The plastidial pentose phosphate pathway is essential for postglobular embryo development in Arabidopsis.

Large numbers of genes essential for embryogenesis in Arabidopsis encode enzymes of plastidial metabolism. Disruption of many of these genes results in embryo arrest at the globular stage of development. However, the cause of lethality is obscure. We examined the role of the plastidial oxidative pentose phosphate pathway (OPPP) in embryo development. In nonphotosynthetic plastids the OPPP produces reductant and metabolic intermediates for central biosynthetic processes. Embryos with defects in various steps in the oxidative part of the OPPP had cell division defects and arrested at the globular stage, revealing an absolute requirement for the production via these steps of ribulose-5-phosphate. In the nonoxidative part of the OPPP, ribulose-5-phosphate is converted to ribose-5-phosphate (R5P)-required for purine nucleotide and histidine synthesis-and subsequently to erythrose-4-phosphate, which is required for synthesis of aromatic amino acids. We show that embryo development through the globular stage specifically requires synthesis of R5P rather than erythrose-4-phosphate. Either a failure to convert ribulose-5-phosphate to R5P or a block in purine nucleotide biosynthesis beyond R5P perturbs normal patterning of the embryo, disrupts endosperm development, and causes early developmental arrest. We suggest that seed abortion in mutants unable to synthesize R5P via the oxidative part of the OPPP stems from a lack of substrate for synthesis of purine nucleotides, and hence nucleic acids. Our results show that the plastidial OPPP is essential for normal developmental progression as well as for growth in the embryo.

Iminosugar inhibitors of carbohydrate-active enzymes that underpin cereal grain germination and endosperm metabolism.

Starch is a major energy store in plants. It provides most of the calories in the human diet and, as a bulk commodity, it is used across broad industry sectors. Starch synthesis and degradation are not fully understood, owing to challenging biochemistry at the liquid/solid interface and relatively limited knowledge about the nature and control of starch degradation in plants. Increased societal and commercial demand for enhanced yield and quality in starch crops requires a better understanding of starch metabolism as a whole. Here we review recent advances in understanding the roles of carbohydrate-active enzymes in starch degradation in cereal grains through complementary chemical and molecular genetics. These approaches have allowed us to start dissecting aspects of starch degradation and the interplay with cell-wall polysaccharide hydrolysis during germination. With a view to improving and diversifying the properties and uses of cereal grains, it is possible that starch degradation may be amenable to manipulation through genetic or chemical intervention at the level of cell wall metabolism, rather than simply in the starch degradation pathway per se.

12-oxo-phytodienoic acid accumulation during seed development represses seed germination in Arabidopsis.

Arabidopsis thaliana COMATOSE (CTS) encodes an ABC transporter involved in peroxisomal import of substrates for ß-oxidation. Various cts alleles and mutants disrupted in steps of peroxisomal ß-oxidation have previously been reported to exhibit a severe block on seed germination. Oxylipin analysis on cts, acyl CoA oxidase1 acyl CoA oxidase2 (acx1 acx2), and keto acyl thiolase2 dry seeds revealed that they contain elevated levels of 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA), and JA-Ile. Oxylipin and transcriptomic analysis showed that accumulation of these oxylipins occurs during late seed maturation in cts. Analysis of double mutants generated by crossing cts with mutants in the JA biosynthesis pathway indicate that OPDA, rather than JA or JA-Ile, contributes to the block on germination in cts seeds. We found that OPDA was more effective at inhibiting wild-type germination than was JA and that this effect was independent of CORONATINE INSENSITIVE1 but was synergistic with abscisic acid (ABA). Consistent with this, OPDA treatment increased ABA INSENSITIVE5 protein abundance in a manner that parallels the inhibitory effect of OPDA and OPDA+ABA on seed germination. These results demonstrate that OPDA acts along with ABA to regulate seed germination in Arabidopsis.

Altered starch turnover in the maternal plant has major effects on Arabidopsis fruit growth and seed composition.

Mature seeds of both the high-starch starch-excess1 (sex1) mutant and the almost starchless phosphoglucomutase1 mutant of Arabidopsis (Arabidopsis thaliana) have 30% to 40% less lipid than seeds of wild-type plants. We show that this is a maternal effect and is not attributable to the defects in starch metabolism in the embryo itself. Low lipid contents and consequent slow postgerminative growth are seen only in mutant embryos that develop on maternal plants with mutant phenotypes. Mutant embryos that develop on plants with wild-type starch metabolism have wild-type lipid contents and postgerminative growth. The maternal effect on seed lipid content is attributable to carbohydrate starvation in the mutant fruit at night. Fruits on sex1 plants grow more slowly than those on wild-type plants, particularly at night, and have low sugars and elevated expression of starvation genes at night. Transcript levels of the transcription factor WRINKLED1, implicated in lipid synthesis, are reduced at night in sex1 but not in wild-type seeds, and so are transcript levels of key enzymes of glycolysis and fatty acid synthesis. sex1 embryos develop more slowly than wild-type embryos. We conclude that the reduced capacity of mutant plants to convert starch to sugars in leaves at night results in low nighttime carbohydrate availability in the developing fruit. This in turn reduces the rate of development and expression of genes encoding enzymes of storage product accumulation in the embryo. Thus, the supply of carbohydrate from the maternal plant to the developing fruit at night can have an important influence on oilseed composition and on postgerminative growth.

The plastidial glucose-6-phosphate/phosphate antiporter GPT1 is essential for morphogenesis in Arabidopsis embryos.

The glucose-6-phosphate/phosphate antiporter GPT1 is a major route of entry of carbon into non-photosynthetic plastids. To discover its importance in oilseeds, we used a seed-specific promoter to generate lines of Arabidopsis thaliana with reduced levels of GPT1 in developing embryos. Strong reductions resulted in seed abortion at the end of the globular stage of embryo development, when proplastids in normal embryos differentiate and acquire chlorophyll. Seed abortion was partly dependent on the light level during silique development. Embryos in seeds destined for abortion failed to undergo normal morphogenesis and were 'raspberry-like' in appearance. They had ultrastructural and biochemical defects including proliferation of peroxisomes and starch granules, and altered expression of genes involved in starch turnover and the oxidative pentose phosphate pathway. We propose that GPT1 is necessary for early embryo development because it catalyses import into plastids of glucose-6-phosphate as the substrate for NADPH generation via the oxidative pentose phosphate pathway. We suggest that low NADPH levels during plastid differentiation and chlorophyll synthesis may result in generation of reactive oxygen species and triggering of embryo cell death.

Starch turnover in developing oilseed embryos.

*Starch accumulates early during embryo development in Arabidopsis and oilseed rape, then disappears during oil accumulation. Little is known about the nature and importance of starch metabolism in oilseed embryos. *Histochemical and quantitative measures of starch location and content were made on developing seeds and embryos from wild-type Arabidopsis plants, and from mutants lacking enzymes of starch synthesis and degradation with established roles in leaf starch turnover. Feeding experiments with [(14)C]sucrose were used to measure the rate of starch synthesis in oilseed rape embryos within intact siliques. *The patterns of starch turnover in the developing embryo are spatially and temporally complex. Accumulation is associated with zones of cell division. Study of mutant plants reveals a major role in starch turnover for glucan, water dikinase (absent from the sex1 mutant) and isoforms of beta-amylase (absent from various bam mutants). Starch is synthesized throughout the period of its accumulation and loss in embryos within intact siliques of oilseed rape. *We suggest that starch turnover is functionally linked to cell division and differentiation rather than to developmental or storage functions specific to embryos. The pathways of embryo starch metabolism are similar in several respects to those in Arabidopsis leaves.

Plastidial glycolysis in developing Arabidopsis embryos.

During oilseed embryo development, carbon from sucrose is utilized for fatty acid synthesis in the plastid. The role of plastidial glycolysis in Arabidopsis embryo oil accumulation was investigated. Genes encoding enolases (ENO) and phosphoglyceromutases (PGlyM) were identified, and activities and subcellular locations were established by expression of recombinant and green fluorescent protein (GFP)-fusion proteins. Mutant Arabidopsis plants lacking putative plastidial isoforms were characterized with respect to isoform composition and embryo oil content. In the developing embryo, ENO1 and ENO2 account for most or all of the plastidial and cytosolic ENO activity, respectively, and PGLYM1 accounts for most or all of the plastidial PGlyM activity. The eno1 and pglym1 mutants, in which plastidic ENO and PGlyM activities were undetectable, had wild-type amounts of seed oil at maturity. It is concluded that although plastids of developing Arabidopsis embryos have the capacity to carry out the lower part of the glycolytic pathway, the cytosolic glycolytic pathway alone is sufficient to support the flux from 3-phosphoglycerate to phosphoenolpyruvate required for oil production. The results highlight the importance for oil production of translocators that facilitate interchange of glycolytic intermediates between the cytosol and the plastid stroma.

A chemical genetic screen reveals that iminosugar inhibitors of plant glucosylceramide synthase inhibit root growth in Arabidopsis and cereals.

Iminosugars are carbohydrate mimics that are useful as molecular probes to dissect metabolism in plants. To analyse the effects of iminosugar derivatives on germination and seedling growth, we screened a library of 390 N-substituted iminosugar analogues against Arabidopsis and the small cereal Eragrostis tef (Tef). The most potent compound identified in both systems, N-5-(adamantane-1-yl-ethoxy)pentyl- L-ido-deoxynojirimycin (L-ido-AEP-DNJ), inhibited root growth in agar plate assays by 92% and 96% in Arabidopsis and Tef respectively, at 10 µM concentration. Phenocopying the effect of L-ido-AEP-DNJ with the commercial inhibitor (PDMP) implicated glucosylceramide synthase as the target responsible for root growth inhibition. L-ido-AEP-DNJ was twenty-fold more potent than PDMP. Liquid chromatography-mass spectrometry (LC-MS) analysis of ceramide:glucosylceramide ratios in inhibitor-treated Arabidopsis seedlings showed a decrease in the relative quantity of the latter, confirming that glucosylceramide synthesis is perturbed in inhibitor-treated plants. Bioinformatic analysis of glucosylceramide synthase indicates gene conservation across higher plants. Previous T-DNA insertional inactivation of glucosylceramide synthase in Arabidopsis caused seedling lethality, indicating a role in growth and development. The compounds identified herein represent chemical alternatives that can overcome issues caused by genetic intervention. These inhibitors offer the potential to dissect the roles of glucosylceramides in polyploid crop species.

The Maltase Involved in Starch Metabolism in Barley Endosperm Is Encoded by a Single Gene.

During germination and early seedling growth of barley (Hordeum vulgare), maltase is responsible for the conversion of maltose produced by starch degradation in the endosperm to glucose for seedling growth. Despite the potential relevance of this enzyme for malting and the production of alcoholic beverages, neither the nature nor the role of maltase is fully understood. Although only one gene encoding maltase has been identified with certainty, there is evidence for the existence of other genes and for multiple forms of the enzyme. It has been proposed that maltase may be involved directly in starch granule degradation as well as in maltose hydrolysis. The aim of our work was to discover the nature of maltase in barley endosperm. We used ion exchange chromatography to fractionate maltase activity from endosperm of young seedlings, and we partially purified activity for protein identification. We compared maltase activity in wild-type barley and transgenic lines with reduced expression of the previously-characterised maltase gene Agl97, and we used genomic and transcriptomic information to search for further maltase genes. We show that all of the maltase activity in the barley endosperm can be accounted for by a single gene, Agl97. Multiple forms of the enzyme most likely arise from proteolysis and other post-translational modifications.

Cell wall degradation is required for normal starch mobilisation in barley endosperm.

Starch degradation in barley endosperm provides carbon for early seedling growth, but the control of this process is poorly understood. We investigated whether endosperm cell wall degradation is an important determinant of the rate of starch degradation. We identified iminosugar inhibitors of enzymes that degrade the cell wall component arabinoxylan. The iminosugar 1,4-dideoxy-1, 4-imino-l-arabinitol (LAB) inhibits arabinoxylan arabinofuranohydrolase (AXAH) but does not inhibit the main starch-degrading enzymes α- and ß-amylase and limit dextrinase. AXAH activity in the endosperm appears soon after the onset of germination and resides in dimers putatively containing two isoforms, AXAH1 and AXAH2. Upon grain imbibition, mobilisation of arabinoxylan and starch spreads across the endosperm from the aleurone towards the crease. The front of arabinoxylan degradation precedes that of starch degradation. Incubation of grains with LAB decreases the rate of loss of both arabinoxylan and starch, and retards the spread of both degradation processes across the endosperm. We propose that starch degradation in the endosperm is dependent on cell wall degradation, which permeabilises the walls and thus permits rapid diffusion of amylolytic enzymes. AXAH may be of particular importance in this respect. These results provide new insights into the mobilization of endosperm reserves to support early seedling growth.

Isolation and characterisation of phytase from dormant Corylus avellana seeds.

Phytase (myo-inositol-1,2,3,4,5,6-hexakisphosphate phosphohydrolase, EC 3.1.3.26), which catalyses the step-wise hydrolysis of phytic acid, was purified from cotyledons of dormant Corylus avellana L. seeds. The enzyme was separated from the major soluble acid phosphatase by successive (NH4)(2)SO(4) precipitation, gel filtration and cation exchange chromatography resulting in a 300-fold purification and yield of 7.5%. The native enzyme positively interacted with Concanavalin A suggesting that it is putatively glycosylated. After size exclusion chromatography and SDS-PAGE it was found to be a monomeric protein with molecular mass 72+/-2.5 kDa. The hazel enzyme exhibited optimum activity for phytic acid hydrolysis at pH 5 and, like other phytases, had broad substrate specificity. It exhibited the lowest Km (162 microM) and highest specificity constant (V(max)/Km) for phytic acid, indicating that this is the preferred in vivo substrate. It required no metal ion as a co-factor, while inorganic phosphate and fluoride competitively inhibited enzymic activity (Ki=407 microM and Ki=205 microM, respectively).

The Pto kinase of tomato, which regulates plant immunity, is repressed by its myristoylated N terminus.

Specific recognition of the Pseudomonas syringae effector proteins AvrPto and AvrPtoB in tomato is mediated by Pto kinase resulting in induction of defense responses, including hypersensitive cell death via a signaling pathway requiring the nucleotide-binding leucine-rich repeats protein Prf. Pto is a myristoylated protein, and N-myristoylation is required for signaling. Here we demonstrated a role for N-myristoylation in controlling Pto kinase activity. A myristoylated peptide corresponding to Pto residues 2-10 significantly impaired the kinase activity of N-truncated Pto. We show that kinase inhibition was specific to the myristoylated form of the peptide and that free myristate supplied in trans was a potent suppressor of Pto kinase activity. Thus, myristate, but not Pto residues 2-10, contributes to suppression of kinase activity in vitro. Accordingly, elimination of the in vivo myristoylation potential of Pto de-repressed kinase activity. The increased potency of free myristate relative to the myristoylated N-peptide inhibitor suggested that the peptide moiety is antagonistic to repression by myristate. Suppression of related protein kinases by myristate declined with similarity to Pto, and the inhibitory activity could be attributed to hydrophobicity. We present evidence that inhibition of Pto by the myristoylated N-peptide is mediated through a previously identified surface regulatory patch. The data show a role for negative regulation of Pto by N-myristoylation, in addition to the previously demonstrated positive role, and are consistent with a model in which the acylated N terminus is sequestered in the catalytic cleft prior to release by Pto activation.

Tomato Pto encodes a functional N-myristoylation motif that is required for signal transduction in Nicotiana benthamiana.

Pto kinase of tomato (Lycopersicon esculentum) confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato expressing avrPto or avrPtoB. Pto interacts directly with these type-III secreted effectors, leading to induction of defence responses including the hypersensitive response (HR). Signalling by Pto requires the nucleotide-binding site-leucine-rich repeat (NBS-LRR) protein Prf. Little is known of how Pto is controlled prior to or during stimulation, although kinase activity is required for Avr-dependent activation. Here we demonstrate a role for the N-terminus in signalling by Pto. N-terminal residues outside the kinase domain were required for induction of the HR in Nicotiana benthamiana. The N-terminus also contributed to both AvrPto-binding and phosphorylation abilities. Pto residues 1-10 comprise a consensus motif for covalent attachment of myristate, a hydrophobic 14-carbon saturated fatty acid, to the Gly-2 residue. Several lines of evidence indicate that this motif is important for Pto function. A heterologous N-myristoylation motif complemented N-terminal deletion mutants of Pto for Prf-dependent signalling. Signalling by wild-type and mutant forms of Pto was strictly dependent on the Gly-2 residue. The N-myristoylation motif of Pto complemented the cognate motif of AvrPto for avirulence function and membrane association. Furthermore, Pto was myristoylated in vivo dependent on the presence of Gly-2. The subcellular localization of Pto was independent of N-myristoylation, indicating that N-myristoylation is required for some function other than membrane affinity. Consistent with this idea, AvrPtoB was also found to be a soluble protein. The data indicate an important role(s) for the myristoylated N-terminus in Pto signalling.

The tomato NBARC-LRR protein Prf interacts with Pto kinase in vivo to regulate specific plant immunity.

Immunity in tomato (Solanum lycopersicum) to Pseudomonas syringae bacteria expressing the effector proteins AvrPto and AvrPtoB requires both Pto kinase and the NBARC-LRR (for nucleotide binding domain shared by Apaf-1, certain R gene products, and CED-4 fused to C-terminal leucine-rich repeats) protein Prf. Pto plays a direct role in effector recognition within the host cytoplasm, but the role of Prf is unknown. We show that Pto and Prf are coincident in the signal transduction pathway that controls ligand-independent signaling. Pto and Prf associate in a coregulatory interaction that requires Pto kinase activity and N-myristoylation for signaling. Pto interacts with a unique Prf N-terminal domain outside of the NBARC-LRR domain and resides in a high molecular weight recognition complex dependent on the presence of Prf. In this complex, both Pto and Prf contribute to specific recognition of AvrPtoB. The data suggest that the role of Pto is confined to the regulation of Prf and that the bacterial effectors have evolved to target this coregulatory molecular switch.

Phytic acid mobilization is an early response to chilling of the embryonic axes from dormant oilseed of hazel (Corylus avellana).

Dormancy of hazel (Corylus avellana L.) seeds is alleviated by a chilling treatment during which cytological, hormonal, and biochemical changes occur. Phytic acid and phosphate mobilization have been examined during this treatment. Phytic acid accounted for 0.7% and up to 3.2% of dry weight in axiferous and cotyledonary tissue, respectively. Phytic acid levels in embryonic axes were reduced by 60% within the first 3 weeks of chilling, with little subsequent change, in contrast to warm-imbibed tissue where levels did not change significantly. In cotyledons, phytic acid was mobilized to a lesser extent. Phosphate levels expressed on a fresh weight basis remained almost unaltered suggesting either the operation of a homeostatic mechanism for intracellular concentration or rapid utilization due to active metabolism. Phytase activity increased during stratification in both axiferous and cotyledonary tissue. The initial rise observed was associated with dormancy alleviation, since it occurred before the realization of full germination potential by the seeds and not in warm-imbibed tissue. Protein bodies were isolated from hazel seeds by non-aqueous density gradients. Phytase activity was closely associated with the purified organelles, where phytic acid was located by light microscopy. Overall, these findings suggest that phytic acid mobilization by phytase and previously described processes associated with protein bodies, such as considerable proteolysis, are early participants in the plethora of events leading to seed dormancy relief and germination in hazel.

Isolation and partial characterisation of acid phosphatase isozymes from dormant oilseed of Corylus avellana L.

The acid phosphatase (orthophosphoric-monoester phosphohydrolase, EC 3.1.3.2) complement from dormant hazel (Corylus avellana L.) seeds was found to exhibit significant electrophoretic heterogeneity partially attributable to the presence of distinct molecular forms. In axiferous tissue, total acid phosphatase activity increased in a biphasic fashion during chilling, a treatment necessary to alleviate seed dormancy. Three acid phosphatase isozymes were isolated from cotyledons of dormant hazel seeds by successive ammonium sulphate precipitation, size-exclusion, Concanavalin A affinity, cation- and anion-exchange chromatographies resulting in 75-, 389- and 191-fold purification (APase1, APase2, APase3, respectively). The three glycosylated isoforms were isolated to catalytic homogeneity as determined by electrophoretic, kinetic and heat-inactivation studies. The native acid phosphatase complement of hazel seeds had an apparent Mr of 81.5 +/- 3.5 kDa as estimated by size-exclusion chromatography, while the determined pI values were 5.1 (APase1), 6.9 (APase2) and 7.3 (APase3). The optimum pH for p-nitrophenyl phosphate hydrolysis was pH 3 (APase1), pH 5.6 (APase2) and pH 6 (APase3). The hazel isozymes hydrolysed a variety of phosphorylated substrates in a non-specific manner, exhibiting low Km and the highest specificity constant (Vmax/ Km) for pyrophosphate. They were not primary phytases since they could not initiate phytic acid hydrolysis, while APase2 and APase3 had significant phospho-tyrosine phosphatase activity. Inorganic phosphate was a competitive inhibitor, while activity was significantly impaired in the presence of vanadate and fluoride.

A patch of surface-exposed residues mediates negative regulation of immune signaling by tomato Pto kinase.

Tomato (Lycopersicon esculentum) Pto kinase specifically recognizes the Pseudomonas effector proteins AvrPto and AvrPtoB, leading to induction of defense responses and hypersensitive cell death. Structural modeling of Pto combined with site-directed mutagenesis identified a patch of surface-exposed residues required for native regulation of signaling. Mutations in this area resulted in constitutive gain-of-function (CGF) forms of Pto that activated AvrPto-independent cell death via the cognate signaling pathway. The patch overlaps the peptide binding region of the kinase catalytic cleft and is part of a broader region required for interaction with bacterial effectors. We propose that the negative regulatory patch is normally occupied by a peptide that represses Pto signaling. Furthermore, we found that Pto kinase activity was required for Avr-dependent activation but dispensable for signaling by CGF forms of Pto. This suggests that Pto signals by a conformational change rather than phosphorylation of downstream substrates in the defense signaling pathway.