Celebrating 20 Years of The Plant Journal
Since its inception twenty years ago, The Plant Journal has developed into one of the premier journals in the basic plant sciences. During this period, plant molecular biology has come of age, plant genomics has emerged as a new driving force for discovery, and work on model species such as Arabidopsis and rice has shaped our understanding of basic plant functions. The papers published in The Plant Journal bear witness to these developments in the plant sciences, and to highlight this we have decided to assemble a ―virtual‖ special issue that brings together some of the highest impact papers published in The Plant Journal.
Virtual Issue | 20th Anniversary | July 2011 www.theplantjournal.com Introduction Introduction Celebrating 20 years of The Plant journal Christoph Benning Since its inception twenty years ago, The Plant Journal has developed into one of the premier journals in the basic plant sciences. During this period, plant molecular biology has come of age, plant genomics has emerged as a new driving force for discovery, and work on model species such as Arabidopsis and rice has shaped our understanding of basic plant functions. The papers published in The Plant Journal bear witness to these developments in the plant sciences, and to highlight this we have decided to assemble a virtual special issue that brings together some of the highest impact papers published in The Plant Journal. For simplicity, we have chosen to focus on the most highly cited primary research articles in each year, as well as the five most highly cited Technical Advance papers published in The Plant Journal. Of course, citation alone is but one measure of impact. Thus, the collection of papers presented here does not necessarily provide a representative cross section of the many papers describing fundamental discoveries that The Plant Journal has published during its twenty-year history. Nevertheless, it is certainly a collection of classic works in the plant sciences, demonstrating the progress made in our discipline in recent times. The Plant Journal is proud to have been a key publication for the dissemination of this important research, and as new trends continue to emerge over the next twenty years, the editors and staff are ready to adjust, provide the appropriate venue for those at the cutting edge, and to build upon its already very strong reputation as one of the leading journals in the field. Read Online Technical Advance Floral dip: a simplified method forAgrobacterium-mediated transformation ofArabidopsis thaliana Steven J. Clough and Andrew F. Bent The Agrobacterium vacuum infiltration method has made it possible to transform Arabidopsis thaliana without plant tissue culture or regeneration. In the present study, this method was evaluated and a substantially modified transformation method was developed. The laborintensive vacuum infiltration process was eliminated in favor of simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77. Sucrose and surfactant were critical to the success of the floral dip method. Plants inoculated when numerous immature floral buds and few siliques were present produced transformed progeny at the highest rate. Plant tissue culture media, the hormone benzylamino purine and pH adjustment were unnecessary, and Agrobacterium could be applied to plants at a range of cell densities. Repeated application ofAgrobacterium improved transformation rates and overall yield of transformants approximately twofold. Covering plants for 1 day to retain humidity after inoculation also raised transformation rates twofold. Multiple ecotypes were transformable by this method. The modified method should facilitate high-throughput transformation of Arabidopsis for efforts such as T-DNA gene tagging, positional cloning, or attempts at targeted gene replacement. Read Online Technical Advance Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA Yukoh Hiei, Shozo Ohta, Toshihiko Komari andTakashi Kumashiro A large number of morphologically normal, fertile, transgenic rice plants were obtained by co-cultivation of rice tissues with Agrobacterium tumefaciens. The efficiency of transformation was similar to that obtained by the methods used routinely for transformation of dicotyledons with the bacterium. Stable integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis of transformants in the R0, R1 and R2 generations. Sequence analysis revealed that the boundaries of the T-DNA in transgenic rice plants were essentially identical to those in transgenic dicotyledons. Calli induced from scutella were very good starting materials. A strain of A. tumefaciens that carried a so-called super-binary` vector gave especially high frequencies of transformation of various cultivars of japonica rice that included Koshihikari, which normally shows poor responses in tissue culture. Read Online Technical Advance A procedure for mapping Arabidopsis mutations using co-dominant ecotypespecific PCR-based markers Andrzej Konieczny and Frederick M. Ausubel A set of mapping markers have been designed for Arabidopsis thaliana that correspond to DNA fragments amplifed by the polymerase chain reaction (PCR). The ecotype of origin of these amplified fragments can be determined by cleavage with a restriction endo-nuclease. Specifically, 18 sets of PCR primers were synthesized, each of which amplifies a single mapped DNA sequence from the Columbia and Landsberg erecta ecotypes. Also identifed was at least one restriction endonuclease for each of these PCR products that generates ecotype-specific digestion patterns. Using these co-dominant cleaved amplified polymorphic sequences (CAPS), an Arabidopsis gene can be unambiguously mapped to one of the 10 Arabidopsis chromosome arms in a single cross using a limited number of F2 progeny. Read Online Technical Advance Efficient isolation and mapping of Arabidopsis thaliana T-DNA insert junctions by thermal asymmetric interlaced PCR Yao-Guang Liu, Norihiro Mitsukawa,Teruko Oosumi and Robert F. Whittier Thermal asymmetric interlaced (TAIL-) PCR is an efficient technique for amplifying insert ends from yeast artificial chromosome (YAC) and P1 clones. Highly specific amplification is achieved without resort to complex manipulations before or after PCR. The adaptation of this method for recovery and mapping of genomic sequences flanking T-DNA insertions in Arabidopsis thaliana is described. Insertion-specific products were amplified from 183 of 190 tested T-DNA insertion lines. Reconstruction experiments indicate that the technique can recover single-copy sequences from genomes as complex as common wheat (1.5 � 1010 bp). RFLPs were screened using 122 unique flanking sequence probes, and the insertion sites of 26 T-DNA transgenic lines were determined on an RFLP map. These lines, whose mapped T-DNA insertions confer hygromycin resistance, can be used for fine-scale mapping of linked phenotypic loci. Read Online Technical Advance Construction of integrated genetic linkage maps by means of a new computer package: Join Map Piet Stam A computerized procedure to construct integrated genetic maps is presented. The computer program (Join Map) can handle raw data from F2s, backcrosses and recombinant inbred lines, as well as listed pair-wise recombination frequencies. The procedure is useful for combining linkage data that have been collected in different experiments; the result is a mathematical alignment of the distinct genetic maps. Data from single experiments can be dealt with as well. In view of the fast growing amount of linkage information for molecular markers, which is often being generated by different research groups, integrated maps provide useful information on the map position of genes and DNA markers. The procedure performs a sequential build-up of the map and, at each step, a numerical search for the best fitting order of markers. Weighted least squares is used for the estimation of map distances. Read Online Volume 1 | Issue 3 | 1991 Developmentally Regulated Epitopes Of Cellsurface Arabinogalactan Proteins And Their Relation To Root-tissue Pattern-formation Knox, Jp; Linstead, Pj; Peart, J; Cooper, C; Roberts, K Article Two polymorphic forms of an extracellular arabinogalactan protein (AGP1 and AGP2), obtained from the conditioned media of two carrot suspension-cultured cell lines, have been identified in terms of binding of anti-plasma membrane bodies JIM4 and MAC207. AGP1 and AGP2 have been used as immunogens to generate further anti-AGP monoclonal antibodies. JIM14 identified an epitope carried by AGP2 and also by glycoproteins of low molecular weight localized to the plant cell wall. In addition, further antibodies (JIM13 and JIM15) identified carbohydrate epitopes of the AGPs that also occur on plasma membrane glycoproteins and are expressed patterns of cells that reflect cell position at the carrot root apex. Indirect immunofluorescence microscopy indicated that JIM13 recognized the surface of cells forming the epidermis and cells marking the region and axis of the future xylem. JIM15 recgnized a pattern of cells directly complementary to the JIM13 pattern. The panel of anti-AGP monoclonal antibodies now available indicates groups of cells within the root meristem that may reflect an early pre-pattern of the tissues of the mature root structure and suggests extensive modulation of cell surface AGPs during cell development and the positioning of cells within the apex. Read Online Volume 2 | Issue 4 | 1992 Article Lotus japonicus, an autogamous, diploid legume species for classical and molecular genetics Kurt Handberg and Jens Stougaard In the Leguminosae plant family, few of the individual plant species have been used for plant molecular biology research. Among the species investigated no obvious representative model` legume has emerged. Here a member of the tribe Loteae, Lotus japonicus (Regel) Larsen is proposed as a candidate. L. japonicus is a diploid, autogamous species, with a good seed set, and a generation time of approximately 3 months. The haploid genome consists of six chromosomes and the genome size was estimated to be relatively small (0.5 pg per haploid complement). L. japonicus is susceptible to Agrobacterium tumefaciens and transgenic plants can be regenerated after hygromycin or kanamycin selection. Tissue culture conditions and procedures for transformation and regeneration are described. Stable transformation is demonstrated by segregation of the hygromycin selectable marker after selfing of transgenic plants or test crosses. The possibility of mapping polymorphic DNA markers inbred lines of L. japonicus is also discussed. Read Online Volume 3 | Issue 6 | 1993 Article Regulation of the expression of rbcS and other photosynthetic genes by carbohydrates: a mechanism for the `sink regulation' of photosynthesis? Anne Krapp, Bettina Hofmann, Christian Sch�fer and Mark Stitt These experiments were carried out to investigate whether accumulation of carbohydrate leads to decreased expression of genes involved in photosynthesis. Addition of glucose to autotrophic cell suspension cultures of Chenopodium led to a large and reversible decrease of the steady state transcript levels of rbcS, cab and atp-& within 5 h, but did not decrease 18S rRNA or transcript for two glycolytic enzymes. Run-on transcription in isolated nuclei showed that transcription rate had been decreased. [35S]Methionine feeding showed that de novo synthesis of Rubisco was inhibited. Decreased rbcS transcript was also found after feeding glucose to detached leaves, and in transgenic plants expressing invertase in the apoplast to inhibit phloem transport, and in leaves on intact tobacco and potato plants which were cold-girdled to decrease export. The decrease of rbcS transcript level occurred within 12 h of coldgirdling. Comparison of carbohydrate content and rbcS transcript level indicated that carbohydrate content per se is not the direct signal for regulation of gene expression. Feeding of transported analogues indicates that metabolism rather than transport of the sugars is required. Over-expression of rbcS was found in low CO2, again indicating metabolic control of expression. It is proposed that photosynthetic gene expression is inhibited by metabolic factors related to high carbohydrate content, and that this represents a basic mechanism for the sink regulation` of photosynthesis. Read Online Volume 6 | Issue 3 | 1994 Article Manipulation of lignin quality by downregulation of cinnamyl alcohol dehydrogenase Claire Halpin et al. The composition of lignin in tobacco stems has been altered by genetic engineering. Antisense expression of sequences encoding cinnamyl alcohol dehydrogenase (CAD), the enzyme catalysing the final step in lignin precursor synthesis, leads to the production of a modified lignin in otherwise normal plants. Although Klason and acetyl bromide lignin determinations show little quantitative change in lignin deposition in CAD antisense plants, a number of qualitative changes have been identified. The lignin is altered in both composition and structure and is more susceptible to chemical extraction. Consistent with a block in CAD activity, antisense plants incorporate less cinnamyl alcohol monomers and more cinnamyl aidehyde monomers into lignin than corresponding control plants. Antisense plants with very low levels of CAD activity also show a novel phenotype with the appearance of a red-brown colour in xylem tissues. A similar phenotype is correlated with altered lignification and improved digestibility in brownmidrib mutants of maize and sorghum. The improved chemical extractability of lignin in CAD antisense plants supports a role for this technology in improving the pulp and papermaking value of forest trees while the similarity with brown-midrib mutants suggests a route to more digestible forage crops. Read Online Volume 8 | Issue 1 | 1995 Enhanced quantitative resistance against fungal disease by combinatorial expression of different barley antifungal proteins in transgenic tobacco Guido Jach et al. cDNAs encoding three proteins from barley (Hordeum vulgare), a class-II chitinase (CHI), a class-II -1,3-glucanase (GLU) and a Type-I ribosomeinactivating protein (RIP) were expressed in tobacco plants under the control of the CaMV 35S-promoter. High-level expression of the transferred genes was detected in the transgenic plants by Northern and Western blot analysis. The leader peptides in CHI and GLU led to accumulation of these proteins in the intercellular space of tobacco leaves. RIP, which is naturally deposited in the cytosol of barley endosperm cells, was expressed either in its original cytosolic form or fused to a plant secretion peptide (spRIP). Fungal infection assays revealed that expression of the individual genes in each case resulted in an increased protection against the soilborne fungal pathogen Rhizoctonia solani, which infects a range of plant species including tobacco. To create a situation similar to multi-gene` tolerance, which traditional breeding experience has shown to provide crops with a longerlasting protection, several of these antifungal genes were combined and protection against fungal attack resulting from their co-expression in planta was evaluated. Transgenic tobacco lines were generated with tandemly arranged genes coding for RIP and CHI as well as GLU and CHI. The performance of tobacco plants co-expressing the barley transgenes GLU/ CHI or CHI/RIP in a Rhizoctonia solani infection assay revealed significantly enhanced protection against fungal attack when compared with the protection levels obtained with corresponding isogenic lines expressing a single barley transgene to a similar level. The data indicate synergistic protective interaction of the co-expressed anti-fungal proteins in vivo. Article Read Online Volume 10 | Issue 1 | 1996 Benzothiadiazole induces disease resistance in Arabidopsis by activation of the systemic acquired resistance signal transduction pathway Kay A. Lawton et al. Article Benzothiadiazole (BTH) is a novel chemical activator of disease resistance in tobacco, wheat and other important agricultural plants. In this report, it is shown that BTH works by activating SAR in Arabidopsis thaliana. BTH-treated plants were resistant to infection by turnip crinkle virus, Pseudomonas syringae pv tomato` DC3000 and Peronospora parasitica. Chemical treatment induced accumulation of mRNAs from the SAR-associated genes, PR-1, PR-2 and PR-5. BTH treatment induced both PR-1 mRNA accumulation and resistance against P. parasitica in the ethylene response mutants, etr1 and ein2, and in the methyl jasmonateinsensitive mutant, jar1, suggesting that BTH action is independent of these plant hormones. BTH treatment also induced both PR-1 mRNA accumulation and P. parasitica resistance in transgenic Arabidopsis plants expressing the nahG gene, suggesting that BTH action does not require salicylic acid accumulation. However, because BTH-treatment failed to induce either PR-1 mRNA accumulation or P. parasitica resistance in the non-inducible immunity mutant, nim1, it appears that BTH activates the SAR signal transduction pathway. Read Online Volume 10 | Issue 1 | 1996 Article A benzothiadiazole derivative induces systemic acquired resistance in tobacco Leslie Friedrich et al. Systemic acquired resistance (SAR) is a pathogen-induced disease resistance response in plants that is characterized by broad spectrum disease control and an associated coordinate expression of a set of SAR genes. Benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester (BTH) is a novel synthetic chemical capable of inducing disease resistance in a number of dicotyledenous and monocotyledenous plant species. In this report, the response of tobacco plants to BTH treatment is characterized and the fact that it controls disease by activating SAR is demonstrated. BTH does not cause an accumulation of salicylic acid (SA), an intermediate in the SAR signal transduction pathway. As BTH also induces disease resistance and gene expression in transgenic plants expressing the nahG gene, it appears to activate the SAR signal transduction pathway at the site of or downstream of SA accumulation. BTH, SA and TMV induce the PR-1a promoter using similar cis-acting elements and gene expression is blocked by cycloheximide treatment. Thus, BTH induces SAR based on all of the physiological and biochemical criteria that define SAR in tobacco. Read Online Volume 11 | Issue 1 | 1997 Subcellular localization of H2O2 in plants. H2O2 accumulation in papillae and hypersensitive response during the barley--powdery mildew interaction Hans Thordal-Christensen, Ziguo Zhang, Yangdou Wei and David B. Collinge Article Active oxygen species (AOS) are believed to have important roles in plants in general and in plant--pathogen interactions in particular. They are believed to be involved in signal transduction, cell wall reinforcement, hypersensitive response (HR) and phytoalexin production, and to have direct antimicrobial effects. Since current methods are inadequate for localizing AOS in intact plant tissue, most studies have been conducted using cell suspension culture/elicitors systems. 3,3-diaminobenzidine (DAB) polymerizes instantly and locally as soon as it comes into contact with H2O2 in the presence of peroxidase, and it was found that, by allowing the leaf to take up this substrate, in-vivo and in-situ detection of H2O2 can be made at subcellular levels. This method was successfully used to detect H2O2 in developing papillae and surrounding haloes (cell wall appositions) and whole cells of barley leaves interacting with the powdery mildew fungus. Thus, H2O2 can be detected in the epidermal cell wall subjacent to the primary germ tube from 6 h after inoculation, and subjacent to the appressorium from 15 h. The earliest time point for observation of H2O2 in relation to epidermal cells undergoing HR is 15 h after inoculation, first appearing in the zones of attachment to the mesophyll cells underneath, and eventually in the entire epidermal cell. Furthermore, it was observed that proteins in papillae and HR cells are cross-linked, a process believed to be fuelled by H2O2. This cross-linking reinforces the apposition, presumably assisting the arrest of the pathogen. Read Online Volume 15 | Issue 3 | 1998 Article Stacks on tracks: the plant Golgi apparatus traffics on an actin/ER network Petra Boevink, Karl Oparka, Simon Santa Cruz, Barry Martin, Alan Betteridge and Chris Hawes We have visualized the relationship between the endoplasmic reticulum (ER) and Golgi in leaf cells of Nicotiana clevelandii by expression of two Golgi proteins fused to green fluorescent protein (GFP). A fusion of the trans-membrane domain (signal anchor sequence) of a rat sialyl transferase to GFP was targeted to the Golgi stacks. A second construct that expressed the Arabidopsis H/KDEL receptor homologue aERD2, fused to GFP, was targeted to both the Golgi apparatus and ER, allowing the relationship between these two organelles to be studied in living cells for the first time. The Golgi stacks were shown to move rapidly and extensively along the polygonal cortical ER network of leaf epidermal cells, without departing from the ER tubules. Co-localization of F-actin in the GFP-expressing cells revealed an underlying actin cytoskeleton that matched precisely the architecture of the ER network, while treatment of cells with the inhibitors cytochalasin D and N-ethylmaleimide revealed the dependency of Golgi movement on actin cables. These observations suggest that the leaf Golgi complex functions as a motile system of actindirected stacks whose function is to pick up products from a relatively stationary ER system. Also, we demonstrate for the first time in vivo brefeldin A-induced retrograde transport of Golgi membrane protein to the ER. Read Online Volume 18 | Issue 3 | 1999 Article Plants have a sensitive perception system for the most conserved domain of bacterial flagellin Georg Felix, Juliana D. Duran, Sigrid Volko and Thomas Boller The flagellum is an important virulence factor for bacteria pathogenic to animals and plants. Here we demonstrate that plants have a highly sensitive chemoperception system for eubacterial flagellins, specifically targeted to the most highly conserved domain within its N terminus. Synthetic peptides comprising 15�22 amino acids of this domain acted as elicitors of defence responses at sub-nanomolar concentrations in cells of tomato and several other plant species. Peptides comprising only the central 8 to 11 amino acids of the active domain had no elicitor activity but acted as specific, competitive inhibitors in tomato cells. These antagonists suppressed the plant`s response to flagellin, crude bacterial extracts and living bacterial cells. Thus, plants have a highly sensitive and selective perception system for the flagellin of motile eubacteria. Read Online Volume 23 | Issue 3 | 2000 Article Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants Yusuke Saijo, Shingo Hata, Junko Kyozuka, Ko Shimamoto and Katsura Izui A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stressresponsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement. Read Online Volume 28 | Issue 4 | 2001 Article Sites and homeostatic control of auxin biosynthesis in Arabidopsis during vegetative growth Karin Ljung, Rishikesh P. Bhalerao and G�ran Sandberg The distribution and biosynthesis of indole-3-acetic acid (IAA) was investigated during early plant development in Arabidopsis. The youngest leaves analysed, less than 0.5 mm in length, contained 250 pg mg-1 of IAA and also exhibited the highest relative capacity to synthesize this hormone. A decrease of nearly one hundred-fold in IAA content occurred as the young leaves expanded to their full size, and this was accompanied by a clear shift in both pool size and IAA synthesis capacity. The correlation between high IAA content and intense cell division was further verified in tobacco leaves, where a detailed analysis revealed that dividing mesophyll tissue contained ten-fold higher IAA levels than tissue growing solely by elongation. We demonstrated that all parts of the young Arabidopsis plant can potentially contribute to the auxin needed for growth and development, as not only young leaves, but also all other parts of the plant such as cotyledons, expanding leaves and root tissues have the capacity to synthesize IAA de novo. We also observed that naphthylphthalamic acid (NPA) treatment induced tissue-dependent feedback inhibition of IAA biosynthesis in expanding leaves and cotyledons, but intriguingly not in young leaves or in the root system. This observation supports the hypothesis that there is a sophisticated tissue-specific regulatory mechanism for auxin biosynthesis. Finally, a strict requirement for maintaining the pool sizes of IAA was revealed as reductions in leaf expansion followed both decreases and increases in the IAA levels in developing leaves. This indicates that leaves are not only important sources for IAA synthesis, but that normal leaf expansion depends on rigorous control of IAA homeostasis. Read Online Volume 31 | Issue 3 | 2002 Monitoring the expression profiles of 7000 Arabidopsis genes under drought, cold and high-salinity stresses using a full-length cDNA microarray Motoaki Seki et al. Article Full-length cDNAs are essential for functional analysis of plant genes in the post-sequencing era of the Arabidopsis genome. Recently, cDNA microarray analysis has been developed for quantitative analysis of global and simultaneous analysis of expression profiles. We have prepared a full-length cDNA microarray containing 7000 independent, full-length cDNA groups to analyse the expression profiles of genes under drought, cold (low temperature) and high-salinity stress conditions over time. The transcripts of 53, 277 and 194 genes increased after cold, drought and high-salinity treatments, respectively, more than fivefold compared with the control genes. We also identified many highly drought-, cold- or highsalinity- stress-inducible genes. However, we observed strong relationships in the expression of these stress-responsive genes based on Venn diagram analysis, and found 22 stress-inducible genes that responded to all three stresses. Several gene groups showing different expression profiles were identified by analysis of their expression patterns during stress-responsive gene induction. The cold-inducible genes were classified into at least two gene groups from their expression profiles. DREB1A was included in a group whose expression peaked at 2 h after cold treatment. Among the drought, cold or high-salinity stress-inducible genes identified, we found 40 transcription factor genes (corresponding to 11% of all stress-inducible genes identified), suggesting that various transcriptional regulatory mechanisms function in the drought, cold or high-salinity stress signal transduction pathways. Read Online Volume 33 | Issue 4 | 2003 OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt- and cold-responsive gene expression Joseph G. Dubouzet et al. Article The transcription factors DREBs/CBFs specifically interact with the dehydration-responsive element/C-repeat (DRE/CRT) cis-acting element (core motif: G/ACCGAC) and control the expression of many stressinducible genes in Arabidopsis. In rice, we isolated five cDNAs for DREB homologs: OsDREB1A, OsDREB1B, OsDREB1C, OsDREB1D, and OsDREB2A. Expression of OsDREB1A and OsDREB1B was induced by cold, whereas expression of OsDREB2A was induced by dehydration and high-salt stresses. The OsDREB1A and OsDREB2A proteins specifically bound to DRE and activated the transcription of the GUS reporter gene driven by DRE in rice protoplasts. Over-expression of OsDREB1A in transgenic Arabidopsis induced over-expression of target stress-inducible genes of Arabidopsis DREB1A resulting in plants with higher tolerance to drought, high-salt, and freezing stresses. This indicated that OsDREB1A has functional similarity to DREB1A. However, in microarray and RNA blot analyses, some stress-inducible target genes of the DREB1A proteins that have only ACCGAC as DRE were not over-expressed in the OsDREB1A transgenic Arabidopsis. The OsDREB1A protein bound to GCCGAC more preferentially than to ACCGAC whereas the DREB1A proteins bound to both GCCGAC and ACCGAC efficiently. The structures of DREB1-type ERF/AP2 domains in monocots are closely related to each other as compared with that in the dicots. OsDREB1A is potentially useful for producing transgenic monocots that are tolerant to drought, high-salt, and/or cold stresses. Read Online Volume 37 | Issue 5 | 2004 Article A wheat gene encoding an aluminumactivated malate transporter Takayuki Sasaki et al. The major constraint to plant growth in acid soils is the presence of toxic aluminum (Al) cations, which inhibit root elongation. The enhanced Al tolerance exhibited by some cultivars of wheat is associated with the Aldependent efflux of malate from root apices. Malate forms a stable complex with Al that is harmless to plants and, therefore, this efflux of malate forms the basis of a hypothesis to explain Al tolerance in wheat. Here, we report on the cloning of a wheat gene, ALMT1 (aluminumactivated malate transporter), that co-segregates with Al tolerance in F2 and F3 populations derived from crosses between near-isogenic wheat lines that differ in Al tolerance. The ALMT1 gene encodes a membrane protein, which is constitutively expressed in the root apices of the Altolerant line at greater levels than in the near-isogenic but Al-sensitive line. Heterologous expression of ALMT1 in Xenopus oocytes, rice and cultured tobacco cells conferred an Al-activated malate efflux. Additionally, ALMT1 increased the tolerance of tobacco cells to Al treatment. These findings demonstrate that ALMT1 encodes an Al-activated malate transporter that is capable of conferring Al tolerance to plant cells. Read Online Volume 42 | Issue 2 | 2005 Article Functional genomics by integrated analysis of metabolome and transcriptome of Arabidopsis plants over-expressing an MYB transcription factor Takayuki Tohge et al. The integration of metabolomics and transcriptomics can provide precise information on gene-to-metabolite networks for identifying the function of unknown genes unless there has been a post-transcriptional modification. Here, we report a comprehensive analysis of the metabolome and transcriptome of Arabidopsis thaliana over-expressing the PAP1 gene encoding an MYB transcription factor, for the identification of novel gene functions involved in flavonoid biosynthesis. For metabolome analysis, we performed flavonoid-targeted analysis by high-performance liquid chromatography-mass spectrometry and non-targeted analysis by Fouriertransform ion-cyclotron mass spectrometry with an ultrahigh-resolution capacity. This combined analysis revealed the specific accumulation of cyanidin and quercetin derivatives, and identified eight novel anthocyanins from an array of putative 1800 metabolites in PAP1 over-expressing plants. The transcriptome analysis of 22 810 genes on a DNA microarray revealed the induction of 38 genes by ectopic PAP1 over-expression. In addition to well-known genes involved in anthocyanin production, several genes with unidentified functions or annotated with putative functions, encoding putative glycosyltransferase, acyltransferase, glutathione S-transferase, sugar transporters and transcription factors, were induced by PAP1. Two putative glycosyltransferase genes (At5g17050 and At4g14090) induced by PAP1 expression were confirmed to encode flavonoid 3-O-glucosyltransferase and anthocyanin 5-O-glucosyltransferase, respectively, from the enzymatic activity of their recombinant proteins in vitro and results of the analysis of anthocyanins in the respective T-DNA-inserted mutants. The functional genomics approach through the integration of metabolomics and transcriptomics presented here provides an innovative means of identifying novel gene functions involved in plant metabolism. Read Online Volume 45 | Issue 1 | 2006 Article ABA-induced NO generation and stomatal closure in Arabidopsis are dependent on H2O2 synthesis Jo Bright, Radhika Desikan, John T. Hancock, Iain S. Weir and Steven J. Neil Nitric oxide (NO) and hydrogen peroxide (H2O2) are key signalling molecules produced in response to various stimuli and involved in a diverse range of plant signal transduction processes. Nitric oxide and H2O2 have been identified as essential components of the complex signalling network inducing stomatal closure in response to the phytohormone abscisic acid (ABA). A close inter-relationship exists between ABA and the spatial and temporal production and action of both NO and H2O2 in guard cells. This study shows that, in Arabidopsis thaliana guard cells, ABA-mediated NO generation is in fact dependent on ABA-induced H2O2 production. Stomatal closure induced by H2O2 is inhibited by the removal of NO with NO scavenger, and both ABA and H2O2 stimulate guard cell NO synthesis. Conversely, NO-induced stomatal closure does not require H2O2 synthesis nor does NO treatment induce H2O2 production in guard cells. Tungstate inhibition of the NO-generating enzyme nitrate reductase (NR) attenuates NO production in response to nitrite in vitro and in response to H2O2 and ABA in vivo. Genetic data demonstrate that NR is the major source of NO in guard cells in response to ABA-mediated H2O2 synthesis. In the NR double mutant nia1, nia2 both ABA and H2O2 fail to induce NO production or stomatal closure, but in the nitric oxide synthase deficient Atnos1 mutant, responses to H2O2 are not impaired. Importantly, we show that in the NADPH oxidase deficient double mutant atrbohD/F, NO synthesis and stomatal closure to ABA are severely reduced, indicating that endogenous H2O2 production induced by ABA is required for NO synthesis. In summary, our physiological and genetic data demonstrate a strong inter-relationship between ABA, endogenous H2O2 and NO-induced stomatal closure. Read Online Volume 50 | Issue 2 | 2007 The AtGenExpress global stress expression data set: protocols, evaluation and model data analysis of UV-B light, drought and cold stress responses Joachim Kilian et al. Article The tolerance responses of plants to many abiotic stresses are conjectured to be controlled by complex gene networks. In the frame of the AtGenExpress project a comprehensive Arabidopsis thaliana genome transcript expression study was performed using the Affymetrix ATH1 microarray in order to understand these regulatory networks in detail. In contrast to earlier studies, we subjected, side-by-side and in a highresolution kinetic series, Arabidopsis plants, of identical genotype grown under identical conditions, to different environmental stresses comprising heat, cold, drought, salt, high osmolarity, UV-B light and wounding. Furthermore, the harvesting of tissue and RNA isolation were performed in parallel at the same location using identical experimental protocols. Here we describe the technical performance of the experiments. We also present a general overview of environmental abiotic stress-induced gene expression patterns and the results of a model bioinformatics analysis of gene expression in response to UV-B light, drought and cold stress. Our results suggest that the initial transcriptional stress reaction of Arabidopsis might comprise a set of core environmental stress response genes which, by adjustment of the energy balance, could have a crucial function in various stress responses. In addition, there are indications that systemic signals generated by the tissue exposed to stress play a major role in the coordination and execution of stress responses. In summary, the information reported provides a prime reference point and source for the subsequent exploitation of this important resource for research into plant abiotic stress. Read Online Volume 53 | Issue 5 | 2008 Article MicroRNA399 is a long-distance signal for the regulation of plant phosphate homeostasis Bikram Datt Pant, Anja Buhtz, Julia Kehr and WolfR�diger Scheible The presence of microRNA species in plant phloem sap suggests potential signaling roles by long-distance regulation of gene expression. Proof for such a role for a phloem-mobile microRNA is lacking. Here we show that phosphate (Pi) starvation-induced microRNA399 (miR399) is present in the phloem sap of two diverse plant species, rapeseed and pumpkin, and levels are strongly and specifically increased in phloem sap during Pi deprivation. By performing micro-grafting experiments using Arabidopsis, we further show that chimeric plants constitutively overexpressing miR399 in the shoot accumulate mature miR399 species to very high levels in their wild-type roots, while corresponding primary transcripts are virtually absent in roots, demonstrating shoot-to-root transport. The chimeric plants exhibit (i) down-regulation of the miR399 target transcript (PHO2), which encodes a critical component for maintenance of Pi homeostasis, in the wild-type root, and (ii) Pi accumulation in the shoot, which is the phenotype of pho2 mutants, miR399 over-expressers or chimeric plants with a genetic knock-out of PHO2 in the root. Hence the transported miR399 molecules retain biological activity. This is a demonstration of systemic control of a biological process, i.e. maintenance of plant Pi homeostasis, by a phloem-mobile microRNA. Read Online Volume 57 | Issue 3 | 2009 Article Ferritins control interaction between iron homeostasis and oxidative stress in Arabidopsis Karl Ravet, Brigitte Touraine, Jossia Boucherez, JeanFran�ois Briat, Fr�d�ric Gaymard and Fran�oise Cellier Ferritin protein nanocages are the main iron store in mammals. They have been predicted to fulfil the same function in plants but direct evidence was lacking. To address this, a loss-of-function approach was developed in Arabidopsis. We present evidence that ferritins do not constitute the major iron pool either in seeds for seedling development or in leaves for proper functioning of the photosynthetic apparatus. Loss of ferritins in vegetative and reproductive organs resulted in sensitivity to excess iron, as shown by reduced growth and strong defects in flower development. Furthermore, the absence of ferritin led to a strong deregulation of expression of several metal transporters genes in the stalk, overaccumulation of iron in reproductive organs, and a decrease in fertility. Finally, we show that, in the absence of ferritin, plants have higher levels of reactive oxygen species, and increased activity of enzymes involved in their detoxification. Seed germination also showed higher sensitivity to pro-oxidant treatments. Arabidopsis ferritins are therefore essential to protect cells against oxidative damage. Read Online Volume 61 | Issue 2 | 2010 Article PYR/PYL/RCAR family members are major in-vivo ABI1 protein phosphatase 2C-interacting proteins in Arabidopsis Noriyuki Nishimura et al. Abscisic acid (ABA) mediates resistance to abiotic stress and controls developmental processes in plants. The group-A PP2Cs, of which ABI1 is the prototypical member, are protein phosphatases that play critical roles as negative regulators very early in ABA signal transduction. Because redundancy is thought to limit the genetic dissection of early ABA signalling, to identify redundant and early ABA signalling proteins, we pursued a proteomics approach. We generated YFP-tagged ABI1 Arabidopsis expression lines and identified in vivo ABI1-interacting proteins by mass-spectrometric analyses of ABI1 complexes. Known ABA signalling components were isolated including SnRK2 protein kinases. We confirm previous studies in yeast and now show that ABI1 interacts with the ABA-signalling kinases OST1, SnRK2.2 and SnRK2.3 in plants. Interestingly, the most robust in planta ABI1-interacting proteins in all LC-MS/MS experiments were nine of the 14 PYR/PYL/RCAR proteins, which were recently reported as ABA-binding signal transduction proteins, providing evidence for in vivo PYR/PYL/RCAR interactions with ABI1 in Arabidopsis. ABI1�PYR1 interaction was stimulated within 5 min of ABA treatment in Arabidopsis. Interestingly, in contrast, PYR1 and SnRK2.3 coimmunoprecipitated equally well in the presence and absence of ABA. To investigate the biological relevance of the PYR/PYLs, we analysed pyr1/pyl1/pyl2/pyl4 quadruple mutant plants and found strong insensitivities in ABA-induced stomatal closure and ABA-inhibition of stomatal opening. These findings demonstrate that ABI1 can interact with several PYR/PYL/RCAR family members in Arabidopsis, that PYR1�ABI1 interaction is rapidly stimulated by ABA in Arabidopsis and indicate new SnRK2 kinasePYR/PYL/RCAR interactions in an emerging model for PYR/PYL/RCARmediated ABA signalling. Read Online Read Online