The LS locus of pea encodes the gibberellin biosynthesis enzyme ent-kaurene synthase A

Gibberellins (GAs) are hormones required for several aspects of plant development, including internode elongation and seed development in pea (Pisum sativum L.). The first committed step in the GA biosynthesis pathway is the conversion of geranylgeranyl diphosphate (GGDP) to ent-kaurene via copalyl diphosphate (CDP). These two reactions are catalyzed by the cyclases ent-kaurene synthase A (KSA) and ent-kaurene synthase B (KSB), respectively. Previous genetic and biochemical analysis of the GA-responsive ls-1 mutant of pea suggested that GA levels are reduced in a developmental- and organ-specific manner due to reduced GA biosynthesis. Analysis of cell-free enzyme preparations from WT and ls-1 embryos at contact point reveals that ls-1 reduces the activity of KSA but not KSB. To characterize the ls-1 mutation in more detail, a cDNA coding for a pea KSA was cloned and shown to be encoded by the LS locus. The ls-1 mutation results from an intronic G to A substitution that causes impaired RNA splicing. To determine the activity of the KSAs encoded by the LS and ls-1 alleles, a new in vitro assay for combined KSA and KSB activity has been developed using the KSB gene of pumpkin. Using recombinant WT KSA and KSB fusion proteins, GGDP is converted to ent-kaurene in vitro. Based on the sequence of RT-PCR products, three different truncated KSA proteins are predicted to exist in ls-1 plants. The most abundant mutant KSA protein does not possess detectable activity in vitro. Nevertheless, the ls-1 allele is not null and is able to encode at least a partially functional KSA since a more severe ls allele has been identified. The ls-1 mutation has played a key role in identifying a role for GAs in pea seed development in the first few days after fertilization, but not in older seeds. KSA expression in seeds is developmentally regulated and parallels overall GA biosynthesis, suggesting that KSA expression may play an important role in the regulation of GA biosynthesis and seed development.     

Gibberellin biosynthesis in fungi: genes, enzymes, evolution, and impact on biotechnology

Gibberellins (GAs) constitute a large family of tetracyclic diterpenoid carboxylic acids, some members of which function as growth hormones in higher plants. As well as being phytohormones, GAs are also present in some fungi and bacteria. In recent years, GA biosynthetic genes from Fusarium fujikuroi and Arabidopsis thaliana have been cloned and well characterised. Although higher plants and the fungus both produce structurally identical GAs, there are important differences indicating that GA biosynthetic pathways have evolved independently in higher plants and fungi. The fact that horizontal gene transfer of GA genes from the plant to the fungus can be excluded, and that GA genes are obviously missing in closely related Fusarium species, raises the question of the origin of fungal GA biosynthetic genes. Besides characterisation of F. fujikuroi GA pathway genes, much progress has been made in the molecular analysis of regulatory mechanisms, especially the nitrogen metabolite repression controlling fungal GA biosynthesis. Basic research in this field has been shown to have an impact on biotechnology. Cloning of genes, construction of knock-out mutants, gene amplification, and regulation studies at the molecular level are powerful tools for improvement of production strains. Besides increased yields of the final product, GA₃, it is now possible to produce intermediates of the GA biosynthetic pathway, such as ent-kaurene, ent-kaurenoic acid, and GA₁₄, in high amounts using different knock-out mutants. This review concentrates mainly on the fungal biosynthetic pathway, the genes and enzymes involved, the regulation network, the biotechnological relevance of recent studies, and on evolutionary aspects of GA biosynthetic genes.     

Over-expression of a tobacco homeobox gene, NTH15 , decreases the expression of a gibberellin biosynthetic gene encoding GA 20-oxidase

Ectopic expression of the homeobox gene, NTH15 ( Nicotiana tabacum homeobox 15) in transgenic tobacco leads to abnormal leaf and flower morphology, accompanied by a decrease in the content of the active gibberellin, GA₁. Quantitative analysis of intermediates in the GA biosynthetic pathway revealed that the step from GA₁₉ to GA₂₀ was blocked in transgenic tobacco plants overexpressing NTH15 . To investigate the relationship between the expression of NTH15 and genes involved in GA biosynthesis, we isolated three cDNA clones from tobacco encoding two types of GA 20-oxidase and a 3β-hydroxylase. RNA gel blot analysis revealed that the expression of one gene ( Ntc12 , encoding GA 20-oxidase), which in wild-type tobacco plants was abundantly expressed in leaves, was strongly suppressed in the transformants. The expression level of Ntc12 decreased with increasing severity of phenotype of transgenic tobacco leaves. The abnormal leaf morphology was largely overcome by treatment with GA₂₀ or GA₁ but not by GA₁₉. These data strongly suggest that overexpression of NTH15 inhibits the expression of Ntc12 , resulting in reduced levels of active GA and abnormal leaf morphology in transgenic tobacco plants. In situ hybridization in wild-type tobacco revealed that expression of Ntc12 occurred mainly in the rib meristem, cells surrounding the procambium and in leaf primordia. Expression was not seen in the tunica, corpus and procambium, tissues in which NTH15 was predominantly expressed. The contrasting expression patterns of these genes may reflect their antagonistic functions in the formation of lateral organs from the shoot apical meristem.     

The gene encoding tobacco gibberellin 3beta-hydroxylase is expressed at the site of GA action during stem elongation and flower organ development

Gibberellin 3beta-hydroxylase catalyzes the final step in the biosynthetic pathway leading to the plant hormone gibberellin (GA) and, therefore, the in vivo localization of this enzyme should give a direct indication of the site of synthesis of bioactive GAs in plants. We have isolated a cDNA clone, Nty (Nicotiana tabacum GA 3beta-hydroxylase), which encodes a putative GA 3beta-hydroxylase, by RT-PCR using RNA from tobacco shoot apices. Functional analysis, using an NTY protein expressed in Escherichia coli, revealed that Nty encoded an active GA 3beta-hydroxylase. A high expression level of Nty was observed in shoot apices, flowers, roots, young internodes but not in leaves or seeds. We performed more detailed expression analyses using in situ hybridization and histochemical analyses of the GUS activity in transgenic tobacco plants carrying an Nty promoter:GUS fusion gene. These studies revealed that expression of Nty was restricted to specific regions, including actively dividing and elongating cells in the various organs; rib meristem and elongation zones of shoot apices, tapetum and pollen grains in developing anthers and root tips, which are consistent with the sites of GA action. It is proposed that GA actions depend on the modulation of endogenous bioactive GA levels through the regulation of GA 3beta-hydroxylase expression in situ.     

Temporal and spatial control of expression of anthocyanin biosynthetic genes in developing flowers of Antirrhinum majus

The co-ordination of expression of anthocyanin biosynthetic genes was studied in developing flowers. Four genes encoding enzymes operating late in the anthocyanin biosynthetic pathway are induced together during flower development but the early steps appear to be induced more rapidly. Co-ordination of expression could imply a common regulatory mechanism controlling the expression of metabolically related genes. The data presented here show that while four genes may share such a mechanism for the control of their expression during flower development, different control processes regulate the early steps of the pathway. Spatially, gene expression is patterned across the flower and appears to be very similar for all the biosynthetic genes. However, the observed influence of the regulatory gene Delila shows that the spatial co-ordination of gene expression must involve more than one regulatory system. Delila itself appears to have a dual function, being required for activation of expression of the later genes in the flower tube but repressing chalcone synthase gene expression in the mesophyll of the corolla lobes. It is postulated that common signals induce the expression of genes in the pathway during flower development. The data presented here suggest that the same regulatory mechanism interprets these signals for four of the genes encoding the later biosynthetic enzymes, but that different or modified mechanisms interpret the signals to control expression of chalcone synthase and chalcone isomerase genes in Antirrhinum flowers.     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

Characterization of cis-regulatory regions responsible for developmental regulation of the gibberellin biosynthetic gene GA1 in Arabidopsis thaliana

The Arabidopsis GA1 gene encodes copalyl diphosphate synthase, which catalyzes the first committed step in the gibberellin biosynthetic pathway. Previous studies indicated that the expression pattern of the GA1 gene is tissue-specific and cell-type-specific during development. Here we showed that expression of GA1 cDNA driven by the 2.4 kb 5-upstream sequence plus the GA1 genomic coding region into the third exon was able to rescue the ga1-3 mutant phenotype. To understand the mechanism controlling GA1 gene expression, cis-regulatory regions in the GA1 promoter were identified by promoter deletion analysis with the GA1--glucuronidase (GUS) gene fusion system. The second intron and the region from –1391 to –997, with respect to the translation initiation site, positively regulate overall GA1-GUS expression level in all tissues examined. Several additional regulatory regions are involved in GA1-GUS expression in all the stages except in seeds: two positive regulatory regions in the first intron and the sequence between –425 and –207, and a negative regulatory region between –1848 and –1391. We also found that the region between –997 and –796 is essential for a high level of GA1 expression in developing seeds.     

The role of a class III gibberellin 2‐oxidase in tomato internode elongation

A network of environmental inputs and internal signaling controls plant growth, development and organ elongation. In particular, the growth‐promoting hormone gibberellin (GA) has been shown to play a significant role in organ elongation. The use of tomato as a model organism to study elongation presents an opportunity to study the genetic control of internode‐specific elongation in a eudicot species with a sympodial growth habit and substantial internodes that can and do respond to external stimuli. To investigate internode elongation, a mutant with an elongated hypocotyl and internodes but wild‐type petioles was identified through a forward genetic screen. In addition to stem‐specific elongation, this mutant, named tomato internode elongated ‐1 (tie‐1) is more sensitive to the GA biosynthetic inhibitor paclobutrazol and has altered levels of intermediate and bioactive GAs compared with wild‐type plants. The mutation responsible for the internode elongation phenotype was mapped to GA2oxidase 7, a class III GA 2‐oxidase in the GA biosynthetic pathway, through a bulked segregant analysis and bioinformatic pipeline, and confirmed by transgenic complementation. Furthermore, bacterially expressed recombinant TIE protein was shown to have bona fide GA 2‐oxidase activity. These results define a critical role for this gene in internode elongation and are significant because they further the understanding of the role of GA biosynthetic genes in organ‐specific elongation.     

夏枯草PvGGPPS基因的克隆和诱导表达分析

为探究夏枯草中GGPPS基因的生物学特性及功能,该文在夏枯草转录组测序的基础上设计特异性引物,采用逆转录PCR技术获得夏枯草中GGPPS基因的全长核苷酸序列,并进行生物信息学分析;采用qPCR法分析PvGGPPS基因在不同外源性物质诱导下在夏枯草果穗中的表达量以及该基因在夏枯草不同组织中的表达量。结果表明:PvGGPPS基因开放阅读框1 092 bp,编码363个氨基酸,理论分子量为38 815.68 D,等电点为5.69。PvGGPPS蛋白具有异戊烯基焦磷酸合酶家族的特征结构域。系统进化树表明PvGGPPS蛋白与丹参、毛喉鞘蕊花GGPPS蛋白具有较高的亲缘关系。qPCR分析表明,PvGGPPS基因在叶中表达量高于果穗及茎。对果穗施加7种外源性物质处理24 h后,GA3处理组该基因表达量升高。PvGGPPS基因在夏枯草不同组织中表达量差异较大,且受外源物质诱导表达。该研究结果为进一步研究PvGGPPS基因对夏枯草萜类成分合成途径中的功能及表达调控奠定基础。     

Light modulates the spatial patterns of 3-hydroxy-3-methylglutaryl coenzyme A reductase gene expression in Arabidopsis thaliana

Although the coordinated biosynthesis of isoprenoid compounds is thought to be essential to the normal processes of plant growth and development, the mechanisms that regulate the mevalonate pathway in plants are not well understood. As the first committed step in the pathway, the conversion of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) to mevalonic acid by HMG CoA reductase and the regulation of the genes encoding this enzyme have been implicated in the network that controls isoprenoid biosynthesis in higher plants. Using histochemical staining for β-glucuronidase, as well as conventional RNA hybridization analysis, the temporal and spatial regulation of HMG1, one of the genes encoding HMG CoA reductase in the crucifer Arabidopsis thaliana, has been characterized. Furthermore, the HMG1 promoter is shown to be differentially responsive to illumination in different organs, and promoter activation by light deprivation is confined primarily to immature leaves. In contrast, expression of the HMG1 gene in roots is confined to the elongation zone and is not responsive to illumination. Light-mediated regulation of HMG1 expression is shown to be an organ-autonomous response that depends on direct illumination, and environmental cues regarding light do not appear to be exchanged between different organs in Arabidopsis. These studies reveal several new features of HMG1 regulation, and indicate that the high levels of HMG CoA reductase expression detected in immature leaves may be primarily attributed to the dark-induced expression of HMG1, and that HMG1 is expressed at low levels throughout the plant in response to light. Thus, environmental cues interact with the developmental program to define the pattern of HMG1 gene expression in Arabidopsis.     

Biosynthesis of gibberellins in Gibberella fujikuroi: biomolecular aspects

Gibberellins (GAs) are a large family of isoprenoid plant hormones, some of which are bioactive growth regulators, controlling seed germination, stem elongation, and flowering. The rice pathogen Gibberella fujikuroi (mating population C) is able to produce large amounts of GAs, especially the bioactive compounds gibberellic acid (GA₃) and its precursors, GA₄ and GA₇. The main steps of the biosynthetic pathway have long been established from the identification of intermediates in wild-type G. fujikuroi and mutant strains. However, the genetics of the fungus have been rather under-developed, and molecular genetic studies of the GA pathway started just recently. The progress in researching GA biosynthesis in the last 2 years resulted primarily from development of the molecular tools, e.g. transformation systems for the fungus, and cloning the genes encoding GA biosynthesis enzymes, such as the bifunctional ent-copalyl diphosphate/kaurene synthase and several cytochrome P450 monooxygenases. The availability of these genes opened new horizons both for detailed study of the pathway and the regulation mechanisms at the molecular level, and for modern strain improvement programs. This review gives a short overview of the well-known physiological and biochemical studies and concentrates mainly on the new molecular genetic data from GA research, including new information on the regulation of GA biosynthesis. Received: 15 February 1999 / Received revision: 16 April 1999 / Accepted: 16 April 1999     

Regulation of the gibberellin pathway by auxin and DELLA proteins

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 GA₂₀ showed that in Arabidopsis, as in pea, auxin can promote the accumulation of bioactive GA.     

Down regulation of StGA3ox genes in potato results in altered GA content and affect plant and tuber growth characteristics

GA biosynthesis and catabolism has been shown to play an important role in regulating tuberization in potato. Active GAs are inactivated in the stolon tips shortly after induction to tuberization. Overexpression of a GA inactivation gene results in an earlier tuberization phenotype, while reducing expression of the same gene results in delayed tuberization. In addition, overexpression of genes involved in GA biosynthesis results in delayed tuberization, while decreased expression of those genes results in earlied tuberization. The final step in GA biosynthesis is catalysed by StGA3ox1 and StGA3ox2 activity, that convert inactive forms of GA into active GA₁ and GA₄. In this study we cloned StGA3ox2 gene in an RNAi construct and used this construct to transform potato plants. The StGA3ox2 silenced plants were smaller and had shorter internodes. In addition, we assayed the concentrations of various GAs in the transgenic plants and showed an altered GA content. No difference was observed on the time point of tuber initiation. However, the transgenic clones had increased number of tubers with the same yield, resulting in smaller average tuber weight. In addition, we cloned the promoter of StGA3ox2 to direct expression of the GUS reporter gene to visualize the sites of GA biosynthesis in the potato plant. Finally, we discuss how changes of several GA levels can have an impact on shoot, stolon and tuber development, as well as the possible mechanisms that mediate feed-forward and feed-back regulation loops in the GA biosynthetic pathway in potato.