GA Biosynthetic Pathway Research Articles

Inactivation Pathway of Diterpenoid Regulator in the Moss Physcomitrium patens

AbstractThe endogenous levels of plant hormones, including gibberellins (GAs), are strictly regulated and maintained during growth and development in seed plants. The regulation of endogenous levels of bioactive GAs is mediated by the mechanisms of their biosynthesis and inactivation. The moss Physcomitrium patens harbors a partial GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid (KA). Recently, we have identified ent-3β-hydroxy kaurenoic acid (3OH-KA) as a biologically active metabolite of KA to control the protonemal cell differentiation. In addition, ent-2α-hydroxy kaurenoic acid catalyzed by KA 2-oxidase (KA2ox) was also identified as inactive product. Although the activation and inactivation pathways from KA have been identified, the inactivation pathway of 3OH-KA remains to be elucidated. Considering the GA inactivation mechanism of flowering plants, in which GA2ox hydroxylates the C-2 position of GAs as part of the biosynthetic pathway, it was presumed that 3OH-KA was converted to 2,3-dihydroxy KA by PpKA2ox; however, this work shows that PpKA2ox undergoes hydroxylation at the C-16 position to synthesize a new compound ent-3β,16β-dihydroxy kaurenoic acid (3,16diOH-KA) from 3OH-KA. The protonemal cell differentiation activity of 3,16diOH-KA was low, and 3,16diOH-KA was detected in wild-type strains. These results indicate that 3,16diOH-KA was the major inactivating metabolite of 3OH-KA.

Important Factors Controlling Gibberellin Homeostasis in Plant Height Regulation.

Plant height is an important agronomic trait that is closely associated with crop yield and quality. Gibberellins (GAs), a class of highly efficient plant growth regulators, play key roles in regulating plant height. Increasing reports indicate that transcriptional regulation is a major point of regulation of the GA pathways. Although substantial knowledge has been gained regarding GA biosynthetic and signaling pathways, important factors contributing to the regulatory mechanisms homeostatically controlling GA levels remain to be elucidated. Here, we provide an overview of current knowledge regarding the regulatory network involving transcription factors, noncoding RNAs, and histone modifications involved in GA pathways. We also discuss the mechanisms of interaction between GAs and other hormones in plant height development. Finally, future directions for applying knowledge of the GA hormone in crop breeding are described.

The intrinsic developmental age signal defines an age-dependent climbing behavior in cucumber

The tendril is a climbing organ in cucurbits and functions in physical support and to avoid shading by neighboring vegetation. However, how cucurbits produce tendrils to obtain climbing ability is largely unknown. In this study, tendril phenotypes were investigated during different developmental stages. Our results revealed that tendril growth exhibited an age-dependent pattern in cucurbits. Tendril growth was inhibited, and the tendril was formed as a short tendril [nonfunctional tendril (nonF-tendril), approximately 0.1 cm] during the seedling stage. In contrast, enhanced cell proliferation and cell expansion led to rapid elongation of the tendril during the climbing stage, and the tendril formed as a functional tendril (F-tendril, approximately 30 cm) to obtain climbing ability. RT-qPCR detection showed that age-dependent tendril growth correlated negatively with the abundance of the conserved age regulator CsmiR156. Defoliation induced CsmiR156 to inhibit CsSPLs, and F-tendril formation and climbing ability were delayed in defoliated cucumbers, which confirmed the role of CsmiR156 in regulating tendril growth in vivo. Additionally, exogenous gibberellin (GA) treatment showed that GA positively regulated tendril growth, and RT-qPCR detection showed that the GA bio-synthetic genes and metabolic genes were affected by age pathway, suggesting that the age pathway depended on GA bio-synthetic and metabolic pathway to regulate cell expansion to determine tendril growth. In summary, our work reveals that change in tendril type is an important marker of phase transition in cucumber, and tendril growth is regulated by an intrinsic developmental age signal, ensuring that the cucumber obtains climbing ability at a suitable age.

TEMPRANILLO homologs in apple regulate flowering time in the woodland strawberry Fragaria vesca

The long juvenile period of fruit trees makes their breeding costly and time-consuming. Therefore, flowering time engineering and shortening the juvenile phase have become a breeding priority for the genetic improvement of fruit tree crops. Many economically valuable fruit trees belong to the Rosaceae family including apples and strawberries. TEMPRANILLO (TEM) acts as a key player in flowering time control through inhibiting FT function. Two genes with high sequence similarity with the Arabidopsis TEM genes were isolated from apple (Malus domestica). Due to the complexity of carrying out functional studies in apple, we characterized their function in woodland strawberry as well as their expression in apple. The expression of MdTEM genes in apple tissues from juvenile plants was dramatically higher than that in the tissues from adult trees. In woodland strawberry, the overexpression of MdTEM genes down-regulated FvFT1, FvGA3OX1, and FvGA3OX2 genes in strawberry. The MdTEM-overexpressing lines exhibited delayed flowering, in terms of days to flowering and the number of leaves at flowering. While, RNAi-mediated silencing of TEM resulted in five days earlier flowering, with a lower number of leaves, a higher trichome density, and in some cases, caused in vitro flowering. According to these results and in silico analyses, it can be concluded that MdTEM1 and MdTEM2 can be considered as orthologs of FvTEM and probably AtTEM genes, which play an important role in regulating the juvenile phase and flowering time through regulating FT and GA biosynthetic pathway.

The qLTG1.1 candidate gene CsGAI regulates low temperature seed germination in cucumber.

The CsGAI gene, identified by map-based, was involved in regulating seed germination in low temperature via the GA and ABA signaling pathways. Low temperature reduces the percentage of seeds germinating and delays seed germinating time, thus posing a threat to cucumber production. However, the molecular mechanism regulating low temperature germination in cucumber is unknown. We here dissected a major quantitative trait locus qLTG1.1 that controls seed germination at low temperature in cucumber. First, we fine-mapped qLTG1.1 to a 46.3-kb interval, containing three candidate genes. Sequence alignment and gene expression analysis identified Csa1G408720 as the gene of interest that was highly expressed in seeds, and encoded a highly conserved, low temperature-regulated DELLA family protein CsGAI. GUS expression analysis indicated that higher promoter activity underscored higher transcriptional expression of the CsGAI gene. Consistent with the known roles of GAI in ABA and GA signaling during germination, genes involved in the GA (CsGA2ox, CsGA3ox) and ABA biosynthetic pathways (CsABA1, CsABA2, CsAAO3 and CsNCED) were found to be differently regulated in the tolerant and sensitive genotypes under low temperatures, and this was reflected in differences in their ratio of GA-to-ABA. Based on these data, we proposed a working model explaining how CsGAI integrates the GA and ABA signaling pathways, to regulate cucumber seed germination at low temperature, thus providing new insights into this mechanism.

A high-throughput visual screening method for p-hydroxybenzoate hydroxylase to increase phenolic compounds biosynthesis

BackgroundGallic acid (GA) and pyrogallol are phenolic hydroxyl compounds and have diverse biological activities. Microbial-based biosynthesis, as an ecofriendly method, has been used for GA and pyrogallol production. In GA and pyrogallol biosynthetic pathways, the low hydroxylation activity of p-hydroxybenzoate hydroxylase (PobA) towards 3,4-dihydroxybenzoic acid (3,4-DHBA) limited the high-level biosynthesis of GA and pyrogallol.ResultsThis work reported a high activity PobA mutant (Y385F/T294A/V349A PobA) towards 3,4-DHBA. This mutant was screened out from a PobA random mutagenesis library through a novel naked eye visual screening method. In vitro enzyme assay showed this mutant has a kcat/Km of 0.059 μM−1 s−1 towards 3,4-DHBA, which was 4.92-fold higher than the reported mutant (Y385F/T294A PobA). Molecular docking simulation provided the possible catalytic mechanism explanation of the high activity mutant. Expression of this mutant in E. coli BW25113 (Fʹ) can generate 840 ± 23 mg/L GA from 1000 mg/L 3,4-DHBA. After that, this mutant was assembled into a de novo GA biosynthetic pathway. Subsequently, this pathway was introduced into a 3,4-DHBA-producing strain (E. coli BW25113 (Fʹ)ΔaroE) to achieve 301 ± 15 mg/L GA production from simple carbon sources. Similarly, assembling this mutant into a de novo pyrogallol biosynthetic pathway enabled 129 ± 15 mg/L pyrogallol production.ConclusionsThis work established an efficient screening method and generated a high activity PobA mutant. Assembling this mutant into de novo GA and pyrogallol biosynthetic pathways achieved the production of these two compounds from glucose. Besides, this mutant has great potential for the production of GA or pyrogallol derivatives. The screening method could be used for other GA biosynthesis-related enzymes.

The HB40-JUB1 transcriptional regulatory network controls gibberellin homeostasis in Arabidopsis

The gibberellins (GAs) are phytohormones that play fundamental roles in almost every aspect of plant growth and development. Although GA biosynthetic and signaling pathways are well understood, the mechanisms that control GA homeostasis remain largely unclear in plants. Here, we demonstrate that the homeobox transcription factor (TF) HB40 of the HD-Zip family regulates GA content at two additive control levels in Arabidopsis thaliana. We show that HB40 expression is induced by GA and in turn reduces the levels of endogenous bioactive GAs by simultaneously reducing GA biosynthesis and increasing GA deactivation. Consistently, HB40 overexpression leads to typical GA-deficiency traits, such as small rosettes, reduced plant height, delayed flowering, and male sterility. By contrast, a loss-of-function hb40 mutation enhances GA-controlled growth. Genome-wide RNA sequencing combined with molecular-genetic analyses revealed that HB40 directly activates the transcription of JUNGBRUNNEN1 (JUB1), a key TF that represses growth by suppressing GA biosynthesis and signaling. HB40 also activates genes encoding GA 2-oxidases (GA2oxs), which are major GA-catabolic enzymes. The effect of HB40 on plant growth is ultimately mediated through the induction of nuclear growth-repressing DELLA proteins. Collectively, our results reveal the important role of the HB40-JUB1 regulatory network in controlling GA homeostasis during plant growth.

Tailored biosynthesis of gibberellin plant hormones in yeast

The application of small amounts of natural plant growth hormones, such as gibberellins (GAs), can increase the productivity and quality of many vegetable and fruit crops. However, gibberellin growth hormones usage is limited by the high cost of their production, which is currently based on fermentation of a natural fungal producer Fusarium fujikuroi that produces a mix of several GAs. We explored the potential of the oleaginous yeast Yarrowia lipolytica to produce specific profiles of GAs. Firstly, the production of the GA-precursor ent-kaurenoic acid (KA) at 3.75 mg/L was achieved by expression of biosynthetic enzymes from the plant Arabidopsis thaliana and upregulation of the mevalonate (MVA) pathway.We then built a GA4-producing strain by extending the GA-biosynthetic pathway and upregulating the MVA-pathway further, resulting in 17.29 mg/L GA4. Additional expression of the F. fujikoroi GA-biosynthetic enzymes resulted in the production of GA7 (trace amounts) and GA3 (2.93 mg/L). Lastly, through protein engineering and the expression of additional KA-biosynthetic genes, we increased the GA3-production 4.4-fold resulting in 12.81 mg/L. The developed system presents a promising resource for the recombinant production of specific gibberellins, identifying bottlenecks in GA biosynthesis, and discovering new GA biosynthetic genes. ClassificationBiological Sciences, Applied Biological Sciences.

Combinatorial synthetic pathway fine-tuning and cofactor regeneration for metabolic engineering of Escherichia coli significantly improve production of D-glucaric acid

D-glucaric acid (GA) has been identified as among promising biotechnological alternatives to oil-based chemicals. GA and its derivatives are widely used in food additives, dietary supplements, drugs, detergents, corrosion inhibitors and biodegradable materials. The increasing availability of a GA market is improving the cost-effectiveness and efficiency of various biosynthetic pathways. In this study, an engineered Escherichia coli strain GA10 was constructed by systematic metabolic engineering. This involved redirecting metabolic flux into the GA biosynthetic pathways, blocking the conversion pathways of d-glucuronic acid (GlcA) and GA into by-products, introducing an in situ NAD+ regeneration system and fine-tuning the activity of the key enzyme, myo-inositol oxygenase (Miox). Subsequently, the culture medium was optimized to achieve the best performance of the GA10 strain. GA was produced at 5.35 g/L (extracellular and intracellular), with a maximized yield of ∼0.46 mol/mol on d-glucose and glycerol, by batch fermentation. This work demonstrates efficient biosynthetic pathways of GA in E. coli by metabolic engineering and should accelerate the application of GA biosynthetic pathways in industrial processes.

The Multifunctional Dioxygenases of Gibberellin Synthesis.

Gibberellin (GA) hormones regulate the development of plants and their responses to environmental signals. The final part of GA biosynthesis is catalyzed by multifunctional 2-oxoglutarate-dependent dioxygenases, which are encoded by multigene families. According to their enzymatic properties and physiological functions, GA-oxidases are classified as anabolic or catabolic enzymes. Together they allow complex regulation of the GA biosynthetic pathway, which adapts the specific hormonal needs of a plant during development and interaction with its environment. In this review, we combine recent advances in enzymatic characterization of the multifunctional GA-oxidases, in particular, from cucumber and Arabidopsis that have been most comprehensively investigated.

OsbHLH073 Negatively Regulates Internode Elongation and Plant Height by Modulating GA Homeostasis in Rice.

Internode elongation is one of the key agronomic traits determining a plant’s height and biomass. However, our understanding of the molecular mechanisms controlling internode elongation is still limited in crop plant species. Here, we report the functional identification of an atypical basic helix-loop-helix transcription factor (OsbHLH073) through gain-of-function studies using overexpression (OsbHLH073-OX) and activation tagging (osbhlh073-D) lines of rice. The expression of OsbHLH073 was significantly increased in the osbhlh073-D line. The phenotype of osbhlh073-D showed semi-dwarfism due to deficient elongation of the first internode and poor panicle exsertion. Transgenic lines overexpressing OsbHLH073 confirmed the phenotype of the osbhlh073-D line. Exogenous gibberellic acid (GA3) treatment recovered the semi-dwarf phenotype of osbhlh073-D plants at the seedling stage. In addition, quantitative expression analysis of genes involving in GA biosynthetic and signaling pathway revealed that the transcripts of rice ent-kaurene oxidases 1 and 2 (OsKO1 and OsKO2) encoding the GA biosynthetic enzyme were significantly downregulated in osbhlh073-D and OsbHLH073-OX lines. Yeast two-hybrid and localization assays showed that the OsbHLH073 protein is a nuclear localized-transcriptional activator. We report that OsbHLH073 participates in regulating plant height, internode elongation, and panicle exsertion by regulating GA biosynthesis associated with the OsKO1 and OsKO2 genes.

PbGA20ox2 Regulates Fruit Set and Induces Parthenocarpy by Enhancing GA4 Content.

Fruit set and development occur following successful fertilization. Parthenocarpy, a valuable trait in some self-incompatible species, produces seedless fruit without fertilization. Gibberellin (GA) is a crucial hormone in fruit-set regulation and development. While investigating the development of parthenocarpy in pear (Pyrus bretschneideri Rehd.), we determined that GA 20-oxidases (GA20ox) may play key roles in seedless pear fruit development. Sequence analysis revealed three PbGA20ox genes: PbGA20ox1, PbGA20ox2, and PbGA20ox3. We analyzed the expression patterns of candidate genes and found that PbGA20ox2 levels significantly changed in pollinated fruits. Tissue-specific expression assays revealed that PbGA20ox2 is highly expressed in young fruit and leaves. Subcellular localization assays showed it was located in the cytoplasm, nucleus, and plasma membrane. Overexpressed PbGA20ox2 tomato plants were taller and had longer hypocotyls and internodes, and the emasculated flowers produced parthenocarpic fruit. In pear, the transient overexpression of PbGA20ox2 promoted fruit development and delayed the drop of nonpollinated fruit. Furthermore, the fruit of PbGA20ox2-overexpressing tomato and transient PbGA20ox2-overexpressing pear had increased GA4 (but not GA3 and GA1) contents. This result provided evidence that PbGA20ox2 was necessary for GA4-dependent pear fruit development. Our study revealed that PbGA20ox2 altered the GA biosynthetic pathway and enhanced GA4 synthesis, thereby promoting fruit set and parthenocarpic fruit development.

Activation of the Transcription of BrGA20ox3 by a BrTCP21 Transcription Factor Is Associated with Gibberellin-Delayed Leaf Senescence in Chinese Flowering Cabbage during Storage.

Several lines of evidence have implicated the involvement of the phytohormone gibberellin (GA) in modulating leaf senescence in plants. However, upstream transcription factors (TFs) that regulate GA biosynthesis in association with GA-mediated leaf senescence remain elusive. In the current study, we report the possible involvement of a TEOSINTE BRANCHED1/CYCLOIDEA/PCF (TCP) TF BrTCP21 in GA-delayed leaf senescence in Chinese flowering cabbage. Exogenous GA3 treatment maintained a higher value of maximum PSII quantum yield (Fv/Fm) and total chlorophyll content, accompanied by the repression of the expression of senescence-associated genes and chlorophyll catabolic genes, which led to the delay of leaf senescence. A class I member of TCP TFs BrTCP21, was further isolated and characterized. The transcript level of BrTCP21 was low in senescing leaves, and decreased following leaf senescence, while GA3 could keep a higher expression level of BrTCP21. BrTCP21 was further found to be a nuclear protein and exhibit trans-activation ability through transient-expression analysis in tobacco leaves. Intriguingly, the electrophoretic mobility shift assay (EMSA) and transient expression assay illustrated that BrTCP21 bound to the promoter region of a GA biosynthetic gene BrGA20ox3, and activated its transcription. Collectively, these observations reveal that BrTCP21 is associated with GA-delayed leaf senescence, at least partly through the activation of the GA biosynthetic pathway. These findings expand our knowledge on the transcriptional mechanism of GA-mediated leaf senescence.

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.

2,4-D-induced parthenocarpy in pear is mediated by enhancement of GA4 biosynthesis.

Parthenocarpy, the productions of seedless fruit without pollination or fertilization, is a potentially desirable trait in many commercially grown fruits, especially in pear, which is self-incompatible. Phytohormones play important roles in fruit set, a process crucial for parthenocarpy. In this study, 2,4-dichlorophenoxyacetic acid (2,4-D), an artificially synthesized plant growth regulator with functions similar to auxin, was found to induce parthenocarpy in pear. Histological observations revealed that 2,4-D promoted cell division and expansion, which increased cortex thickness, but the effect was weakened by paclobutrazol (PAC), a gibberellin (GA) biosynthesis inhibitor. Phenotypic differences in pear may therefore be due to different GA contents. Hormone testing indicated that 2,4-D mainly induced the production of bioactive GA4 , rather than GA3. Three key oxidase genes function in the GA biosynthetic pathway: GA20ox, GA3ox and GA2ox. In a pear group treated with only 2,4-D, PbGA20ox2-like and PbGA3ox-1 were significantly upregulated. When treated with 2,4-D supplemented with PAC, however, expression levels of these genes were significantly downregulated. Additionally, PbGA2ox1-like and PbGA2ox2-like expression levels were significantly downregulated in pear treated with either 2,4-D only or 2,4-D supplemented with PAC. We thus hypothesize that 2,4-D can induce parthenocarpy by enhancing GA4 biosynthesis.

One-pot two-strain system based on glucaric acid biosensor for rapid screening of myo-inositol oxygenase mutations and glucaric acid production in recombinant cells

The development of D-glucaric acid (GA) production in recombinant cells has leapt forward in recent years, and higher throughput screening and selection of better-performing recombinant cells or biocatalysts is in current demand. A biosensor system which converts GA concentration into fluorescence signal in Escherichia coli was developed in 2016, but its application has rarely been reported. Herein, an effective high-throughput screening approach independent of special-purpose devices such as microfluidic platforms was established and tentatively applied. In this one-pot two-strain system, GA producers—bacterial or yeast cells containing the GA biosynthetic pathway—were sorted with the help of another E. coli strain acting as a GA biosensor. The identification of highly active mutants of myo-inositol oxygenase through this system validates its effectiveness in sorting E. coli cells. Subsequently, accurate ranking of the GA synthesis capacity of a small library of Saccharomyces cerevisiae strains containing distinct GA synthesis pathways demonstrated that this optimized one-pot two-strain system may also be used for eukaryotic producer strains. These results will assist in research into metabolic engineering for GA production and development of biosensor applications.

Exogenous GA3 application altered morphology, anatomic and transcriptional regulatory networks of hormones in Eucalyptus grandis.

Gibberellins (GAs) play a key role in plant growth and development including cell elongation, cell expansion, and xylem differentiation. Eucalyptus are the world's most widely planted hardwood trees providing fiber and energy. However, the roles of GAs in Eucalyptus remain unclear and their effects on xylem development remain to be determined. In this study, E. grandis plants were treated with 0.10mgL-1 GA3 and/or paclobutrazol (PAC, a GA inhibitor). The growth of shoot and root were recorded, transverse sections of roots and stems were stained using toluidine blue, and expression levels of genes related to hormone response and secondary cell wall biosynthesis were analyzed by quantitative real-time PCR. The results showed that GA3 dramatically promoted the length of shoot and root, but decreased the diameter of root and stem. Exogenous GA3 application also significantly promoted xylem development in both stem and root. Expression analysis revealed that exogenous GA3 application altered the transcript levels of genes related to the GA biosynthetic pathway and GA signaling, as well as genes related to auxin, cytokinin, and secondary cell wall. These findings suggest that GAs may interact with other hormones (such as auxin and cytokinin) to regulate the expression of secondary cell wall biosynthesis genes and trigger xylogenesis in Eucalyptus plants.

Effect of Treatment with Gibberellin, Gibberellin Biosynthesis Inhibitor, and Auxin on Steviol Glycoside Content in Stevia rebaudiana Bertoni

Steviol glycosides (SGs) are natural compounds present in Stevia rebaudiana (Bertoni) Bertoni, which are approximately 200–400 times sweeter than sucrose. A biosynthetic pathway of SGs shares a biosynthetic pathway of gibberellin (GA) through a formation of ent-kaurenoic acid. Previous studies have demonstrated that SG-related genes are upregulated by exogenous GA treatment. However, the transcription level of UGT85C2, which is involved in a glycosylation step to steviol from steviolmonoside and suggests a rate-limiting step in the SGs biosynthesis, remains controversial in GA treatments. To have a detailed understanding of the relationship between SGs and GAs, a concentration gradient of exogenous GA3, GA inhibitor daminozide (DAM), and naphthaleneacetic acid (NAA) was used for treatment by the leaf infiltration method. Results showed that transcription level of UGT85C2 and the total number of stevioside and rebaudioside-A induced by GA3, DAM, or NAA treatment were significantly upregulated compared to an untreated group. These results suggest that the GA biosynthetic pathway affects the number of SGs and that hormonal crosstalk occurs between GA and auxin, which changes the number of SGs in S. rebaudiana. Controlling the GA biosynthesis could be considered to modulate the yield of SGs.

Exogenous auxin represses soybean seed germination through decreasing the gibberellin/abscisic acid (GA/ABA) ratio

Auxin is an important phytohormone which mediates diverse development processes in plants. Published research has demonstrated that auxin induces seed dormancy. However, the precise mechanisms underlying the effect of auxin on seed germination need further investigation, especially the relationship between auxins and both abscisic acid (ABA) and gibberellins (GAs), the latter two phytohormones being the key regulators of seed germination. Here we report that exogenous auxin treatment represses soybean seed germination by enhancing ABA biosynthesis, while impairing GA biogenesis, and finally decreasing GA1/ABA and GA4/ABA ratios. Microscope observation showed that auxin treatment delayed rupture of the soybean seed coat and radicle protrusion. qPCR assay revealed that transcription of the genes involved in ABA biosynthetic pathway was up-regulated by application of auxin, while expression of genes involved in GA biosynthetic pathway was down-regulated. Accordingly, further phytohormone quantification shows that auxin significantly increased ABA content, whereas the active GA1 and GA4 levels were decreased, resulting insignificant decreases in the ratiosGA1/ABA and GA4/ABA.Consistent with this, ABA biosynthesis inhibitor fluridone reversed the delayed-germination phenotype associated with auxin treatment, while paclobutrazol, a GA biosynthesis inhibitor, inhibited soybean seed germination. Altogether, exogenous auxin represses soybean seed germination by mediating ABA and GA biosynthesis.

Elucidation of gibberellin biosynthesis in bacteria reveals convergent evolution.

Gibberellins (GAs) are crucial phytohormones involved in many aspects of plant growth and development, including plant-microbe interactions, which has led to GA production by plant-associated fungi and bacteria as well. While the GA biosynthetic pathways in plants and fungi have been elucidated and found to have arisen independently through convergent evolution, little has been uncovered about GA biosynthesis in bacteria. Some nitrogen-fixing, symbiotic, legume-associated rhizobia, including Bradyrhizobium japonicum-the symbiont of soybean-and Sinorhizobium fredii-a broad-host-nodulating species-contain a putative GA biosynthetic operon, or gene cluster. Through functional characterization of five unknown genes, we demonstrate that this operon encodes the enzymes necessary to produce GA9, thereby elucidating bacterial GA biosynthesis. The distinct nature of these enzymes indicates that bacteria have independently evolved a third biosynthetic pathway for GA production. Furthermore, our results also reveal a central biochemical logic that is followed in all three convergently evolved GA biosynthetic pathways.