Ataxia telangiectasia-mutated and Rad3-related (ATR) plays a central role in cell-cycle regulation, transmitting DNA damage signals to downstream effectors of cell-cycle progression. In animals, ATR is an essential gene. Here, we find that Arabidopsis (Arabidopsis thaliana) atrÿ/ÿ mutants were viable, fertile, and phenotypically wild-type in the absence of exogenous DNA damaging agents but exhibit altered expression of AtRNR1 (ribonucleotide reductase large subunit) and alteration of some damage-induced cell-cycle checkpoints. atr mutants were hypersensitive to hydroxyurea (HU), aphidicolin, and UV-B light but only mildly sensitive to g-radiation. G2 arrest was observed in response to g-irradiation in both wild-type and atr plants, albeit with slightly different kinetics, suggesting that ATR plays a secondary role in response to double-strand breaks. G2 arrest also was observed in wild-type plants in response to aphidicolin but was defective in atr mutants, resulting in compaction of nuclei and subsequent cell death. By contrast, HU-treated wild-type and atr plants arrested in G1 and showed no obvious signs of cell death. We propose that, in plants, HU invokes a novel checkpoint responsive to low levels of deoxynucleotide triphosphates. These results demonstrate the important role of cell-cycle checkpoints in the ability of plant cells to sense and cope with problems associated with DNA replication.

SummaryDespite the recent discovery that trehalose synthesis is widespread in higher plants very little is known about its physiological signi®cance. Here we report on an Arabidopsis mutant (tps1), disrupted in a gene encoding the ®rst enzyme of trehalose biosynthesis (trehalose-6-phosphate synthase). The tps1 mutant is a recessive embryo lethal. Embryo morphogenesis is normal but development is retarded and stalls early in the phase of cell expansion and storage reserve accumulation. TPS1 is transiently up-regulated at this same developmental stage and is required for the full expression of seed maturation marker genes (2S2 and OLEOSN2). Sucrose levels also increase rapidly in seeds during the onset of cell expansion. In Saccharomyces cerevisiae trehalose-6-phosphate (T-6-P) is required to regulate sugar in¯ux into glycolysis via the inhibition of hexokinase and a de®ciency in TPS1 prevents growth on sugars (Thevelein and Hohmann, 1995). The growth of Arabidopsis tps1±1 embryos can be partially rescued in vitro by reducing the sucrose level. However, T-6-P is not an inhibitor of AtHXK1 or AtHXK2. Nor does reducing hexokinase activity rescue tps1±1 embryo growth. Our data establish for the ®rst time that an enzyme of trehalose metabolism is essential in plants and is implicated in the regulation of sugar metabolism/embryo development via a different mechanism to that reported in S. cerevisiae.

In contrast to yeast or mammalian cells, little is known about the signaling responses to DNA damage in plants. We previously characterized AtATM , an Arabidopsis homolog of the human ATM gene, which is mutated in ataxia telangiectasia, a chromosome instability disorder. The Atm protein is a protein kinase whose activity is induced by DNA damage, particularly DNA double-strand breaks. The phosphorylation targets of Atm include proteins involved in DNA repair, cell cycle control, and apoptosis. Here, we describe the isolation and functional characterization of two Arabidopsis mutants carrying a T-DNA insertion in AtATM . Arabidopsis atm mutants are hypersensitive to ␥ -radiation and methylmethane sulfonate but not to UV-B light. In correlation with the radiation sensitivity, atm mutants failed to induce the transcription of genes involved in the repair and/or detection of DNA breaks upon irradiation. In addition, atm mutants are partially sterile, and we show that this effect is attributable to abundant chromosomal fragmentation during meiosis. Interestingly, the transcription of DNA recombination genes during meiosis was not dependent on AtATM , and meiotic recombination occurred at the same rate as in wild-type plants, raising questions about the function of AtAtm during meiosis in plants. Our results demonstrate that AtATM plays a central role in the response to both stress-induced and developmentally programmed DNA damage.

In the wild tomato Solanum habrochaites, the Sst2 locus on chromosome 8 is responsible for the biosynthesis of several class II sesquiterpene olefins by glandular trichomes. Analysis of a trichome-specific EST collection from S. habrochaites revealed two candidate genes for the synthesis of Sst2-associated sesquiterpenes. zFPS encodes a protein with homology to Z-isoprenyl pyrophosphate synthases and SBS (for Santalene and Bergamotene Synthase) encodes a terpene synthase with homology to kaurene synthases. Both genes were found to cosegregate with the Sst2 locus. Recombinant zFPS protein catalyzed the synthesis of Z,Z-FPP from isopentenylpyrophosphate (IPP) and dimethylallylpyrophosphate (DMAPP), while coincubation of zFPS and SBS with the same substrates yielded a mixture of olefins identical to the Sst2-associated sesquiterpenes, including (+)-a-santalene, (+)-endo-b-bergamotene, and (2)-endo-a-bergamotene. In addition, headspace analysis of tobacco (Nicotiana sylvestris) plants expressing zFPS and SBS in glandular trichomes afforded the same mix of sesquiterpenes. Each of these proteins contains a putative plastid targeting sequence that mediates transport of a fused green fluorescent protein to the chloroplasts, suggesting that the biosynthesis of these sesquiterpenes uses IPP and DMAPP from the plastidic DXP pathway. These results provide novel insights into sesquiterpene biosynthesis and have general implications concerning sesquiterpene engineering in plants.

A new system for insertional mutagenesis based on the maize Enhancer/Suppressor-mutator ( En/Spm ) element was introduced into Arabidopsis. A single T-DNA construct carried a nonautonomous defective Spm (d Spm ) element with a phosphinothricin herbicide resistance ( BAR ) gene, a transposase expression cassette, and a counterselectable gene. This construct was used to select for stable d Spm transpositions. Treatments for both positive ( BAR ) and negative selection markers were applicable to soil-grown plants, allowing the recovery of new transpositions on a large scale. To date, a total of 48,000 lines in pools of 50 have been recovered, of which ‫ف‬ 80% result from independent insertion events. DNA extracted from these pools was used in reverse genetic screens, either by polymerase chain reaction (PCR) using primers from the transposon and the targeted gene or by the display of insertions whereby inverse PCR products of insertions from the DNA pools are spotted on a membrane that is then hybridized with the probe of interest. By sequencing PCR-amplified fragments adjacent to insertion sites, we established a sequenced insertion-site database of 1200 sequences. This database permitted a comparison of the chromosomal distribution of transpositions from various T-DNA locations.

SummaryInventors in the field of mechanical and electronic engineering can access multitudes of components and, thanks to standardization, parts from different manufacturers can be used in combination with each other. The introduction of BioBrick standards for the assembly of characterized DNA sequences was a landmark in microbial engineering, shaping the field of synthetic biology. Here, we describe a standard for Type IIS restriction endonuclease-mediated assembly, defining a common syntax of 12 fusion sites to enable the facile assembly of eukaryotic transcriptional units. This standard has been developed and agreed by representatives and leaders of the international plant science and synthetic biology communities, including inventors, developers and adopters of Type IIS cloning methods. Our vision is of an extensive catalogue of standardized, characterized DNA parts that will accelerate plant bioengineering.

Tomato breeding has been tremendously efficient in increasing fruit quality and quantity but did not focus on improving herbivore resistance. The biosynthetic pathway for the production of 7-epizingiberene in a wild tomato was introduced into a cultivated greenhouse variety with the aim to obtain herbivore resistance. 7-Epizingiberene is a specific sesquiterpene with toxic and repellent properties that is produced and stored in glandular trichomes. We identified 7-epizingiberene synthase (ShZIS) that belongs to a new class of sesquiterpene synthases, exclusively using Z-Z-farnesyl-diphosphate (zFPP) in plastids, probably arisen through neo-functionalization of a common ancestor. Expression of the ShZIS and zFPP synthases in the glandular trichomes of cultivated tomato resulted in the production of 7-epizingiberene. These tomatoes gained resistance to several herbivores that are pests of tomato. Hence, introduction of this sesquiterpene biosynthetic pathway into cultivated tomatoes resulted in improved herbivore resistance.metabolic engineering | terpenoid | white fly | spider mite | promoter H erbivorous insects pose serious problems in agricultural production areas. On commercial tomato, whiteflies, spider mites, and aphids are major pests, not only by the feeding damage they cause, but also because they transmit devastating viruses that can cause losses up to 100% (1). Production of tomato, a crop with considerable economic importance, reached 145,751,507 tons worldwide in 2010, representing a value of 53.3 billion dollars (www.fao.org). In general, wild tomatoes are far less attractive to pests than cultivated tomatoes due to an elevated or qualitatively different production of an array of defense compounds such as alkaloids, phenolic compounds, and terpenes.Toxic and repellent compounds are mostly produced in glandular trichomes, autonomous epidermal protrusions specialized in efficient production, storage, and release of defense compounds (2). These glands have long been regarded as economically important and challenging targets for bioengineering (2). Confining metabolic changes to trichomes ensures that the plant metabolism and crop yield remains unaffected.Terpenes constitute the largest and most diverse class of plantproduced secondary compounds that maintain a variety of biological functions and applications, including anticancer (taxol) and antimalarial (artemisin) drugs, in flavor and fragrance industry, and for crop improvement through enhanced pest resistance. Wild tomato sesquiterpenes and derivatives have been implicated in defense against herbivores (3-5). Sesquiterpenes are C 15 -terpenes predominantly derived from precursors of the cytosolic mevalonate pathway where the C 5 -isoprene units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) are assembled into C 15 E-E-farnesyl diphosphate (FPP) by FPP synthase (FPS) and converted by specific sesquiterpene synthases. However, it was recently shown that some wild tomato species contain a sesquiterpene pathway that is confined to ...

Glandular trichomes are metabolic cell factories with the capacity to produce large quantities of secondary metabolites. Little is known about the connection between central carbon metabolism and metabolic productivity for secondary metabolites in glandular trichomes. To address this gap in our knowledge, we performed comparative metabolomics, transcriptomics, proteomics, and 13 C-labeling of type VI glandular trichomes and leaves from a cultivated (Solanum lycopersicum LA4024) and a wild (Solanum habrochaites LA1777) tomato accession. Specific features of glandular trichomes that drive the formation of secondary metabolites could be identified. Tomato type VI trichomes are photosynthetic but acquire their carbon essentially from leaf sucrose. The energy and reducing power from photosynthesis are used to support the biosynthesis of secondary metabolites, while the comparatively reduced Calvin-Benson-Bassham cycle activity may be involved in recycling metabolic CO 2 . Glandular trichomes cope with oxidative stress by producing high levels of polyunsaturated fatty acids, oxylipins, and glutathione. Finally, distinct mechanisms are present in glandular trichomes to increase the supply of precursors for the isoprenoid pathways. Particularly, the citrate-malate shuttle supplies cytosolic acetyl-CoA and plastidic glycolysis and malic enzyme support the formation of plastidic pyruvate. A model is proposed on how glandular trichomes achieve high metabolic productivity.