclase. Subsequent oxidation steps are catalyzed by P450s, leading to biologically active GA1. GA biosynthesis in plants is divided into three steps involving soluble enzymes and membrane-bound cytochrome P450. The activation of plant GAs is catalyzed by soluble 2-oxoglutarate-dependent dioxygenases, which is in contrast to the catalysis of fungal GA biosynthesis. This difference suggests that the origin of fungal GA biosynthesis is evolutionally independent of that in plants.

This study tested the hypothesis that gibberellin levels were responsible for the superior growth habit of hybrids (i.e., heterosis). If this were true, plants reduced in their capacity to produce gibberellin, such as maize plants homozygous for dwarf1 (d1), should display a lesser heterotic response. The d1 mutation was introgressed into two inbred lines of maize, B73 and Mo17, for seven generations. Plants segregating for the dwarf phenotype were produced both by self-fertilizing the introgressed inbred lines and by making reciprocal crosses between them to produce hybrids. Measurements were made of several physical traits. The results indicated that the hybrid dwarf plants experienced no loss of heterosis relative to their normal siblings. These results exclude the possibility that modulation of bioactive gibberellins is a major underlying basis of the heterotic response.

The regulation of gene expression at the transcription initiation level is highly complex and requires the presence of multiple transcription factors. These transcription factors are often proteins or peptides that bind to the so-called cis-acting elements, which are present in the promoter regions and conserved among different species. In order to predict these cis-acting elements, a computer program called PRECISE (Prediction of REgulatory CIS-acting Elements) was developed. The power of the tool lies in its user-friendly interface and in the possibility of using empirical motif frequency tables to filter through the many discovered motifs. The tools to create the empirical motif frequency table (e.g., from a whole genome sequence) are included in the package. In the first case study, the upstream regions of all the genes in the Arabidopsis genome were used to create an empirical motif frequency table and a set of 64 upstream sequences of genes known to be involved in starch metabolism was subjected to analysis by PRECISE. The 20 motifs with the highest specificity in the selected set were analyzed in more detail. Of these 20 motifs, 15 showed a very high or complete homology to the sequences of known cis-acting elements. These cis-acting elements are regulated by light, auxin, and abscisic acid, and confer specific expression in sink organs such as leaves and seeds. All these factors have been shown to play an important role in starch biosynthesis. In the second case study, the upstream regions of 16 genes whose transcription is induced by gibberellins (GA) in Arabidopsis were analyzed with PRECISE and compared to the motifs present in the PLACE database. Among the most promising motifs found by PRECISE were 6 of the 17 known GA motifs. These results indicate the power of the PRECISE software package in the prediction of regulatory elements.

To clarify the role of gibberellins in the seed development of Arabidopsis, we investigated the sites where gibberellins are synthesized and induce alpha-amylase genes. The spatial and temporal expression of the genes encoding gibberellin biosynthetic enzymes and alpha-amylases was examined by reverse transcription-PCR (RT-PCR) and in situ hybridization. The mRNAs of AtGA20ox2, AtGA20ox3 and AtGA3ox4 began to be detectable 5-7 d after pollination. In situ hybridization showed that these genes were expressed almost simultaneously around starch granules in the outer integument, preceding the disappearance of those granules. AtGA20ox2 and AtGA3ox4 but not AtGA20ox3 also showed their signals at the rim of the developing embryo. The alpha-amylase gene, Amy3, which responded to gibberellin, was mainly expressed in the developing seed, spatially overlapping with the expression of AtGA20ox2 and AtGA3ox4. These results suggest that gibberellins function in at least two sites of the seed: the outer integument and part of the embryo. We examined the phenotypes of a T-DNA insertion line of AtGA3ox4 and observed the following: (i) a decrease of alpha-amylase gene transcripts in young siliques; (ii) delay of starch degradation in the outer integument; (iii) disarrangement of the seed surface structure; and (iv) abnormal swelling pattern of polysaccharides after imbibition by the mature seed. These characteristics are phenotypes of plants under gibberellin starvation, because the abnormalities could be almost overcome with applied gibberellin, and the gibberellin-treated mutant was indistinguishable from the wild type. These results strongly suggest that gibberellins in the outer integument would be required for the normal formation of the Arabidopsis seed coat.

We have isolated a severe dwarf transposon (Ds) insertion mutant in rice (Oryza sativa L.), which could be differentiated early in the seedling stage by reduced shoot growth and dark green leaves, and later by severe dwarfism and failure to initiate flowering. These mutants, however, showed normal seed germination and root growth. One of the sequences flanking Ds, rescued from the mutant, was of a chromosome 4-located putative ent-kaurene synthase (KS) gene, encoding the enzyme catalyzing the second step of the gibberellin (GA) biosynthesis pathway. Dwarf mutants were always homozygous for this Ds insertion and no normal plants homozygous for this mutation were recovered in the segregating progeny, indicating that the Ds insertion mutation is recessive. As mutations in three recently reported rice GA-responsive dwarf mutant alleles and the dwarf mutation identified in this study mapped to the same locus, we designate the corresponding gene OsKS1. The osks1 mutant seedlings were responsive to exogenous gibberellin (GA3). OsKS1 transcripts of about 2.3 kb were detected in leaves and stem of wild-type plants, but not in germinating seeds or roots, suggesting that OsKS1 is not involved in germination or root growth. There are at least five OsKS1-like genes in the rice genome, four of which are also represented in rice expressed sequence tag (EST) databases. All OsKS1-like genes are transcribed with different expression patterns. ESTs corresponding to all six OsKS genes are represented in other cereal databases including barley, wheat and maize, suggesting that they are biologically active.

To examine the role of gibberellin hormones (GAs) in tolerance to apical meristem damage (AMD), we characterized the reaction norms of several GA-deficient and insensitive mutants of Arabidopsis thaliana in response to AMD and compared them to those of the wild type, Landsberg, from which they were derived. We included 'natural' genotypes of A. thaliana--accessions with shorter lab histories--in order to evaluate how representative Landsberg is of other genotypes. The GA mutations did not alter the level of tolerance to AMD, which was consistent with equal compensation for all genotypes. Generally, the reaction norms to AMD did not differ among the GA mutants themselves, or between the GA mutants and Landsberg. The GA mutations did affect the overall phenotypes of the plants, but these effects were not simply related to whether the mutation was early or late in the biochemical pathways. The GA-insensitive mutant was phenotypically different from the GA-deficient mutants and from Landsberg. The natural populations differed significantly from Landsberg, particularly in attributes related to size and inflorescence production, one more example of the need for researchers to be careful when generalizing the results of studies based upon laboratory strains. Our results indicate that early-flowering genotypes of A. thaliana can be remarkably tolerant to AMD, and that GA deficiency/insensitivity does not hinder tolerance to AMD, at least in this genetic background. Moreover, we confirm that mutations at regulatory loci can have noncatastrophic effects on fitness, as recently found by other investigators.

The expressions of gibberellin (GA) biosynthetic gene in both of the upright and the weeping types of Japanese flowering cherry (Prunus spachiana) were investigated. A part of the GA 3-beta hydroxylase gene of Prunus spachiana (Ps3bhx), which synthesizes active GAs, were amplified by real-time RT-PCR technique. The accumulation of Ps3bhx mRNA in the weeping type were much more than that in the upright type.

The phytohormone gibberellin (GA) regulates the development and fertility of Arabidopsis flowers. The mature flowers of GA-deficient mutant plants typically exhibit reduced elongation growth of petals and stamens. In addition, GA-deficiency blocks anther development, resulting in male sterility. Previous analyses have shown that GA promotes the elongation of plant organs by opposing the function of the DELLA proteins, a family of nuclear growth repressors. However, it was not clear that the DELLA proteins are involved in the GA-regulation of stamen and anther development. We show that GA regulates cell elongation rather than cell division during Arabidopsis stamen filament elongation. In addition, GA regulates the cellular developmental pathway of anthers leading from microspore to mature pollen grain. Genetic analysis shows that the Arabidopsis DELLA proteins RGA and RGL2 jointly repress petal, stamen and anther development in GA-deficient plants, and that this function is enhanced by RGL1 activity. GA thus promotes Arabidopsis petal, stamen and anther development by opposing the function of the DELLA proteins RGA, RGL1 and RGL2.

The hormone gibberellin (GA) plays an important role in modulating diverse processes throughout plant development. In recent years, significant progress has been made in the identification of upstream GA signaling components and trans- and cis-acting factors that regulate downstream GA-responsive genes in higher plants. GA appears to derepress its signaling pathway by inducing proteolysis of GA signaling repressors (the DELLA proteins). Recent evidence indicates that the DELLA proteins are targeted for degradation by an E3 ubiquitin ligase SCF complex through the ubiquitin-26S proteasome pathway.

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.

Recently, six genes of the gibberellin (GA) biosynthesis gene cluster in Gibberella fujikuroi were cloned and the functions of five of these genes were determined. Here we describe the function of the sixth gene, P450-3, and the cloning and functional analysis of a seventh gene, orf3, located at the left border of the gene cluster. We have thereby defined the complete GA biosynthesis gene cluster in this fungus. The predicted amino acid sequence of orf3 revealed no close homology to known proteins. High performance liquid chromatography and gas chromatography-mass spectrometry analyses of the culture fluid of knock-out mutants identified GA1 and GA4, rather than GA3 and GA7, as the major C19-GA products, suggesting that orf3 encodes the GA4 1,2-desaturase. This was confirmed by transformation of the SG139 mutant, which lacks the GA biosynthesis gene cluster, with the desaturase gene renamed des. The transformants converted GA4 to GA7, and also metabolized GA9 (3-deoxyGA4) to GA120 (1,2-didehydroGA9), but the 2alpha-hydroxylated compound GA40 was the major product in this case. We demonstrate also by gene disruption that P450-3, one of the four cytochrome P450 monooxygenase genes in the GA gene cluster, encodes the 13-hydroxylase, which catalyzes the conversion of GA7 to GA3, in the last step of the pathway. This enzyme also catalyzes the 13-hydroxylation of GA4 to GA1. Disruption of the des gene in an UV-induced P450-3 mutant produced a double mutant lacking both desaturase and 13-hydroxylase activities that accumulated high amounts of the commercially important GA4. The des and P450-3 genes differ in their regulation by nitrogen metabolite repression. In common with the other five GA biosynthesis genes, expression of the desaturase gene is repressed by high amounts of nitrogen in the culture medium, whereas P450-3 is the only gene in the cluster not repressed by nitrogen.

Gibberellin (GA) plays an important role in the induction of germination of photoblastic lettuce (Lactuca sativa L. cv. Grand Rapids) seeds. We have previously shown that gene expression of a GA 3-oxidase (Ls3h1) increased after a red light treatment, resulting in an increase in the endogenous content of GA1, bioactive GA. Since the metabolism of GAs is also important for determining the endogenous levels of bioactive GAs, cDNAs encoding GA 2-oxidases (LsGA2ox1 and LsGA2ox2, for L. sativa GA 2-oxidase), which catalyze the deactivation of GAs, were isolated from lettuce seeds to investigate the regulation of these genes by light. An expression analysis shows that the mRNA levels of both enzymes was not markedly altered under different light conditions during germination. However, the amount of LsGA2ox2 transcripts was decreased to approximately half the level by red light. This reduction might play a role in the increase in GA1 level by red light in the lettuce seeds.

The spectacular increases in wheat and rice yields during the 'Green Revolution', were enabled by the introduction of dwarfing traits into the plants. Now, identification of the genes responsible for these traits shows that they interfere with the action or production of the gibberellin (GA) plant hormones. We knew that the wheat Rht genes encode growth repressors that are normally suppressed by GA, and recent work shows that the rice sd1 gene encodes a defective enzyme in the GA-biosynthetic pathway.

Seed germination is promoted by gibberellin (GA) in many plant species. Several GA signalling factors are known to induce the expression of genes encoding enzymes that mobilise food reserves, including starches, proteins and lipids, stored in the endosperm during seed germination. However, these factors do not control seed germination. Two recent reports have indicated that RGL1 and RGL2, both homologous to the GA-response height-regulating factors GAI/RGA/RHT/d8/SLR1/SLN1, are repressors of seed germination in Arabidopsis. These reports provide new clues as to how GA controls seed germination. The induction of RGL2 expression by imbibition and its repression by GA are of particular interest because they imply that RGL2 acts as an integrator of environmental and endogenous cues for germination.

The chronic food shortage that was feared after the rapid expansion of the world population in the 1960s was averted largely by the development of a high-yielding semi-dwarf variety of rice known as IR8, the so-called rice 'green revolution'. The short stature of IR8 is due to a mutation in the plant's sd1 gene, and here we identify this gene as encoding an oxidase enzyme involved in the biosynthesis of gibberellin, a plant growth hormone. Gibberellin is also implicated in green-revolution varieties of wheat, but the reduced height of those crops is conferred by defects in the hormone's signalling pathway.

Dwarf plants are useful in research because multiple plants can be grown in a small area. Rice (Oryza sativa L.) is especially important since its relatively simple genome has recently been sequenced. We are characterizing a gibberellic acid (GA) mutant of rice (japonica cv 'Shiokari,' line N-71) that is extremely dwarf (20 cm tall). Unfortunately, this GA mutation is associated with poor germination (70%) under aerobic conditions. Neither exogenous GA nor a dormancy-breaking heat treatment improved germination. However, 95% germination was achieved by germinating the seeds anaerobically, either in a pure N2 environment or submerged in unstirred tap water. The anaerobic conditions appear to break a mild post-harvest dormancy in this rice cultivar., (Copyright 2002 Crop Science Society of America.)

The genes of the gibberellin (GA) biosynthesis pathway in Gibberella fujikuroi are organized in a gene cluster consisting of at least seven genes. Here we report the cloning and characterization of smt, a gene encoding a membrane transporter of the major facilitator super-family 1, which is located next to the GA gene cluster. Since pathway-specific transporters occur frequently in prokaryotic and fungal antibiotic and toxin clusters, smt was thought to be involved in GA secretion. The gene is expressed in mycelium grown under GA-production conditions, but not when the GA biosynthesis is repressed by high amounts of ammonium. To investigate the function of SMT, gene replacement experiments were performed. The smt-mutants did not show any reduction in the GA yield; and gibberellic acid or its precursors did not influence the gene expression. However, sugar alcohols, such as myo-inositol, sorbitol and mannitol, induced the expression of smt. The results demonstrate that the smt gene does not play an essential role in biosynthesis and secretion of GAs in G. fujikuroi, despite the location adjacent to the GA gene cluster.

The antagonism between gibberellins (GA) and abscisic acid (ABA) is an important factor regulating the developmental transition from embryogenesis to seed germination. In barley aleurone layers, the expression of genes encoding alpha-amylases and proteases is induced by GA but suppressed by ABA. It has been shown that an ABA-induced protein kinase, PKABA1, mediates the ABA suppression of alpha-amylase expression. Using a barley aleurone transient expression system, we have now localized the site of action of PKABA1 relative to other signal transduction components governing the expression of alpha-amylase. The expression of alpha-amylase can be transactivated by the transcription factor GAMyb, which is itself induced by GA. A truncated GAMyb containing the DNA binding domain but lacking the transactivation domain prevents the GA induction of alpha-amylase, further supporting the notion that GAMyb mediates the GA induction of alpha-amylase expression. Although ABA and PKABA1 strongly inhibit the GA induction of alpha-amylase, they have no effect on GAMyb-transactivated alpha-amylase expression. Using a GAMyb promoter--beta-glucuronidase construct, we also show that both ABA and PKABA1 repress the GA induction of GAMyb. In the slender mutant, GAMyb and alpha-amylase are highly expressed, even in the absence of GA. However, this constitutive expression can still be inhibited by ABA, PKABA1, or an inhibitor of cGMP synthesis. On the basis of these observations, we suggest that PKABA1 acts upstream from the formation of functional GAMyb but downstream from the site of action of the Slender gene product. Because PKABA1 inhibits the GA induction of the GAMyb promoter--beta-glucuronidase construct, it appears that at least part of the action of PKABA1 is to downregulate GAMyb at the transcriptional level.

To identify genes targeted by the tobacco KNOX homeodomain protein, Nicotiana tabacum homeobox 15 (NTH15), we have generated an inducible system using the human glucocorticoid receptor. In this system, steroid treatment strictly induced NTH15 function and immediately suppressed the expression of a gibberellin (GA) biosynthetic gene encoding GA 20-oxidase (Ntc12) and also resulted in a decrease in bioactive GA levels. The repression of Ntc12 was observed even when indirect effects were blocked by cycloheximide. NTH15 mRNA was present in corpus cells of the shoot apical meristem (SAM), whereas Ntc12 mRNA was observed in leaf primordia and rib meristem but not in the corpus. Recombinant NTH15 protein strongly bound to a 5-bp dyadsymmetric sequence, GTGAC, in the first intron of Ntc12 in vitro. Mutation of this sequence in the Ntc12 gene abolished the NTH15-dependent suppression of Ntc12 in the corpus of the SAM. Our results indicate that NTH15 directly represses Ntc12 expression in the corpus of the wild-type SAM to maintain the indeterminate state of corpus cells. The suppression of NTH15 within cells at the flanks of the SAM permits GA biosynthesis, which promotes organized cell proliferation and consequently induces the determination of cell fate.

Dwarf mutants of pea (Pisum sativum), with impaired gibberellin (GA) biosynthesis in the shoot, were studied to determine whether the roots of these genotypes had altered elongation and GA levels. Mutations na, lh-2, and ls-1 reduced GA levels in root tips and taproot elongation, although in lh-2 and ls-1 roots the reduction in elongation was small (less than 15%). The na mutation reduced taproot length by about 50%. The roots of na plants elongated in response to applied GA(1) and recombining na with mutation sln (which blocks GA catabolism) increased GA(1) levels in root tips and completely restored normal root development. In shoots, Mendel's le-1 mutation impairs the 3beta-hydroxylation of GA(20) to the bioactive GA(1), resulting in dwarfism. However, GA(1) and GA(20) levels were normal in le-1 roots, as was root development. The null mutation le-2 also did not reduce root GA levels or elongation. The results support the theory that GAs are important for normal root elongation in pea, and indicate that a 3beta-hydroxylase gene other than LE operates in pea roots.

The Arabidopsis hypocotyl, together with hormone mutants and chemical inhibitors, was used to study the role of auxin in cell elongation and its possible interactions with ethylene and gibberellin. When wild-type Arabidopsis seedlings were grown on media containing a range of auxin concentrations, hypocotyl growth was inhibited. However, when axr1-12 and 35S-iaaL (which have reduced auxin response and levels, respectively) were grown in the same conditions, auxin was able to promote hypocotyl growth. In contrast, auxin does not promote hypocotyl growth of axr3-1, which has phenotypes that suggest an enhanced auxin response. These results are consistent with the hypothesis that auxin levels in the wild-type hypocotyl are optimal for elongation and that additional auxin is inhibitory. When ethylene responses were reduced using either the ethylene-resistant mutant etr1 or aminoethoxyvinylglycine, an inhibitor of ethylene synthesis, auxin responses were unchanged, indicating that auxin does not inhibit hypocotyl elongation through ethylene. To test for interactions between auxin and gibberellin, auxin mutants were grown on media containing gibberellin and gibberellin mutants were grown on media containing auxin. The responses were found to be the same as wild-type Arabidopsis seedlings in all cases. In addition, 1 microM of the auxin transport inhibitor 1-naphthylphthalmic acid does not alter the response of wild-type seedlings to gibberellin. Double mutants were made between gibberellin and auxin mutants and the phenotypes of these appear additive. These results indicate that auxin and gibberellin are acting independently in hypocotyl elongation. Thus auxin, ethylene, and gibberellin each regulate hypocotyl elongation independently.

A fusion genetic strategy was used to identify gibberellin (GA) signaling mutants in transgenic Arabidopsis expressing the beta-glucuronidase (GUS) and firefly luciferase (LUC) reporter genes under control of the GA-responsive GASA1 promoter. Initial analyses determined the spatial and temporal patterns of reporter expression, and showed that reporter induction by GA was antagonized by ABA. gamma-Irradiated M2 progeny with altered reporter activities were identified by LUC bioimaging followed by GUS assays and northern hybridization of the endogenous GASA1 mRNA. Genetic analysis showed that three mutants, which overexpressed both reporters and endogenous GASA1, were caused by recessive (goe1 and goe2, for GASA over-expressed) and semi-dominant (goe3) mutations at different loci. These mutants are altered in their sensitivity to GA and the GA biosynthetic inhibitor paclobutrazol, and in the expression of several GA signaling related genes.