Plant Biotechnology Institute

Over 225,000 independent Agrobacterium transferred DNA (T-DNA) insertion events in the genome of the reference plant Arabidopsis thaliana have been created that represent near saturation of the gene space. The precise locations were determined for more than 88,000 T-DNA insertions, which resulted in the identification of mutations in more than 21,700 of the approximately 29,454 predicted Arabidopsis genes. Genome-wide analysis of the distribution of integration events revealed the existence of a large integration site bias at both the chromosome and gene levels. Insertion mutations were identified in genes that are regulated in response to the plant hormone ethylene.

An annotated reference sequence representing the hexaploid bread wheat genome in 21 pseudomolecules has been analyzed to identify the distribution and genomic context of coding and noncoding elements across the A, B, and D subgenomes. With an estimated coverage of 94% of the genome and containing 107,891 high-confidence gene models, this assembly enabled the discovery of tissue- and developmental stage-related coexpression networks by providing a transcriptome atlas representing major stages of wheat development. Dynamics of complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. This community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding.

Abscisic acid (ABA) regulates numerous developmental processes and adaptive stress responses in plants. Many ABA signaling components have been identified, but their interconnections and a consensus on the structure of the ABA signaling network have eluded researchers. Recently, several advances have led to the identification of ABA receptors and their three-dimensional structures, and an understanding of how key regulatory phosphatase and kinase activities are controlled by ABA. A new model for ABA action has been proposed and validated, in which the soluble PYR/PYL/RCAR receptors function at the apex of a negative regulatory pathway to directly regulate PP2C phosphatases, which in turn directly regulate SnRK2 kinases. This model unifies many previously defined signaling components and highlights the importance of future work focused on defining the direct targets of SnRK2s and PP2Cs, dissecting the mechanisms of hormone interactions (i.e., cross talk) and defining connections between this new negative regulatory pathway and other factors implicated in ABA signaling.

The Bioperl project is an international open-source collaboration of biologists, bioinformaticians, and computer scientists that has evolved over the past 7 yr into the most comprehensive library of Perl modules available for managing and manipulating life-science information. Bioperl provides an easy-to-use, stable, and consistent programming interface for bioinformatics application programmers. The Bioperl modules have been successfully and repeatedly used to reduce otherwise complex tasks to only a few lines of code. The Bioperl object model has been proven to be flexible enough to support enterprise-level applications such as EnsEMBL, while maintaining an easy learning curve for novice Perl programmers. Bioperl is capable of executing analyses and processing results from programs such as BLAST, ClustalW, or the EMBOSS suite. Interoperation with modules written in Python and Java is supported through the evolving BioCORBA bridge. Bioperl provides access to data stores such as GenBank and SwissProt via a flexible series of sequence input/output modules, and to the emerging common sequence data storage format of the Open Bioinformatics Database Access project. This study describes the overall architecture of the toolkit, the problem domains that it addresses, and gives specific examples of how the toolkit can be used to solve common life-sciences problems. We conclude with a discussion of how the open-source nature of the project has contributed to the development effort. [Supplemental material is available online at www.genome.org . Bioperl is available as open-source software free of charge and is licensed under the Perl Artistic License ( http://www.perl.com/pub/a/language/misc/Artistic.html ). It is available for download at http://www.bioperl.org . Support inquiries should be addressed to bioperl-l@bioperl.org .]

Xie and colleagues previously isolated the Arabidopsis COI1 gene that is required for response to jasmonates (JAs), which regulate root growth, pollen fertility, wound healing, and defense against insects and pathogens. In this study, we demonstrate that COI1 associates physically with AtCUL1, AtRbx1, and either of the Arabidopsis Skp1-like proteins ASK1 or ASK2 to assemble ubiquitin-ligase complexes, which we have designated SCF(COI1). COI1(E22A), a single amino acid substitution in the F-box motif of COI1, abolishes the formation of the SCF(COI1) complexes and results in loss of the JA response. AtRbx1 double-stranded RNA-mediated genetic interference reduces AtRbx1 expression and affects JA-inducible gene expression. Furthermore, we show that the AtCUL1 component of SCF(COI1) complexes is modified in planta, where mutations in AXR1 decrease the abundance of the modified AtCUL1 of SCF(COI1) and lead to a reduction in JA response. Finally, we demonstrate that the axr1 and coi1 mutations display a synergistic genetic interaction in the double mutant. These results suggest that the COI1-mediated JA response is dependent on the SCF(COI1) complexes in Arabidopsis and that the AXR1-dependent modification of the AtCUL1 subunit of SCF(COI1) complexes is important for JA signaling.

BACKGROUND: Cannabis sativa has been cultivated throughout human history as a source of fiber, oil and food, and for its medicinal and intoxicating properties. Selective breeding has produced cannabis plants for specific uses, including high-potency marijuana strains and hemp cultivars for fiber and seed production. The molecular biology underlying cannabinoid biosynthesis and other traits of interest is largely unexplored. RESULTS: We sequenced genomic DNA and RNA from the marijuana strain Purple Kush using shortread approaches. We report a draft haploid genome sequence of 534 Mb and a transcriptome of 30,000 genes. Comparison of the transcriptome of Purple Kush with that of the hemp cultivar 'Finola' revealed that many genes encoding proteins involved in cannabinoid and precursor pathways are more highly expressed in Purple Kush than in 'Finola'. The exclusive occurrence of Δ9-tetrahydrocannabinolic acid synthase in the Purple Kush transcriptome, and its replacement by cannabidiolic acid synthase in 'Finola', may explain why the psychoactive cannabinoid Δ9-tetrahydrocannabinol (THC) is produced in marijuana but not in hemp. Resequencing the hemp cultivars 'Finola' and 'USO-31' showed little difference in gene copy numbers of cannabinoid pathway enzymes. However, single nucleotide variant analysis uncovered a relatively high level of variation among four cannabis types, and supported a separation of marijuana and hemp. CONCLUSIONS: The availability of the Cannabis sativa genome enables the study of a multifunctional plant that occupies a unique role in human culture. Its availability will aid the development of therapeutic marijuana strains with tailored cannabinoid profiles and provide a basis for the breeding of hemp with improved agronomic characteristics.

The Arabidopsis NPR1 gene is essential in activating systemic, inducible plant defense responses. To gain a better understanding of NPR1 function, we conducted a yeast two-hybrid screening procedure and identified a differential interaction between NPR1 and all known members of the Arabidopsis TGA family of basic leucine zipper transcription factors. In the electrophoretic mobility shift assay, NPR1 substantially increased the binding of TGA2 to its cognate promoter element (as-1) as well as to a positive salicylic acid-inducible element (LS7) and a negative element (LS5) in the promoter of the pathogenesis-related PR-1 gene. Proteins encoded by npr1 mutants interacted poorly with TGA2 and did not substantially increase TGA2 binding to the as-1, LS5, or LS7 elements, thus establishing a link between the loss of disease resistance and the loss of TGA2 interaction and NPR1-enhanced DNA binding. Coupled with observations that the DNA binding activity of TGA factors is deregulated in npr1 plants, the results suggest that NPR1-mediated DNA binding of TGA2 is critical for activation of defense genes.

The Arabidopsis NPR1 protein is essential for regulating salicylic acid-dependent gene expression during systemic acquired resistance. NPR1 interacts differentially with members of the TGA class of basic domain/Leu zipper transcription factors and regulates their DNA binding activity. Here, we report that although TGA1 does not interact with NPR1 in yeast two-hybrid assays, treatment with salicylic acid induces the interaction between these proteins in Arabidopsis leaves. This phenomenon is correlated with a reduction of TGA1 Cys residues. Furthermore, site-directed mutagenesis of TGA1 Cys-260 and Cys-266 enables the interaction with NPR1 in yeast and Arabidopsis. Together, these results indicate that TGA1 relies on the oxidation state of Cys residues to mediate the interaction with NPR1. An intramolecular disulfide bridge in TGA1 precludes interaction with NPR1, and NPR1 can only stimulate the DNA binding activity of the reduced form of TGA1. Unlike its animal and yeast counterparts, the DNA binding activity of TGA1 is not redox regulated; however, this property is conferred by interaction with the NPR1 cofactor.

There is an increasing awareness that as a result of structural variation, a reference sequence representing a genome of a single individual is unable to capture all of the gene repertoire found in the species. A large number of genes affected by presence/absence and copy number variation suggest that it may contribute to phenotypic and agronomic trait diversity. Here we show by analysis of the Brassica oleracea pangenome that nearly 20% of genes are affected by presence/absence variation. Several genes displaying presence/absence variation are annotated with functions related to major agronomic traits, including disease resistance, flowering time, glucosinolate metabolism and vitamin biosynthesis.

The potential impact of global temperature change on global crop yield has recently been assessed with different methods. Here we show that grid-based and point-based simulations and statistical regressions (from historic records), without deliberate adaptation or CO2 fertilization effects, produce similar estimates of temperature impact on wheat yields at global and national scales. With a 1◦C global temperature increase, global wheat yield is projected to decline between 4.1% and 6.4%. Projected relative temperature impacts from different methods were similar for major wheat-producing countries China, India, USA and France, but less so for Russia. Point-based and grid-based simulations, and to some extent the statistical regressions, were consistent in projecting that warmer regions are likely to suffer more yield loss with increasing temperature than cooler regions. By forming a multi-method ensemble, it was possible to quantify ‘method uncertainty’ in addition to model uncertainty. This significantly improves confidence in estimates of climate impacts on global food security.

Triacylglycerol (TAG) biosynthesis is a principal metabolic pathway in most organisms, and TAG is the major form of carbon storage in many plant seeds. Acyl-CoA:diacylglycerol acyltransferase 1 (DGAT1) is the only acyltransferase enzyme that has been confirmed to contribute to TAG biosynthesis in Arabidopsis thaliana seeds. However, dgat1 null mutants display only a 20 to 40% decrease in seed oil content. To determine whether other enzymes contribute to TAG synthesis, candidate genes were expressed in TAG-deficient yeast, candidate mutants were crossed with the dgat1-1 mutant, and target genes were suppressed by RNA interference (RNAi). An in vivo role for phospholipid:diacylglycerol acyltransferase 1 (PDAT1; At5g13640) in TAG synthesis was revealed in this study. After failing to obtain double homozygous plants from crossing dgat1-1 and pdat1-2, further investigation showed that the dgat1-1 pdat1-2 double mutation resulted in sterile pollen that lacked visible oil bodies. RNAi silencing of PDAT1 in a dgat1-1 background or DGAT1 in pdat1-1 background resulted in 70 to 80% decreases in oil content per seed and in disruptions of embryo development. These results establish in vivo involvement of PDAT1 in TAG biosynthesis, rule out major contributions by other candidate enzymes, and indicate that PDAT1 and DGAT1 have overlapping functions that are essential for normal pollen and seed development of Arabidopsis.

We recently reported the cloning and characterization of an Arabidopsis (ecotype Columbia) diacylglycerol acyltransferase cDNA (Zou et al., 1999) and found that in Arabidopsis mutant line AS11, an ethyl methanesulfonate-induced mutation at a locus on chromosome II designated as Tag1 consists of a 147-bp insertion in the DNA, which results in a repeat of the 81-bp exon 2 in the Tag1 cDNA. This insertion mutation is correlated with an altered seed fatty acid composition, reduced diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) activity, reduced seed triacylglycerol content, and delayed seed development in the AS11 mutant. The effect of the insertion mutation on microsomal acyl-coenzyme A-dependent DGAT is examined with respect to DGAT activity and its substrate specificity in the AS11 mutant relative to wild type. We demonstrate that transformation of mutant AS11 with a single copy of the wild-type Tag1 DGAT cDNA can complement the fatty acid and reduced oil phenotype of mutant AS11. More importantly, we show for the first time that seed-specific over-expression of the DGAT cDNA in wild-type Arabidopsis enhances oil deposition and average seed weight, which are correlated with DGAT transcript levels. The DGAT activity in developing seed of transgenic lines was enhanced by 10% to 70%. Thus, the current study confirms the important role of DGAT in regulating the quantity of seed triacylglycerols and the sink size in developing seeds.

The plant hormone auxin regulates diverse aspects of plant growth and development. We report that in Arabidopsis, auxin response is dependent on a ubiquitin-ligase (E3) complex called SCFTIR1. The complex consists of proteins related to yeast Skp1p and Cdc53p called ASK and AtCUL1, respectively, as well as the F-box protein TIR1. Mutations in either ASK1 or TIR1 result in decreased auxin response. Further, overexpression of TIR1 promotes auxin response suggesting that SCFTIR1 is limiting for the response. These results provide new support for a model in which auxin action depends on the regulated proteolysis of repressor proteins.

Almost 2000 drought-responsive genes were identified in Arabidopsis thaliana under progressive soil drought stress using whole-genome oligonucleotide microarrays. Most of the drought-regulated genes recovered to normal expression levels by 3 h after rewatering. It has previously been shown that the abscisic acid (ABA) analogue (+)-8'-acetylene-ABA (PBI425) hyperinduces many ABA-like changes in gene expression to reveal a more complete list of ABA-regulated genes, and it is demonstrated here that PBI425 produced a correspondingly increased drought tolerance. About two-thirds of drought-responsive genes (1310 out of 1969) were regulated by ABA and/or the ABA analogue PBI425. Analysis of promoter motifs suggests that many of the remaining drought-responsive genes may be affected by ABA signalling. Concentrations of endogenous ABA and its catabolites significantly increased under drought stress and either completely (ABA) or partially (ABA catabolites) recovered to normal levels by 3 h after rehydration. Detailed analyses of drought transcript profiles and in silico comparisons with other studies revealed that the ABA-dependent pathways are predominant in the drought stress responses. These comparisons also showed that other plant hormones including jasmonic acid, auxin, cytokinin, ethylene, brassinosteroids, and gibberellins also affected drought-related gene expression, of which the most significant was jasmonic acid. There is also extensive cross-talk between responses to drought and other environmental factors including light and biotic stresses. These analyses demonstrate that ABA-related stress responses are modulated by other environmental and developmental factors.

The COP9 signalosome is an evolutionary conserved multiprotein complex of unknown function that acts as a negative regulator of photomorphogenic seedling development in Arabidopsis. Here, we show that plants with reduced COP9 signalosome levels had decreased auxin response similar to loss-of-function mutants of the E3 ubiquitin ligase SCFTIR1. Furthermore, we found that the COP9 signalosome and SCFTIR1 interacted in vivo and that the COP9 signalosome was required for efficient degradation of PSIAA6, a candidate substrate of SCFTIR1. Thus, the COP9 signalosome may play an important role in mediating E3 ubiquitin ligase-mediated responses.

Peer reviewed: Yes

Flax (Linum usitatissimum) is an ancient crop that is widely cultivated as a source of fiber, oil and medicinally relevant compounds. To accelerate crop improvement, we performed whole-genome shotgun sequencing of the nuclear genome of flax. Seven paired-end libraries ranging in size from 300 bp to 10 kb were sequenced using an Illumina genome analyzer. A de novo assembly, comprised exclusively of deep-coverage (approximately 94× raw, approximately 69× filtered) short-sequence reads (44-100 bp), produced a set of scaffolds with N(50) =694 kb, including contigs with N(50)=20.1 kb. The contig assembly contained 302 Mb of non-redundant sequence representing an estimated 81% genome coverage. Up to 96% of published flax ESTs aligned to the whole-genome shotgun scaffolds. However, comparisons with independently sequenced BACs and fosmids showed some mis-assembly of regions at the genome scale. A total of 43384 protein-coding genes were predicted in the whole-genome shotgun assembly, and up to 93% of published flax ESTs, and 86% of A. thaliana genes aligned to these predicted genes, indicating excellent coverage and accuracy at the gene level. Analysis of the synonymous substitution rates (K(s) ) observed within duplicate gene pairs was consistent with a recent (5-9 MYA) whole-genome duplication in flax. Within the predicted proteome, we observed enrichment of many conserved domains (Pfam-A) that may contribute to the unique properties of this crop, including agglutinin proteins. Together these results show that de novo assembly, based solely on whole-genome shotgun short-sequence reads, is an efficient means of obtaining nearly complete genome sequence information for some plant species.

Land plants secrete a layer of wax onto their aerial surfaces that is essential for survival in a terrestrial environment. This wax is composed of long-chain, aliphatic hydrocarbons derived from very-long-chain fatty acids (VLCFAs). Using the Arabidopsis expressed sequence tag database, we have identified a gene, designated CUT1, that encodes a VLCFA condensing enzyme required for cuticular wax production. Sense suppression of CUT1 in transgenic Arabidopsis plants results in waxless (eceriferum) stems and siliques as well as conditional male sterility. Scanning electron microscopy revealed that this was a severe waxless phenotype, because stems of CUT1-suppressed plants were completely devoid of wax crystals. Furthermore, chemical analyses of waxless plants demonstrated that the stem wax load was reduced to 6 to 7% of wild-type levels. This value is lower than that reported for any of the known eceriferum mutants. The severe waxless phenotype resulted from the downregulation of both the decarbonylation and acyl reduction wax biosynthetic pathways. This result indicates that CUT1 is involved in the production of VLCFA precursors used for the synthesis of all stem wax components in Arabidopsis. In CUT1-suppressed plants, the C24 chain-length wax components predominate, suggesting that CUT1 is required for elongation of C24 VLCFAs. The unique wax composition of CUT1-suppressed plants together with the fact that the location of CUT1 on the genetic map did not coincide with any of the known ECERIFERUM loci suggest that we have identified a novel gene involved in wax biosynthesis. CUT1 is currently the only known gene with a clearly established function in wax production.

Cyclin-dependent kinase (CDK) inhibitor genes encode low molecular weight proteins which have important functions in cell cycle regulation, development and perhaps also in tumorigenesis. The first plant CDK inhibitor gene ICK1 was recently identified from Arabidopsis thaliana. Although the C-terminal domain of ICK1 contained an important consensus sequence with the mammalian CDK inhibitor p27Kip1, the remainder of the deduced ICK1 sequence showed little similarity to any known CDK inhibitors. In vitro assays showed that recombinant ICK1 exhibited unique kinase inhibitory properties. In the present study we characterized ICK1 in terms of its gene structure, its interaction with both A. thaliana Cdc2a and CycD3, and its induction by the plant growth regulator, abscisic acid (ABA). ICK1 was expressed at a relatively low level in the tissues surveyed. However, ICK1 was induced by ABA, and along with ICK1 induction there was a decrease in Cdc2-like histone H1 kinase activity. These results suggest a molecular mechanism by which plant cell division might be inhibited by ABA. ICK1 clones were also identified from independent yeast two-hybrid screens using the CycD3 construct. The implication that ICK1 protein could interact with both Cdc2a and CycD3 was confirmed by in vitro binding assays. Furthermore, deletion analysis indicated that different regions of ICK1 are required for the interactions with Cdc2a and CycD3. These results provide a mechanistic basis for understanding the role of CDK inhibitors in cell cycle regulation in plant cells.

Artemisinin, a sesquiterpene lactone endoperoxide derived from the plant Artemisia annua, forms the basis of the most important treatments of malaria in use today. In an effort to elucidate the biosynthesis of artemisinin, an expressed sequence tag approach to identifying the relevant biosynthetic genes was undertaken using isolated glandular trichomes as a source of mRNA. A cDNA clone encoding a cytochrome P450 designated CYP71AV1 was characterized by expression in Saccharomyces cerevisiae and shown to catalyze the oxidation of the proposed biosynthetic intermediates amorpha-4,11-diene, artemisinic alcohol and artemisinic aldehyde. The identification of the CYP71AV1 gene should allow for the engineering of semi-synthetic production of artemisinin in appropriate plant or microbial hosts.