Vasilios M. E. Andriotis

12-Oxo-Phytodienoic Acid Accumulation during Seed Development Represses Seed Germination in Arabidopsis

This work reports the unexpected discovery that levels of the oxylipins OPDA, jasmonic acid (JA), and JA-Ile are elevated in mutant seeds that are disrupted in peroxisomal fatty acid metabolism and compromised in germination. It demonstrates that OPDA, rather than JA, works synergistically with ABA as a key regulator of germination. Arabidopsis thaliana COMATOSE (CTS) encodes an ABC transporter involved in peroxisomal import of substrates for β-oxidation. Various cts alleles and mutants disrupted in steps of peroxisomal β-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.

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.

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.

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.

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 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 nucleotidebinding 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.

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 β-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.

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.

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.

Small-Molecule Probes of Plant Glycopolymer Metabolism

Small-molecule probes can be used in conjunction with traditional genetic and biochemical methods to gain a better understanding of metabolic processes. Chemical genetics has been significantly utilized in the areas of plant hormones and herbicide development; chemical probes have become established tools for the dissection of signaling pathways and for agrochemical applications. Herein we discuss the use of small-molecule probes in the field of plant carbohydrate research, with particular focus on noncovalent probes. The use of direct inhibitors such as iminosugars, metabolic incorporation of labels, and indirect inhibition of carbohydrate processing have helped provide insight into key areas of plant carbohydrate metabolism, including starch, cell wall, glycoprotein and glycolipid synthesis, degradation, and spatial organization.