Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids in Euphorbia peplus.

Most macro- and polycyclic Euphorbiaceae diterpenoids derive from the common C20 precursor casbene. While the biosynthetic pathway from casbene to the lathyrane jolkinol C is characterized, pathways to other more complex classes of bioactive diterpenoids remain to be elucidated. A metabolomics-guided transcriptomic approach and a genomics approach that led to the discovery of two casbene-derived diterpenoid gene clusters yielded a total of 68 candidate genes that were transiently expressed in Nicotiana benthamiana for activity toward jolkinol C and other lathyranes. We report two short-chain dehydrogenases/reductases (SDRs), identified by RNA sequencing to be highly expressed in Euphorbia peplus latex. One of these, EpSDR-5, is a C3-ketoreductase, converting jolkinol C to the lathyrane jolkinol E. Gene function of EpSDR-5 was further confirmed by heterologous expression in Saccharomyces cerevisiae. To investigate the in vivo role of EpSDR-5, we established virus-induced gene silencing (VIGS) in E. peplus, resulting in a significant reduction in jatrophanes and a corresponding increase in ingenanes. VIGS of Casbene Synthase results in a major reduction in both jatrophanes and ingenanes, the two most abundant classes of E. peplus diterpenoids. VIGS of CYP71D365 had a similar effect, consistent with the previously determined role of this gene in the pathway to jolkinol C. These results point to jolkinol C being a branch point intermediate in the pathways to ingenanes and jatrophanes with EpSDR-5 responsible for the first step from jolkinol C to jatrophane production.

Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co-transformed with the optimal set of precursor-pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an 'alkenal reductase'-like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13-double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.

MOTHER-OF-FT-AND-TFL1 represses seed germination under far-red light by modulating phytohormone responses in Arabidopsis thaliana.

Seed germination in many plant species is triggered by sunlight, which is rich in the red (R) wavelength and repressed by under-the-canopy light rich in far red (FR). R:FR ratios are sensed by phytochromes to regulate levels of gibberellins (GAs) and abscisic acid (ABA), which induce and inhibit germination respectively. In this study we have discovered that, under FR light conditions, germination is repressed by MOTHER-OF-FT-AND-TFL1 (MFT) through the regulation of the ABA and GA signaling pathways. We also show that MFT gene expression is tightly regulated by light quality. Previous work has shown that under FR light conditions the transcription factor PHYOCHROME-INTERACTING-FACTOR1 (PIF1) accumulates and promotes expression of SOMNUS (SOM) that, in turn, leads to increased ABA and decreased GA levels. PIF1 also promotes expression of genes encoding ABA-INSENSITIVE5 (ABI5) and DELLA growth-repressor proteins, which act in the ABA and GA signaling pathways, respectively. Here we show that MFT gene expression is promoted by FR light through the PIF1/SOM/ABI5/DELLA pathway and is repressed by R light via the transcription factor SPATULA (SPT). Consistent with this, we also show that SPT gene expression is repressed under FR light in a PIF1-dependent manner. Furthermore, transcriptomic analyses presented in this study indicate that MFT exerts its function by promoting expression of known ABA-induced genes and repressing cell wall expansion-related genes.

Production of bioactive diterpenoids in the euphorbiaceae depends on evolutionarily conserved gene clusters.

The Euphorbiaceae produce a diverse range of diterpenoids, many of which have pharmacological activities. These diterpenoids include ingenol mebutate, which is licensed for the treatment of a precancerous skin condition (actinic keratosis), and phorbol derivatives such as resiniferatoxin and prostratin, which are undergoing investigation for the treatment of severe pain and HIV, respectively. Despite the interest in these diterpenoids, their biosynthesis is poorly understood at present, with the only characterized step being the conversion of geranylgeranyl pyrophosphate into casbene. Here, we report a physical cluster of diterpenoid biosynthetic genes from castor (Ricinus communis), including casbene synthases and cytochrome P450s from the CYP726A subfamily. CYP726A14, CYP726A17, and CYP726A18 were able to catalyze 5-oxidation of casbene, a conserved oxidation step in the biosynthesis of this family of medicinally important diterpenoids. CYP726A16 catalyzed 7,8-epoxidation of 5-keto-casbene and CYP726A15 catalyzed 5-oxidation of neocembrene. Evidence of similar gene clustering was also found in two other Euphorbiaceae, including Euphorbia peplus, the source organism of ingenol mebutate. These results demonstrate conservation of gene clusters at the higher taxonomic level of the plant family and that this phenomenon could prove useful in further elucidating diterpenoid biosynthetic pathways.

A Cytochrome P450-Mediated Intramolecular Carbon-Carbon Ring Closure in the Biosynthesis of Multidrug-Resistance-Reversing Lathyrane Diterpenoids.

The Euphorbiaceae produce a wide variety of bioactive diterpenoids. These include the lathyranes, which have received much interest due to their ability to inhibit the ABC transporters responsible for the loss of efficacy of many chemotherapy drugs. The lathyranes are also intermediates in the biosynthesis of range of other bioactive diterpenoids with potential applications in the treatment of pain, HIV and cancer. We report here a gene cluster from Jatropha curcas that contains the genes required to convert geranylgeranyl pyrophosphate into a number of diterpenoids, including the lathyranes jolkinol C and epi-jolkinol C. The conversion of casbene to the lathyranes involves an intramolecular carbon-carbon ring closure. This requires the activity of two cytochrome P450s that we propose form a 6-hydroxy-5,9-diketocasbene intermediate, which then undergoes an aldol reaction. The discovery of the P450 genes required to convert casbene to lathyranes will allow the scalable heterologous production of these potential anticancer drugs, which can often only be sourced in limited quantities from their native plant.

Regulation of Arabidopsis thaliana seed dormancy and germination by 12-oxo-phytodienoic acid.

We previously demonstrated that the oxylipin 12-oxo-phytodienoic acid (OPDA) acts along with abscisic acid to regulate seed germination in Arabidopsis thaliana, but the mechanistic details of this synergistic interaction remain to be elucidated. Here, we show that OPDA acts through the germination inhibition effects of abscisic acid, the abscisic acid-sensing ABI5 protein, and the gibberellin-sensing RGL2 DELLA protein. We further demonstrate that OPDA also acts through another dormancy-promoting factor, MOTHER-OF-FT-AND-TFL1 (MFT). Both abscisic acid and MFT positively feed back into the OPDA pathway by promoting its accumulation. These results confirm the central role of OPDA in regulating seed dormancy and germination in A. thaliana and underline the complexity of interactions between OPDA and other dormancy-promoting factors such as abscisic acid, RGL2, and MFT.

Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA.

Freshly matured seeds exhibit primary dormancy, which prevents germination until environmental conditions are favorable. The establishment of dormancy occurs during seed development and involves both genetic and environmental factors that impact on the ratio of two antagonistic phytohormones: abscisic acid (ABA), which promotes dormancy, and gibberellic acid, which promotes germination. Although our understanding of dormancy breakage in mature seeds is well advanced, relatively little is known about the mechanisms involved in establishing dormancy during seed maturation. We previously showed that the SPATULA (SPT) transcription factor plays a key role in regulating seed germination. Here we investigate its role during seed development and find that, surprisingly, it has opposite roles in setting dormancy in Landsberg erecta and Columbia Arabidopsis ecotypes. We also find that SPT regulates expression of five transcription factor encoding genes: ABA-INSENSITIVE4 (ABI4) and ABI5, which mediate ABA signaling; REPRESSOR-OF-GA (RGA) and RGA-LIKE3 involved in gibberellic acid signaling; and MOTHER-OF-FT-AND-TFL1 (MFT) that we show here promotes Arabidopsis seed dormancy. Although ABI4, RGA, and MFT are repressed by SPT, ABI5 and RGL3 are induced. Furthermore, we show that RGA, MFT, and ABI5 are direct targets of SPT in vivo. We present a model in which SPT drives two antagonistic "dormancy-repressing" and "dormancy-promoting" routes that operate simultaneously in freshly matured seeds. Each of these routes has different impacts and this in turn explains the opposite effect of SPT on seed dormancy of the two ecotypes analyzed here.

A DELLA in disguise: SPATULA restrains the growth of the developing Arabidopsis seedling.

The period following seedling emergence is a particularly vulnerable stage in the plant life cycle. In Arabidopsis thaliana, the phytochrome-interacting factor (PIF) subgroup of basic-helix-loop-helix transcription factors has a pivotal role in regulating growth during this early phase, integrating environmental and hormonal signals. We previously showed that SPATULA (SPT), a PIF homolog, regulates seed dormancy. In this article, we establish that unlike PIFs, which mainly promote hypocotyl elongation, SPT is a potent regulator of cotyledon expansion. Here, SPT acts in an analogous manner to the gibberellin-dependent DELLAs, REPRESSOR OF GA1-3 and GIBBERELLIC ACID INSENSITIVE, which restrain cotyledon expansion alongside SPT. However, although DELLAs are not required for SPT action, we demonstrate that SPT is subject to negative regulation by DELLAs. Cross-regulation of SPT by DELLAs ensures that SPT protein levels are limited when DELLAs are abundant but rise following DELLA depletion. This regulation provides a means to prevent excessive growth suppression that would result from the dual activity of SPT and DELLAs, yet maintain growth restraint under DELLA-depleted conditions. We present evidence that SPT and DELLAs regulate common gene targets and illustrate that the balance of SPT and DELLA action depends on light quality signals in the natural environment.

Expression and manipulation of phosphoenolpyruvate carboxykinase 1 identifies a role for malate metabolism in stomatal closure.

Malate, along with potassium and chloride ions, is an important solute for maintaining turgor pressure during stomatal opening. Although malate is exported from guard cells during stomatal closure, there is controversy as to whether malate is also metabolised. We provide evidence that phosphoenolpyruvate carboxykinase (PEPCK), an enzyme involved in malate metabolism and gluconeogenesis, is necessary for full stomatal closure in the dark. Analysis of the Arabidopsis PCK1 gene promoter indicated that this PEPCK isoform is specifically expressed in guard cells and trichomes of the leaf. Spatially distinct promoter elements were found to be required for post-germinative, vascular expression and guard cell/trichome expression of PCK1. We show that pck1 mutant plants have reduced drought tolerance, and show increased stomatal conductance and wider stomatal apertures compared with the wild type. During light-dark transients the PEPCK mutant plants show both increased overall stomatal conductance and less responsiveness of the stomata to darkness than the wild type, indicating that stomata get 'jammed' in the open position. These results show that malate metabolism is important during dark-induced stomatal closure and that PEPCK is involved in this process.

DELLA-mediated cotyledon expansion breaks coat-imposed seed dormancy.

Seed dormancy is a key adaptive trait in plants responsible for the soil seed bank. The long established hormone-balance theory describes the antagonistic roles of the dormancy promoting plant hormone abscisic acid (ABA), and the germination promoting hormone gibberellin (GA) in dormancy control. Light, temperature, and other dormancy-breaking signals function to modulate the synthesis and perception of these hormones in the seed. However, the way in which these hormones control dormancy in the imbibed seed remains unknown. Here, we show that the DELLA protein regulators of the GA response are required for dormancy and describe a model through which hormone signal integration and dormancy regulation is achieved. We demonstrate that cotyledon expansion precedes radicle emergence during Arabidopsis seed germination and that a striking correlation exists between final seedling cotyledon size and seed dormancy in the DELLA mutants. Furthermore, twelve previously characterized seed-dormancy mutants are also defective in the control of cotyledon size in a manner consistent with their effect on germination potential. We propose that DELLA-mediated, light-, temperature-, and hormone-responsive cotyledon expansion prior to radicle emergence overcomes dormancy imposed by the seed coat and underlies seed-dormancy control in Arabidopsis.

Cold and light control seed germination through the bHLH transcription factor SPATULA.

BACKGROUND: Plants integrate signals from the environment and use these to modify the timing of development according to seasonal cues. Seed germination is a key example of this phenomenon and in Arabidopsis is promoted by the synergistic interaction of light and low temperatures in dormant seeds. This signaling pathway is known to converge on the regulation of the gibberellin (GA) biosynthetic genes GA3 oxidase (GA3ox), whose expression is transcriptionally induced by light and cold in imbibed seeds. However, the molecular basis of this response has until now been unknown. RESULTS: Here we show that the bHLH transcription factor SPATULA is a light-stable repressor of seed germination and mediates the germination response to temperature. Furthermore, SPT is required in dormant seeds for maintaining the repression of GA3ox transcription. We also show that the related protein PIL5 represses seed germination and GA3ox expression in the dark. CONCLUSIONS: We conclude that SPT and PIL5 form part of a regulatory network coupling seed germination and GA3ox expression to light and temperature signaling in the seed.

Arabidopsis ABA INSENSITIVE4 regulates lipid mobilization in the embryo and reveals repression of seed germination by the endosperm.

Regulation of seed germination requires coordinate action by the embryo and surrounding endosperm. We used Arabidopsis thaliana to establish the relative roles of embryo and endosperm in the control of seed germination and seedling establishment. We previously showed that endospermic oil reserves are used postgerminatively via gluconeogenesis to fuel seedling establishment and that lipid breakdown is repressed by abscisic acid (ABA) in embryo but not endosperm tissues. Here, we use RNA amplification to describe the transcriptome of the endosperm and compare the hormone responses of endosperm and embryo tissues. We show that the endosperm responds to both ABA and gibberellin but that ABA in particular regulates nuclear but not plastid-encoded photosynthetic gene expression in the embryo. We also show that ABA INSENSITIVE4 (ABI4) expression is confined to the embryo, accounts for the major differences in embryo response to ABA, and defines a role for ABI4 as a repressor of lipid breakdown. Furthermore, ABI5 expression in the endosperm defines a second region of altered ABA signaling in the micropylar endosperm cap. Finally, embryo and endosperm ABA signaling mutants demonstrate the spatial specificity of ABA action in seed germination. We conclude that the single cell endosperm layer plays an active role in the regulation of seed germination in Arabidopsis.

The Arabidopsis thaliana multifunctional protein gene (MFP2) of peroxisomal beta-oxidation is essential for seedling establishment.

The multifunctional protein (MFP) of peroxisomal beta-oxidation catalyses four separate reactions, two of which (2-trans enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase) are core activities required for the catabolism of all fatty acids. We have isolated and characterized five Arabidopsis thaliana mutants in the MFP2 gene that is expressed predominantly in germinating seeds. Seedlings of mfp2 require an exogenous supply of sucrose for seedling establishment to occur. Analysis of mfp2-1 seedlings revealed that seed storage lipid was catabolized more slowly, long-chain acyl-CoA substrates accumulated and there was an increase in peroxisome size. Despite a reduction in the rate of beta-oxidation, mfp2 seedlings are not resistant to the herbicide 2,4-dichlorophenoxybutyric acid, which is catabolized to the auxin 2,4-dichlorophenoxyacetic acid by beta-oxidation. Acyl-CoA feeding experiments show that the MFP2 2-trans enoyl-CoA hydratase only exhibits activity against long chain (C18:0) substrates, whereas the MFP2 L-3-hydroxyacyl-CoA dehydrogenase is active on C6:0, C12:0 and C18:0 substrates. A mutation in the abnormal inflorescence meristem gene AIM1, the only homologue of MFP2, results in an abnormal inflorescence meristem phenotype in mature plants (Richmond and Bleecker, Plant Cell 11, 1999, 1911) demonstrating that the role of these genes is very different. The mfp2-1 aim1double mutant aborted during the early stages of embryo development showing that these two proteins share a common function that is essential for this key stage in the life cycle.

Sucrose rescues seedling establishment but not germination of Arabidopsis mutants disrupted in peroxisomal fatty acid catabolism.

The Arabidopsis acyl-CoA oxidase (ACX) family comprises isozymes with distinct fatty acid chain-length specificities that together catalyse the first step of peroxisomal fatty acid beta-oxidation. We have isolated and characterized T-DNA insertion mutants in the medium to long-chain (ACX1) and long-chain (ACX2) acyl-CoA oxidases, and show that the corresponding endogenous activities are decreased in the mutants. Lipid catabolism during germination and early post-germinative growth was unaltered in the acx1-1 mutant, but slightly delayed in the acx2-1 mutant, with 3-day-old acx2-1 seedlings accumulating long-chain acyl-CoAs. In acx1-1 and acx2-1, seedling growth and establishment in the absence of an exogenous supply of sucrose was unaffected. Seedlings of the double mutant acx1-1 acx2-1 were unable to catabolize seed storage lipid, and accumulated long-chain acyl-CoAs. The acx1-1 acx2-1 seedlings were also unable to establish photosynthetic competency in the absence of an exogenous carbon supply, a phenotype that is shared with a number of other Arabidopsis mutants disrupted in storage lipid breakdown. Germination frequency of the double mutant was significantly reduced compared with wild-type seeds. This was unaffected by the addition of exogenous sucrose, but was improved by dormancy-breaking treatments such as cold stratification and after-ripening. We show that the acx1-1, acx2-1 and acx1-2 acx2-1 double mutants and the ketoacyl-CoA thiolase-2 (kat2) mutant exhibit a sucrose-independent germination phenotype comparable with that reported for comatose (cts-2), a mutant in a peroxisomal ABC transporter which exhibits enhanced dormancy. This demonstrates an additional role beyond that of carbon provision for the beta-oxidation pathway during germination or in dormant seeds.

The gluconeogenic enzyme phosphoenolpyruvate carboxykinase in Arabidopsis is essential for seedling establishment.

Phosphoenolpyruvate carboxykinase (PEPCK) catalyzes the conversion of oxaloacetate to phosphoenolpyruvate in the gluconeogenic production of sugars from storage oil in germinating oilseeds. Here, we present the results of analysis on PEPCK antisense Arabidopsis plants with a range of enzyme activities from 20% to 80% of wild-type levels. There is a direct correlation between enzyme activity and seedling establishment during early post-germinative growth, thus demonstrating the absolute requirement of PEPCK and gluconeogenesis in this process. Soluble sugar levels in the 35S-PCK1 antisense seedlings are reduced and seedling establishment can be rescued with an exogenous supply of sucrose. We observed an increase in the respiration of acetyl coenzyme A units released from fatty acid beta-oxidation and a corresponding decrease in the production of sugars with decreasing enzyme activity in 2-d-old antisense seedlings. The 35S-PCK1 antisense lines have a more extreme phenotype when compared with Arabidopsis mutants disrupted in the glyoxylate cycle. We conclude that the 35S-PCK1 antisense seedlings are compromised in the ability to use both storage lipid and storage protein through gluconeogenesis to produce soluble sugars.

Reserve mobilization in the Arabidopsis endosperm fuels hypocotyl elongation in the dark, is independent of abscisic acid, and requires PHOSPHOENOLPYRUVATE CARBOXYKINASE1.

Arabidopsis thaliana is used as a model system to study triacylglycerol (TAG) accumulation and seed germination in oilseeds. Here, we consider the partitioning of these lipid reserves between embryo and endosperm tissues in the mature seed. The Arabidopsis endosperm accumulates significant quantities of storage lipid, and this is effectively catabolized upon germination. This lipid differs in composition from that in the embryo and has a specific function during germination. Removing the endosperm from the wild-type seeds resulted in a reduction in hypocotyl elongation in the dark, demonstrating a role for endospermic TAG reserves in fueling skotomorphogenesis. Seedlings of two allelic gluconeogenically compromised phosphoenolpyruvate carboxykinase1 (pck1) mutants show a reduction in hypocotyl length in the dark compared with the wild type, but this is not further reduced by removing the endosperm. The short hypocotyl phenotypes were completely reversed by the provision of an exogenous supply of sucrose. The PCK1 gene is expressed in both embryo and endosperm, and the induction of PCK1:beta-glucuronidase at radicle emergence occurs in a robust, wave-like manner around the embryo suggestive of the action of a diffusing signal. Strikingly, the induction of PCK1 promoter reporter constructs and measurements of lipid breakdown demonstrate that whereas lipid mobilization in the embryo is inhibited by abscisic acid (ABA), no effect is seen in the endosperm. This insensitivity of endosperm tissues is not specific to lipid breakdown because hydrolysis of the seed coat cell walls also proceeded in the presence of concentrations of ABA that effectively inhibit radicle emergence. Both processes still required gibberellins, however. These results suggest a model whereby the breakdown of seed carbon reserves is regulated in a tissue-specific manner and shed new light on phytohormonal regulation of the germination process.

Arabidopsis mutants in short- and medium-chain acyl-CoA oxidase activities accumulate acyl-CoAs and reveal that fatty acid beta-oxidation is essential for embryo development.

The short-chain acyl-CoA oxidase (ACX4) is one of a family of ACX genes that together catalyze the first step of peroxisomal fatty acid beta-oxidation during early, postgerminative growth in oilseed species. Here we have isolated and characterized an Arabidopsis thaliana mutant containing a T-DNA insert in ACX4. In acx4 seedlings, short-chain acyl-CoA oxidase activity was reduced by greater than 98%, whereas medium-chain activity was unchanged from wild type levels. Despite the almost complete loss of short-chain activity, lipid catabolism and seedling growth and establishment were unaltered in the acx4 mutant. However, the acx4 seedlings accumulated high levels (31 mol %) of short-chain acyl-CoAs and showed resistance to 2,4-dichlorophenoxybutyric acid, which is converted to the herbicide and auxin analogue 2,4-dichlorophenoxyacetic acid by beta-oxidation. A mutant in medium-chain length acyl-CoA activity (acx3) (1) shows a similar phenotype to acx4, and we show here that acx3 seedlings accumulate medium-chain length acyl-CoAs (16.4 mol %). The acx3 and acx4 mutants were crossed together, and remarkably, the acx3acx4 double mutants aborted during the first phase of embryo development. We propose that acx3acx4 double mutants are nonviable because they have a complete block in short-chain acyl-CoA oxidase activity. This is the first demonstration of the effects of eliminating (short-chain) beta-oxidation capacity in plants and shows that a functional beta-oxidation cycle is essential in the early stages of embryo development.