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.

A role for the root cap in root branching revealed by the non-auxin probe naxillin.

The acquisition of water and nutrients by plant roots is a fundamental aspect of agriculture and strongly depends on root architecture. Root branching and expansion of the root system is achieved through the development of lateral roots and is to a large extent controlled by the plant hormone auxin. However, the pleiotropic effects of auxin or auxin-like molecules on root systems complicate the study of lateral root development. Here we describe a small-molecule screen in Arabidopsis thaliana that identified naxillin as what is to our knowledge the first non-auxin-like molecule that promotes root branching. By using naxillin as a chemical tool, we identified a new function for root cap-specific conversion of the auxin precursor indole-3-butyric acid into the active auxin indole-3-acetic acid and uncovered the involvement of the root cap in root branching. Delivery of an auxin precursor in peripheral tissues such as the root cap might represent an important mechanism shaping root architecture.

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.

A cytosolic acyltransferase contributes to triacylglycerol synthesis in sucrose-rescued Arabidopsis seed oil catabolism mutants.

Triacylglycerol (TAG) levels and oil bodies persist in sucrose (Suc)-rescued Arabidopsis (Arabidopsis thaliana) seedlings disrupted in seed oil catabolism. This study set out to establish if TAG levels persist as a metabolically inert pool when downstream catabolism is disrupted, or if other mechanisms, such as fatty acid (FA) recycling into TAG are operating. We show that TAG composition changes significantly in Suc-rescued seedlings compared with that found in dry seeds, with 18:2 and 18:3 accumulating. However, 20:1 FA is not efficiently recycled back into TAG in young seedlings, instead partitioning into the membrane lipid fraction and diacylglycerol. In the lipolysis mutant sugar dependent1and the ß-oxidation double mutant acx1acx2 (for acyl-Coenzyme A oxidase), levels of TAG actually increased in seedlings growing on Suc. We performed a transcriptomic study and identified up-regulation of an acyltransferase gene, DIACYLGLYCEROL ACYLTRANSFERASE3 (DGAT3), with homology to a peanut (Arachis hypogaea) cytosolic acyltransferase. The acyl-Coenzyme A substrate for this acyltransferase accumulates in mutants that are blocked in oil breakdown postlipolysis. Transient expression in Nicotiana benthamiana confirmed involvement in TAG synthesis and specificity toward 18:3 and 18:2 FAs. Double-mutant analysis with the peroxisomal ATP-binding cassette transporter mutant peroxisomal ABC transporter1 indicated involvement of DGAT3 in the partitioning of 18:3 into TAG in mutant seedlings growing on Suc. Fusion of the DGAT3 protein with green fluorescent protein confirmed localization to the cytosol of N. benthamiana. This work has demonstrated active recycling of 18:2 and 18:3 FAs into TAG when seed oil breakdown is blocked in a process involving a soluble cytosolic acyltransferase.

AtTPS1-mediated trehalose 6-phosphate synthesis is essential for embryogenic and vegetative growth and responsiveness to ABA in germinating seeds and stomatal guard cells.

Trehalose and associated metabolites are part of the sugar signalling system in plants and have profound effects on development. Disruption of the TREHALOSE 6-PHOSPHATE SYNTHASE (TPS1) gene in Arabidopsis results in delayed embryo growth, altered cell wall morphology and carbon metabolism and abortion at the torpedo stage. Here we investigate the role of the TPS1 gene in post-embryonic development using two approaches. In the first we use the seed-specific ABI3 promoter to drive the TPS1 cDNA during embryo development, resulting in rescue of the embryo-lethal tps1 phenotype. Lack of expression from the ABI3::TPS1 transgene in post-germinative tps1 seedlings results in severe growth arrest, accumulation of soluble sugars and starch and leads to an increase in expression of genes related to ABA signalling. In the second approach we use TILLING (targeted induced local lesions in genomes) to generate three weaker, non-embryo-lethal, alleles (tps1-11, tps1-12 and tps1-13) and use these to demonstrate that the TPS1 protein plays a key role in modulating trehalose 6-phosphate (T6P) levels in vegetative tissues of Arabidopsis. All three weaker alleles give a consistent phenotype of slow growth and delayed flowering. Germination of tps1-11, tps1-12 and tps1-13 is hypersensitive to ABA with the degree of hypersensitivity correlating with the decrease in T6P levels in the different alleles. Stomatal pore aperture is regulated by ABA, and this was found to be affected in tps1-12. Our results show that the TPS1 gene product plays an essential role in regulating the growth of vegetative as well as embryogenic tissue in a mechanism involving ABA and sugar metabolism.

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.

Oil accumulation in leaves directed by modification of fatty acid breakdown and lipid synthesis pathways.

Plant oils in the form of triacylglycerol (TAG) are used for food, industrial feedstock and biofuel manufacture. Although TAG is typically harvested from the fruit or seeds of oil crop species, plants can also accumulate small amounts of TAG in the leaves and other vegetative tissues. Here we show that leaf TAG levels can be increased significantly (10-20 fold) by blocking fatty acid breakdown, particularly during extended dark treatments or leaf senescence in the model plant Arabidopsis. Generation of a double mutant in fatty acid breakdown and diacylglycerol acyltransferase 1 (DGAT1) resulted in a severe vegetative growth phenotype suggesting that partitioning of fatty acids to TAG in leaves is carried out predominantly by this acyltransferase. LEC2, a seed development transcription factor involved in storage product accumulation, was ectopically expressed during senescence in the fatty acid breakdown mutant COMATOSE (cts2). This resulted in accumulation of seed oil type species of TAG in senescing tissue. Our data suggests that recycled membrane fatty acids can be re-directed to TAG by expressing the seed-programme in senescing tissue or by a block in fatty acid breakdown. This work raises the possibility of producing significant amounts of oil in vegetative tissues of biomass crops such as Miscanthus.

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.

Quantification of sugars and sugar phosphates in Arabidopsis thaliana tissues using porous graphitic carbon liquid chromatography-electrospray ionization mass spectrometry.

This work reports the development and optimisation of a negative ion mode on-line LC-ESI-MS/MS method for the sensitive targeted analysis of the key glycolytic intermediates, sugars and sugar phosphates from plants, using a porous graphitic carbon (PGC) stationary phase and an MS compatible mobile phase. Using this newly developed method, separation and detection of a solution of standard compounds is achieved in less than 20min. Target metabolite compounds were identified in plant extracts from their characteristic retention times, and product ion spectra. This on-line PGC-ESI-MS/MS method shows good linearity over the concentration range 0-100microM, selectivity, short analysis time, and limits of detection of 0.1microM for disaccharides trehalose (Tre), sucrose (Suc), and maltose, and 1.5microM for hexose phosphates fructose-6-phosphate (Fru6P), glucose-1-phosphate (Glc1P), and glucose-6-phosphate (Glc6P), and phosphoenolpyruvate (PEP). This paper describes details of our method and its application to the simultaneous quantitative analysis of soluble sugars and sugar phosphates from Arabidopsis thaliana tissues. We have demonstrated the utility of our method for the analysis of biological samples by applying it to the simultaneous quantitation of changes in soluble sugars and sugar phosphates in A. thaliana Columbia-0 (Col-0) and its starchless phosphoglucomutase (pgm) mutant over a 12-h light/12-h dark growth cycle.

Hydrophilic interaction chromatography/electrospray mass spectrometry analysis of carbohydrate-related metabolites from Arabidopsis thaliana leaf tissue.

This work describes the development and application of an on-line liquid chromatography/mass spectrometry (LC/MS) method using hydrophilic interaction chromatography (HILIC) coupled to negative ion mode electrospray ionisation ion trap mass spectrometry (ESI-MS) for the analysis of highly polar carbohydrate-related metabolites commonly found in plants, ranging from reducing and non-reducing sugars and sugar alcohols to sugar phosphates. Using this method, separation and detection of a mixture of eight authentic standard compounds containing glucose (Glc), sucrose (Suc), raffinose, verbascose, mannitol, maltitol, glucose-6-phosphate (Glc6P) and trehalose-6-phosphate (Tre6P) were achieved in less than 15 min. The method is rapid, robust, selective, and sensitive, with limits of detection (LODs) ranging from 0.2 microM obtained for neutral sugars, to 1.0 microM obtained for sugar alcohols, and 2.0 microM obtained for negatively charged sugar phosphates. We have studied the negative ion collision-induced dissociation (CID) fragmentation behaviour of the non-reducing raffinose family oligosaccharides (RFOs) raffinose, stachyose, and verbascose. Mainly Bi and Ci glycosidic and Ai cross-ring structurally informative cleavages are observed. We have applied this HILIC/ESI-MS method for the analysis of Arabidopsis thaliana wild-type Columbia-0 (Col-0) and its starchless phosphoglucomutase mutant (pgm1) leaf extracts. The method was used to quantify Glc, Suc, raffinose, and Glc6P in A. thaliana extracts. Data obtained using this HILIC/ESI-MS method were compared with those obtained using a comparable porous graphitic carbon-based LC/ESI-MS method.

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.

Delayed embryo development in the ARABIDOPSIS TREHALOSE-6-PHOSPHATE SYNTHASE 1 mutant is associated with altered cell wall structure, decreased cell division and starch accumulation.

The tps1 mutant, which is disrupted in the TREHALOSE-6-PHOSPHATE SYNTHASE 1 gene, has been previously characterized as a recessive embryo lethal. tps1 embryos do not develop past late torpedo or early cotyledon stage. We report here that at the ultrastructural, biochemical, and transcriptional levels tps1 exhibits many features typically associated with the maturation phase. The appearance of storage reserve transcripts and organelles follows the same temporal pattern in tps1 and wild-type (WT) embryos in the same silique as does accumulation of storage lipid and protein. The mutant plastids accumulate large starch granules that persist until the end of seed development, in contrast with WT plastids where starch accumulation is transient. The transcriptome of tps1 embryos shows a coordinate downregulation of genes involved in starch and sucrose degradation. Interestingly, genes involved in lipid mobilization and gluconeogenesis are induced in tps1 embryos. The cell walls of tps1 embryos show a remarkable degree of thickening at the ultrastructural level and immunodetection of cell wall components shows that altered deposition of pectins accounts for this altered morphology. Consistent with this at the transcriptome level, genes involved in sugar nucleotide and pectin metabolism are altered in the mutant. The frequency of cell division in tps1 embryos is half that of the wild type at the heart and torpedo stages. These results suggest that TPS1 may play a major role in coordinating cell wall biosynthesis and cell division with cellular metabolism during embryo development.