Plant biology

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Plant biology Paper alert A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in plant biology. • ••

of special interest of outstanding interest

Current Opinion in Plant Biology 2002, 5:467–474 Contents (chosen by) 467 Growth and development (Schneitz and Sablowski) 468 Plant biotechnology (Dunwell) 469 Physiology and metabolism (Fernie and Sweetlove) 470 Biotic interactions (Metraux) 472 Cell signalling (Palme and McAinsh) 473 Gene regulation (Weisshaar) 473 Cell biology (Berger)

Growth and development Selected by Kay Schneitz Entwicklungsbiologie der Pflanzen, Wissenschaftszentrum Weihenstephan, Freising-Weihenstephan, Germany e-mail: [email protected]

•• BRI1/BAK1, a receptor kinase pair mediating brassinosteroid signaling. Nam KH, Li J: Cell 2002, 110:203-212. •• BAK1, an Arabidopsis LRR receptor-like protein kinase, interacts with BRI1 and modulates brassinosteroid signaling. Li J, Wen J, Lease KA, Doke JT, Tax FE, Walker JC: Cell 2002, 110:213-222. Significance: A receptor-like kinase is identified as a component of the brassinosteroid cell-surface receptor complex. Findings: Nam and Li identified BRI1 ASSOCIATED RECEPTOR KINASE 1 (BAK1), a LRRII-class leucine-rich repeat receptorlike kinase (LRR-RLK), as an interactor of LRR-RLK BRASSINOSTEROID INSENSITIVE 1 (BRI1) in a yeast twohybrid screen. Li et al. isolated the dominant mutation bak1-1D in an activation-tagging screen for revertants of the weak bri1-5 allele. The LRRII class includes the Arabidopsis homolog of the Daucus SOMATIC EMBRYOGENESIS RECEPTOR KINASE (SERK). Both papers provide in vitro and in vivo co-immunoprecipitation data that indicate that BRI1 and BAK1 interact directly. BAK1 localized to the plasma membrane and can act as a serine/threonine kinase. The authors showed that transphosphorylation can occur between the two partners, an event that is likely to be important for effector specificity. Genetic data, for example from a phenotypic analysis of bak1 null mutants or plants overexpressing BAK1, strongly indicate a role for BAK1 in brassinosteroid signaling. •• Generation of self-incompatible Arabidopsis thaliana by transfer of two S locus genes from A. lyrata. Nasrallah ME, Liu P, Nasrallah JB: Science 2002, 297:247-249. Significance: The self-compatible species A. thaliana bears the signaling network that underlies the sporophytic self-incompatibility

response (SI) of some members of the Brassicaceae. These findings will form the basis for a molecular investigation of sporophytic SI. Findings: The genes encoding the S-locus receptor-kinase (SRK) and its ligand, S-locus cysteine rich protein (SCR) of A. lyrata haplotype Sb, were isolated and transferred to A. thaliana. The corresponding transgenic plants exhibited a self-incompatible behavior in pollination assays. •• DEMETER, a DNA glycosylase domain protein, is required for endosperm gene imprinting and seed viability in Arabidopsis. Choi Y, Gehring M, Johnson L, Hannon M, Harada JJ, Goldberg RB, Jacobsen SE, Fischer RL: Cell 2002, 110:33-42. Significance: This paper advances our knowledge on the mechanism that underlies imprinting in plants. Findings: Mutants carrying T-DNA insertions in DEMETER (DME) were identified on the basis of parent-of-origin defects in seed viability. Despite these defects, homozygous dme mutants could be obtained, a small percentage of which exhibited various developmental defects such as variation in floral organ number. The DME gene was cloned and the putative protein was shown to carry a DNA glycosylase domain and a basic region with similarity to Xenopus histone H1. DNA glycosylases perform base excisions from DNA. RT-PCR, promoter::GUS and promoter::GFP studies indicated that DME expression is restricted to the synergids and the central cell of the female gametophyte before fertilization. MEDEA (MEA) is an imprinted gene, and its maternal copy is expressed during and required for endosperm development. Several experiments indicated that DME is an upstream positive regulator of MEA expression. MEA mRNA could not be detected using RT-PCR in dme-1 flowers. In addition, female gametophytes carrying a MEA promoter::GFP fusion construct and a mutation in DME did not express MEA::GFP. Furthermore, the ectopic activation of DME in leaves, using a 35S promoter-driven DME transgene, also led to a corresponding misexpression of MEA. Using a PCR-based assay, the authors also showed that DME expression results in nicks in the MEA promoter. The authors propose a model in which DME marks the maternal MEA allele in the female gametophyte before fertilization, thereby allowing sustained MEA expression during endosperm development. Selected by Robert Sablowski John Innes Centre, Norwich, UK e-mail: [email protected]

•• Endogenous and silencing-associated small RNAs in plants. Llave C, Kasschau KD, Rector MA, Carrington JC: Plant Cell 2002, 14:1605-1619. •• MicroRNAs in plants. Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP: Genes Dev 2002, 16:1616-1626. •• Prediction of plant microRNA targets. Rhoades MW, Reinhart BJ, Lim LP, Burge CB, Bartel B, Bartel DP: Cell Immediate 2002, 110:513-520. Significance: MicroRNAs (miRNAs) are RNAs of 20–24 nucleotides in length that can base-pair with mRNAs to control their function. miRNAs are transcribed from non-protein-coding genes and are processed by dicer-related RNAases. The same RNAases are involved in processing the small-interfering RNAs

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(siRNAs) that participate in post-transcriptional gene silencing. Following on from the isolation of miRNAs in Caenorhabditis, Drosophila, mouse and humans, these authors identify miRNAs in plants and suggest that they are important regulators of endogenous mRNAs. Findings: Both Llave et al. and Reinhart et al. used ligation of RNA adaptors and RT-PCR to clone from Arabidopsis numerous RNAs that have the properties expected of miRNAs. Northern blots showed that these small RNAs had different, tissue-specific expression patterns. The majority of the small RNAs analysed (90% in the case of Llave et al.) seemed to originate from genomic sequences outside annotated genes (i.e. from intergenic regions [IGR]); the rest seemed to derive from known genes, transposons and 5S RNA. The predicted IGR transcripts had the potential to form hairpin structures, consistent with the processing of small RNAs from larger double-stranded RNA precursors by a dicer-like RNAse. Indeed, mutation of CARPEL FACTORY, which encodes an Arabidopsis homolog of dicer, prevented the accumulation of three miRNAs that were tested by Reinhart et al.. Eight of the sixteen miRNA-encoding sequences analysed in detail by these authors were present in rice, suggesting their functional conservation. Rhoades et al. looked for potential mRNA targets of Arabidopsis miRNAs, searching for complementarity (with four mismatches or fewer) to known transcripts. A set of random permutations of the miRNA sequences was used to as a control to account for the frequency of random matches. The actual miRNA sequences yielded a much higher frequency of complementarity to mRNAs than did the control group. 70% of the candidate mRNA targets encoded putative transcription factors, including SCARECROW homologs (which were also identified as potential miRNA targets by Llave et al.) and known developmental regulators, such as LEAFY, CUP-SHAPED COTYLEDON2, PHABULOSA and PHAVOLUTA. All three papers discuss the possibility that miRNAs control mRNA processing, translation or stability. • Roles of PIN-FORMED1 and MONOPTEROS in pattern formation of the apical region of the Arabidopsis embryo. Aida M, Vernoux T, Furutani M, Traas J, Tasaka M: Development 2002, 129:3965-3974. Significance: CUP-SHAPED COTYLEDON1 (CUC1), CUC2 and SHOOT MERISTEMLESS (STM) are regulatory genes that are required for the establishment of the shoot meristem and separation of the cotyledons in the Arabidopsis embryo. Previous studies have also implicated auxin in cotyledon separation and in patterning the embryo shoot apex. This paper links both sets of data, showing that genes that are involved in auxin transport and perception (PIN-FORMED1 and MONOPTEROS, respectively) participate in cotyledon separation and shoot-apex patterning, partly by controlling CUC gene expression. Findings: Previous work has shown that cuc1, cuc2 double mutants lack the shoot meristem and that their cotyledons are fused in a cup-like structure, although their vasculature still retains bilateral symmetry. Mutations in the auxin efflux carrier gene PIN-FORMED1 (PIN1) cause variable defects in the positioning and separation of cotyledons. This paper shows that cuc1, cuc2, pin1 triple mutants are radially symmetrical, with complete cotyledon fusion. Expression analysis placed PIN1 upstream of the CUC genes. Whereas PIN1 is expressed normally in cuc1, cuc2 double mutants, cuc expression is altered in the pin1 mutant: the expression domain of CUC1 is expanded, whereas CUC2 expression is reduced. Consistent with the observation that CUC1 expression accounted for most of the

remaining CUC expression in pin1 mutants, the weak cotyledon fusion seen in pin1 mutants is strongly enhanced in pin1, cuc1 but not in pin1 cuc2 double mutants. CUC genes interact with STM during organ separation and meristem establishment. Accordingly, pin1 also enhances the stm mutant phenotype: the double mutant is radially symmetrical, as is the cuc1, cuc2, pin1 triple mutant. Previously characterised mutations in the gene encoding the auxin-response transcription factor MONOPTEROS (MP) disrupted the embryonic apical–basal axis and caused partial cotyledon fusion. The interaction between CUC genes and MP is similar to that between CUC and PIN1: mutation of mp expands CUC1 and diminishes CUC2 expression, whereas the cuc1 mutation strongly enhances the cotyledon fusion defect seen in mp mutants. • SCHIZORIZA controls an asymmetric cell division and restricts epidermal identity in the Arabidopsis root. Mylona P, Linstead P, Martienssen R, Dolan L: Development 2002, 129:4327-4334. Significance: This paper reveals a new regulator of radial patterning in the Arabidopsis root. SCHIZORIZA (SZR) represses epidermal fate in subepidermal cells and restricts the number of subepidermal cell layers. Findings: The recessive szr mutation was initially identified because it had too many root hairs. Closer examination revealed that the extra hairs emerged through the epidermis but originated from subepidermal cells. Additional evidence that the subepidermal cells had acquired epidermal identity came from their expression of epidermal-specific marker genes, whereas they did not express cortex and endodermis markers. Development of the subepidermal root hairs depended on the same regulatory genes (GL2, TTG, CPC) as did the development of normal epidermal hairs. The defect in cell identity occurred before hairs became visible: in the root meristem, an epidermal marker was misexpressed in subepidermal cells, and expression of an early cortex/endodermis marker (which is also expressed in the quiescent centre) became discontinuous and was shifted away from the quiescent centre. In addition to the defect in subepidermal cell identity, szr roots had more layers of ground tissue than wildtype roots. This defect was traced to extra periclinal divisions of the meristem cells that originate the endodermis and cortex. Like the periclinal divisions that happen during normal development, the additional divisions in szr depended on SCARECROW (SCR) and its upstream regulator, SHORT ROOT (SHR). In szr, scr and in szr, shr double mutants, the number of layers of ground tissue was reduced to one. This single subepidermal layer was also mis-specified as epidermal, showing that the cell identity defect of szr is separable from its cell division defect. Although the extra periclinal divisions in szr roots required SCR, they were not caused by increased expression of SCR.

Plant biotechnology Selected by Jim Dunwell University of Reading, Berkshire, UK e-mail: [email protected]

• Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway. Gordon-Kamm W, Dilkes BP, Lowe K, Hoerster G, Sun X, Ross M, Church L, Bunde C, Farrell J, Hill P et al.: Proc Natl Acad Sci USA 2002, 99:11975-11980.

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Significance: The genome of the Mastreviruses encodes a replication-associated protein (RepA) that interacts with members of the plant retinoblastoma-related protein family, which are putative cell cycle regulators. The data reported here demonstrate that RepA can stimulate cell division and callus growth in culture, and can improve transformation in maize. Findings: RepA expression increased the transformation frequency and callus growth rate of high type II maize germplasm, a model genotype. RepA-containing transgenic calli remained embryogenic, were readily regenerated, and produced fertile plants that expressed the transgene in a Mendelian fashion. Transformation frequency increased with the strength of the promoter that was used to drive RepA expression. RepA was also effective in increasing transformation frequency and callus growth rate in elite inbreds. • Selenium speciation in wild-type and genetically modified Se accumulating plants with HPLC separation and ICP-MS/ES-MS detection. Montes-Bayon M, LeDuc DL, Terry N, Caruso JA: J Anal Atom Spectr 2002, 17:872-879. Significance: Some plants have the ability to accumulate selenium (Se), a potentially toxic metal. Amongst the accumulation mechanisms in such tolerant plants is the formation of organoselenium compounds that cannot be incorporated into proteins, thereby avoiding toxicity. Analytical data reported here may be of value in developing transgenic plants for bioremediation, the biological decontamination of polluted soils. Findings: Brassica juncea (Indian mustard) accumulated Se when grown hydroponically in the presence of selenite, selenate, and Se-methionine. In addition, transgenic plants that overexpressed Se-cysteine methyltransferase (SMT) were grown under the same conditions. Se speciation for the three different Se regimes was performed by ion-pairing reversed phase liquid chromatography. The overexpression of SMT led to the formation of Se-methylselenocysteine, a non-protein amino acid that corresponds to increased Se tolerance in the transgenic plants. The authors demonstrated the capability of inductively coupled plasma mass spectrometry (ICP-MS) to pre-screen particular Se species as potential targets for Electrospray-MS. • Molecular breeding of transgenic rice plants expressing a bacterial chlorocatechol dioxygenase gene. Shimizu M, Kimura T, Koyama T, Suzuki K, Ogawa N, Miyashita K, Sakka K, Ohmiya K: Appl Environ Microbiol 2002, 68:4061-4066. Significance: Chloroaromatic compounds are one of the most important environmental pollutants. This work shows that it is possible to breed transgenic plants that degrade such compounds in soil and surface water. Findings: The cbnA gene, which encodes the chlorocatechol dioxygenase gene from Ralstonia eutropha NH9, was introduced into rice plants under the control of a modified cauliflower mosaic virus 35S promoter. Western-blot analysis using anti-CbnA protein indicated that the cbnA gene was expressed in leaf tissue, roots, culms, and seeds. Transgenic rice calluses expressing the cbnA gene converted 3-chlorocatechol to 2-chloromucote efficiently. The growth and morphology of the transgenic plants were indistinguishable from those of control rice plants that harboured only a Ti binary vector. • Expression of a bacterial carotene hydroxylase gene (crtZ) enhances UV tolerance in tobacco. Gotz T, Sandmann G, Romer S: Plant Mol Biol 2002, 50:129-142.

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Significance: It has been suggested that increased levels of UV irradiation associated with climate change may lead to increased damage to crops. Carotenoids are essential components of the photosynthetic apparatus and are involved in plant photoprotection. This study provides evidence of improved photoprotection in transgenic plants that have increased zeaxanthin levels. Findings: The authors generated transgenic tobacco (Nicotiana tabacum L.) plants that expressed a heterologous carotenoid gene, which encoded beta-carotene hydroxylase (crtZ) from Erwinia uredovora under the control of a constitutive promoter. This enzyme is responsible for the conversion of beta-carotene into zeaxanthin. Although the total pigment content of the transgenics was similar to that of control plants, the transformants synthesised zeaxanthin more rapidly and in larger quantities than controls upon transfer to high-intensity white light. Transformed plants that were exposed to UV light also maintained a higher biomass and a greater amount of photosynthetic pigments than did controls.

Physiology and metabolism Selected by Alisdair Fernie* and Lee Sweetlove† University of Oxford, Oxford, UK *e-mail: [email protected] †e-mail: [email protected]

•• Visualization of maltose uptake in living yeast cells by fluorescent nanosensors. Fehr M, Frommer WB, Lalonde S: Proc Natl Acad Sci USA 2002, 99:9846-9851. Significance: This paper highlights the sensitivity of a breakthrough technology, the conversion of aptamer techniques for the tracking of metabolites. Bacterial periplasmic binding proteins are used as an example. Findings: Upon binding to a specific substrate, bacterial periplasmic binding proteins (PBPs) transform their hinge-bend movement into increased fluorescence resonance energy transfer (FRET) between two coupled green fluorescent proteins. Using the maltose-binding protein as a prototype, the authors constructed nanosensors that allowed the in vitro determination of FRET changes in a concentration-dependent fashion. Mutants with different binding affinities were generated, allowing the dynamic in vivo imaging of the increase in cytosolic maltose concentration in single yeast cells. This study opens up the possibility of monitoring dynamic changes in the concentrations of a wide spectrum of solutes at high resolution, and should allow rapid advances in the understanding of transport processes within and between plant cells. • Identification of a novel transporter for dicarboxylates and tricarboxylates in plant mitochondria — bacterial expression, reconstitution, functional characterization, and tissue distribution. Picault N, Palmieri L, Pisano I, Hodges M, Palmieri F: J Biol Chem 2002, 277:24204-24211. Significance: The authors identify and functionally characterise cDNAs from Arabidopsis and tobacco that encode previously unidentified members of the mitochondrial carrier family. They speculate on their role in the co-ordination of compartmented metabolism. Findings: The transport properties of proteins encoded by cDNAs from Arabidopsis and tobacco revealed them to be isoforms of a novel mitochondrial carrier that is capable of transporting both dicarboxylates (such as malate, oxaloacetate,

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oxoglutarate, and maleate) and tricarboxylates (such as citrate, isocitrate, cis-aconitate, and trans-aconitate). This carrier is able to accept only the single protonated form of citrate (H-citrate[2-]) and the unprotonated form of malate (malate[2-]), and catalyses electroneutral exchanges. Oxoglutarate, citrate, and malate act as mutually competitive inhibitors. As the carrier is widely expressed at both the tissue and the species level, and as its expression is influenced by nitrate status, the authors postulate that it plays a role in important plant metabolic functions that require flux of organic acid to or from the mitochondria. Such processes include nitrogen assimilation, the export of reducing equivalents from the mitochondria, and fatty-acid elongation. • Identifying and characterizing plastidic 2-oxoglutarate/ malate and dicarboxylate transporters in Arabidopsis thaliana. Tanaguchi M, Taniguchi Y, Kawasaki M, Takeda S, Kato T, Sato S, Tabata S, Miyake H, Sugiyama T: Plant Cell Physiol 2002, 43:706-717. Significance: The authors provide functional characterisation of three Arabidopsis genes that are homologous to the spinach plastidial oxoglutarate/malate transporter gene. Findings: The authors describe the thorough functional characterisation of the DCT1 and OMT1 (oxoglutarate/malate transporter) genes via the kinetic analysis of yeast-expressed recombinant proteins followed by the subsequent identification of T-DNA insertional mutants. Hence, they provide a comprehensive analysis of the functionality and in vivo function of the DCT1 and OMT1 proteins. Although there is no clear evidence of changes in the transcript levels of these genes under photorespiratory conditions, the observation that the dct1 mutant was unable to grow under these conditions suggests that this carrier is necessary for photorespiratory nitrogen recycling. • Proteomic survey of metabolic pathways in rice. Koller A, Washburn MP, Lange BM, Andon NL, Deciu C, Haynes PA, Hays L, Schieltz D, Ulaszek R, Wei J, Wolters D, Yates JR III: Proc Natl Acad Sci USA 2002, 99:11969-11974. Significance: The authors present the most extensive analysis to date of a plant proteome. This work demonstrates the feasibility and usefulness of large-scale proteomic surveying of rice, an important model monocot species. Findings: 2528 unique rice proteins were identified using either two-dimensional gel electrophoresis followed by tandem mass spectrometry or multi-dimensional protein identification technology (MudPIT). The authors were able to ascertain, by comparative display of expression patterns, that enzymes involved in central metabolism are present in all tissues. A tissue-specific enzyme complement was also identified. For example, tissue-specific and subcellular-compartment-specific isoforms of ADP-glucose pyrophosphorylase were identified, thus providing proteomic confirmation of the presence of distinct regulatory mechanisms that are involved in the metabolism of starch in different tissues. • Transgenic plant cells lacking mitochondrial alternative oxidase have increased susceptibility to mitochondriadependent and -independent pathways of programmed cell death. Robson CA, Vanlerberghe GC: Plant Physiol 2002,129:1908-1920. Significance: The authors highlight a role for the mitochondrial alternative oxidase in programmed cell death.

Findings: Transgenic tobacco plants that lack alternative oxidase (AOX) show increased susceptibility to cell death induced by H2O2, salicylic acid or the protein phosphatase inhibitor cantharidin. In each case, death is accompanied by DNA laddering, a hallmark of programmed cell death (PCD). H2O2 and salicylic acid, but not cantharidin, induce a PCD that involves the mitochondrion (as indicated by the loss of cytochrome c from mitochondria). When treated with an anti-oxidant compound, the transgenic line showed increased resistance to death, leading the authors to propose that AOX may attenuate PCD by preventing chronic mitochondrial oxidative stress. • Starch synthesis in potato tubers is regulated by post-translational redox modification of ADP-glucose pyrophosphorylase: a novel regulatory mechanism linking starch synthesis to sucrose supply. Tiessen A, Hendriks JHM, Stitt M, Branscheid A, Gibon Y, Farré EM, Geigenberger P: Plant Cell 2002, 14:1-24. Significance: The authors demonstrate a novel mechanism to regulate starch synthesis that involves post-translational redox modification of AGPglucose pyrophosphorylase (AGPase). Findings: When potato tubers are detached form the plant they undergo a rapid inhibition of starch synthesis despite maintaining a high AGPase activity and an increased ratio of allosteric activator::inhibitor molecules. This suggests that additional mechanisms must also be involved in regulating starch synthesis. Analysis of the catalytic subunit of AGPase, AGPB, on native gels revealed that AGPB is present as a mixture of monomers and dimers in growing tubers. AGPB becomes completely dimerised when tubers are detached. This dimerisation led to the inactivation of AGPase due to decreased substrate affinity and insensitivity to allosteric effectors. Reversal of dimerisation by the reductant DTT, suggests that dimerisation is redoxregulated. Furthermore, the activation state of AGPase correlated with sucrose content across a range of treatments.

Biotic interactions Selected by Jean-Pierre Metraux University of Fribourg, Fribourg, Switzerland e-mail: [email protected]

High molecular weight RNAs and small interfering RNAs induce systemic posttranscriptional gene silencing in plants. Klahre U, Crété P, Leuenberger SA, Iglesias VA, Meins F: Proc Natl Acad Sci USA 2002, 99:11981-11986. Significance: Double-stranded siRNAs can function as systemic silencing signals and are likely to induce of selfamplifying process. Findings: RNA silencing is an effective mechanism in the defence of plants against viruses. In this paper, the nature of the molecules that can induce systemic RNA silencing was investigated using a positive marker system and real-time monitoring of green fluorescent protein expression. Large sense, antisense, and double-stranded RNAs, as well as doublestranded siRNAs, were delivered biolistically into plant cells and triggered local and systemic silencing. Systemically silenced leaves showed greatly reduced levels of target RNA as well as the accumulation of siRNAs. These findings provide a direct demonstration that double-stranded siRNAs can induce systemic post-transcriptional gene silencing (PTGS) in

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plants. The systemic silencing signals are likely to be either siRNAs themselves or intermediates that are induced by siRNAs, and can induce a self-amplifying process. •• A R2R3-MYB gene, AtMYB30, acts as a positive regulator of the hypersensitive cell death program in plants in response to pathogen attack. Vailleau F, Daniel X, Tronchet M, Montillet JL, Triantaphylidès C, Roby D: Proc Natl Acad Sci USA, 2002, 99:10179-10184. Significance: A MYB-like gene is a strong regulator of the hypersensitive response (HR) to pathogens. Findings: In Arabidopsis and tobacco, the overexpression of AtMYB30, an R2R3-MYB-related gene, has a profound effect on the expression of the HR to pathogens. Plant lines that overexpress AtMYB30 show accelerated HR in response to different avirulent bacterial pathogens. In addition, these lines express HR-like responses to virulent strains, which is associated with increased resistance to various virulent bacterial and biotrophic fungal pathogens. Antisense AtMYB30 lines of Arabidopsis display strongly decreased HR to avirulent bacterial strains, decreased resistance to bacterial pathogens, and altered expression of HR- and defence-related genes. Thus, ATMYB30 acts as a positive regulator of hypersensitive cell death. •• Interaction between domains of a plant NBS–LRR protein in disease resistance-related cell death. Moffett P, Farnham G, Peart J, Baulcombe DC: EMBO J 2002, 21:4511-4519. Significance: The authors propose that the physical interaction between protein domains of a resistance gene is disrupted upon elicitor perception and is necessary to induce a hypersensitive reaction (HR). Findings: The Rx resistance protein from Solanaceae has an amino-terminal coiled-coil (CC) domain, a central nucleotidebinding site (NBS) domain and a carboxy-terminal leucine-rich repeat (LRR) domain. Rx confers resistance to potato virus X, for which the elicitor is the coat protein (CP). Co-expression of Rx and the CP in Nicotiana benthamiana leads to a HR. Coexpression of the LRR and CC–NBS or the CC and NBS-LRR as separate domains also results in a CP-dependent HR. The CC domain can complement a version of Rx that lacks this domain (NBS– LRR). A physical interaction between the LRR and the CC–NBS domains, as well as between the CC and the NBS–LRR domains, was demonstrated in planta. Both interactions were disrupted in the presence of CP. These findings provide direct evidence that an elicitor-induced conformational change in a resistance gene leads to the activation of a disease resistance response. •• A saponin-detoxifying enzyme mediates suppression of plant defences. Bouarab K, Melton R, Peart J, Baulcombe D, Osbourn A: Nature 2002, 418:889-892. Significance: Pathogen-detoxifying enzymes are shown to inactivate antimicrobial plant products. The enzymes convert the antimicrobials into suppressors of induced disease resistance responses. Findings: Pathogen-produced detoxifying enzymes can counter the effect of antimicrobial products that are produced in plants. For example, the tomato leaf spot fungus Septoria lycopersici produces tomatinase, an enzyme that hydrolyses the antimicrobial saponin tomatine. Interestingly, the degradation product of this hydrolysis, β-tomatine, is now reported to act as a suppressor of

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resistance responses that interferes with signal transduction processes leading to plant defences. • Oomycete plant pathogens use electric fields to target roots. Van West P, Morris BM, Reid B, Appiah AA, Osborne MC, Campbell TA, Shepherd SJ, Gow NAR: Mol Plant Microbe Interact 2002, 15:790-798. Significance: Motile spores of oomycete fungi that infect plant roots can use electrical signals generated in roots to sense their host. Findings: Electrical currents and electrical fields generated by electrogenic ion transport at the root surface attract swimming zoospores of oomycete plant pathogens such as Phytophthora or Pythium. Anode- or cathode-seeking zoospore accumulation on root surfaces correlated with their in vitro electrostatic behaviour. The pattern of zoospore accumulation could be modified by altering the electrical field of the roots. This electrical signal could override chemotactic signals. These plant pathogens are thus equipped with a sophisticated electrical and chemical sensing system that mediates their short-range tactic responses. • Arabidopsis son1 is an F-box protein that regulates a novel induced defense response independent of both salicylic acid and systemic acquired resistance. Kim HS, Delaney TP: Plant Cell 2002, 14:1469-1482. Significance: The ubiquitin-proteosome pathway negatively regulates a salicylic-acid-independent pathway. Findings: Systemic acquired resistance (SAR) in Arabidopsis is regulated by salicylic acid through the NIM1/NPR1 protein. To discover new components that are SA-independent, these authors carried out a genetic suppressor screen of mutagenized nim1-1 seedlings, which are highly susceptible to infection by Peronospora parasitica. A mutant, son1 (suppressor of nim1-1), was identified in which resistance to Peronospora and Pseudomonas syringae pv tomato strain DC3000 is fully restored without the induction of SAR-associated genes. The son1 mutant also expresses resistance when combined with a salicylate hydroxylase (nahG) transgene. SON1 encodes a novel protein that contains an F-box motif, an element that is found within the specificity determinant in the E3 ubiquitin-ligase complex. Thus, the ubiquitin-proteosome pathway negatively regulates a novel defence response that is independent of SA. •• The Arabidopsis mutant cev1 links cell wall signaling to jasmonate and ethylene responses. Ellisa C, Karafyllidisa I, Wasternack C, Turner JG: Plant Cell 2002, 14:1557-1566. Significance: A cellulose synthase mutant reveals a link between the cell wall and the induction of pathogen and stress resistance. Findings: The cev1 mutant has constitutive expression of stress response genes and has enhanced resistance to fungal pathogens. This mutant accumulates high levels of jasmonate (JA) and ethylene, and its phenotype is suppressed by mutations that interrupt jasmonate and ethylene signalling. CEV1 was shown to be the cellulose synthase CeSA3. This protein was expressed predominantly in root tissues, and cev1 roots contained less cellulose than wildtype roots. The cev1 phenotype could be reproduced by treatment of wildtype plants with cellulose biosynthesis inhibitors. In addition, the cellulose synthase mutant root swellng1 (rsw1) constitutively expressed the JA-dependent gene VEGETATIVE STORAGE PROTEIN (VSP). These results show that the cell wall can signal stress responses in plants.

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Cell signalling Selected by Klaus Palme Max-Delbrück Laboratory, Köln, Germany e-mail: [email protected]

• The POLARIS gene of arabidopsis encodes a predicted peptide required for root growth and vascular patterning. Casson SA, Chilley PM, Topping JF, Evans I, Souter MA, Lindsey K: Plant Cell 2002, 14:1705-1721. Significance: The cloning of POLARIS reveals a 36-amino-acid peptide that is necessary for correct auxin-cytokinin homeostasis and that modulates root growth and leaf vascular patterning. Findings: The POLARIS (PLS) gene is identified. This gene has an open reading frame (ORF) that is located within a short (approximately 500 nucleotides) auxin-inducible transcript. PLS is predicted to encode a polypeptide of 36 amino acid residues. The gene is predominantly expressed in the roots, it is expressed only at a low level in the aerial parts of the plant. pls mutants exhibit a short-root phenotype and reduced vascularization of leaves. Most interestingly, the roots of these mutants are hyperresponsive to exogenous cytokinins and have elevated levels of cytokinin-inducible genes. When compared to wildtype seedlings, pls seedlings were less responsive to the growthinhibitory effects of exogenous auxin. Ectopic expression of the PLS ORF reduced the inhibition of root growth by exogenous cytokinins and increased leaf vascularization. On the basis of these findings, the authors proposed that PLS is required for correct auxin-cytokinin homeostasis to modulate root growth and leaf vascular patterning. • The identification of CVP1 reveals a role for sterols in vascular patterning. Carland FM, Fujioka S, Takatsuto S, Yoshida S, Nelson T: Plant Cell 2002, 14:2045-2058. Significance: The Arabidopsis mutant cotyledon vascular pattern1 (cvp1) corresponds to a defect in sterol biosynthesis. Findings: A set of cotyledon vascular pattern mutants was isolated recently. One of these mutants (cvp1) resembled the well-characterised phenotype of monopteros, having discontinuous, poorly axialized venation patterns. In cvp1, the vascular cells are not arranged in parallel files and are malformed, suggesting a role for CVP1 in promoting vascular cell polarity and alignment. Cloning of the gene affected in cvp1 revealed it to be STEROL METHYLTRANSFERASE2 (SMT2), which encodes an enzyme in the sterol biosynthetic pathway. SMT2 and another functionally redundant gene (SMT3) act at a branch point in the pathway that mediates sterol and brassinosteroid levels. The SMT2 gene is expressed in a number of developing organs and is regulated by various hormones. As predicted from SMT2 enzymatic activity, the precursors to brassinosteroid are increased at the expense of sterols in cvp1 mutants, identifying a role for sterols in vascular cell polarisation and axialization. • Early embryo development in Fucus distichus is auxin sensitive. Basu S, Sun H, Brian L, Quatrano RL, Muday GK: 130:292-302. Significance: Auxin plays a role in the formation of the apical–basal pattern of Fucus embryos. Findings: The authors set out to determine whether auxin and its polar transport are important during early embryo development and axis establishment of the brown alga Fucus distichus.

Indole-3-acetic acid (IAA) was indeed found in these embryos and accumulated after application of naphthylphthalamic acid (NAA), an inhibitor of IAA efflux, suggesting the presence of an auxin efflux protein complex similar to that found in land plants. In the presence of IAA, Fucus embryos developed multiple, branched rhizoids, indicating that auxin acts in the formation of apical–basal patterns in F. distichus embryo development. •• In planta protein–protein interactions assessed using a nanovirus-based replication and expression system. Aronson MN, Complainville A, Clerot D, Alcalde H, Katui L, Vetten J, Gronenborn B, Timchenko T: Plant J 2002, 31:767-775. Significance: A nanovirus system is established for high level in planta expression of recombinant proteins and protein complexes from plant tissues. Findings: As plant proteomics is gaining momentum, the production of recombinant proteins becomes limiting. This technical advance describes the use of Faba bean necrotic yellow virus as vector for transient replication and gene expression in plants. Several examples illustrate the potential of this technology for in planta protein–protein interaction studies. Selected by Martin R McAinsh Lancaster University, Lancaster, UK e-mail: [email protected]

•• Modulation of an RNA-binding protein by abscisic-acid-activated protein kinase. Li J, Kinoshita T, Pandey S, Ng CK-Y, Gygi SP, Shimazaki K-I, Assmann SM: Nature 2002, 418:793-797. Significance: Phosphorylation-regulated RNA target discrimination by heterogeneous nuclear-binding proteins may be a general phenomenon in eukaryotes. Findings: Abscisic acid (ABA)-activated protein kinase (AAPK) is implicated in rapid ABA signalling in guard cells. The authors identified AAPK-interacting protein 1 (AKIP1), which exhibits sequence homology to heterogeneous nuclear RNA-binding protein A/B (hnRNP A/B), as a substrate for AAPK. They show AAPK and AKIP1 are both present in the guard cell nucleus and that ABA treatment causes the relocalisation of AKIP1 into, and/or the retention of AKIP1 in, nuclear speckles. In addition, they show that the interaction of AKIP1 with dehydrin mRNA, a potential target of AKIP1 that encodes ubiquitous stress-upregulated proteins, is dependent on AKIP1 phosphorylation by AAPK. These observations suggest the involvement of hnRNPs in the modulation of mRNA stability by abiotic stresses, and implicate ABA in the regulation of protein dynamics during rapid changes in nuclear architecture. • The ELF4 gene controls circadian rhythms and flowering time in Arabidopsis thaliana. Doyle MR, Davis SJ, Bastow RM, McWatters HG, Kozma-Bognár L, Nagy F, Millar AJ, Amasino RM: Nature 2002, 419:74-77. Significance: The EARLY FLOWERING 4 (ELF4) gene is shown to have a role in photoperiod perception and circadian regulation. Findings: The authors used circadian outputs, including the expression of chlorophyll a/b-binding protein (CAB) and cold-and circadian-regulated (CCR) genes to study the role of the ELF4 gene in circadian control. These outputs were monitored using luciferase reporter gene fusions and rhythmic leaf movements. elf4 plants quickly lose rhythmicity when transferred to conditions of continuous light (CAB) or dark (CCR2), their rhythms becoming highly variable before the mutants become

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arrhythmic. In addition, the elf4 lesion disrupts the rhythmic expression of the CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) gene, which is thought to function as a component of the central oscillator. The authors conclude that ELF4 functions either in a circadian oscillator or by conferring accuracy and persistence on a separate oscillator. • Evidence for a role for protein tyrosine phosphatase in the control of ion release from the guard cell vacuole in stomatal closure. MacRobbie EAC: Proc Natl Acad Sci USA 2002, 99:11963-11968. Significance: Evidence for a critical role of protein tyrosine phosphatases (PTPases) in the guard cell signalling network. Findings: MacRobbie used a pharmacological approach to investigate the role of protein tyrosine phosphorylation in guard cell signalling. She showed that phenylarsine oxide, a specific inhibitor of PTPases, prevents stomatal closure in response to four closing stimuli: abscisic acid, high external calcium, transfer to dark, and hydrogen peroxide. Phenylarsine oxide also promotes the reopening of closed stomata and strongly inhibits 86Rb+ (an analogue for K+) efflux from the vacuole. A second PTPase inhibitor, 3,4 dephosphatin, and two vanadate derivatives that inhibit PTPases also give results consistent with these observations. These data identify the efflux of Rb(K) at the vacuolar membrane as the sensitive process, and place protein tyrosine phosphorylation at, or downstream of, the calcium signal that is responsible for triggering vacuolar ion efflux. • Repression of stress-response genes by FIERY2, a novel transcriptional regulator in Arabidopsis. Xiong L, Lee H, Ishitani M, Tanaka Y, Stevenson B, Koiwa H, Bressan RA, Hasegawa PM, Zhu J-K: Proc Natl Acad Sci USA 2002, 99:10899-10904. Significance: The authors identify and characterise FIERY2 (FRY2), an important negative regulator of stress gene transcription. Findings: A reporter-gene-aided genetic screen in Arabidopsis was used to study the mechanisms of transcriptional activation of stress and abscisic acid (ABA)-induced genes. Following stress or ABA treatment, fry2 mutants exhibited super-induction of the DRE/CRT class of stress-responsive genes together with physiological responses to stress and ABA that differed from those of wildtype plants. The expression of genes encoding CBF/DREB transcription factors (which activate DRE/CRT genes) is also induced to higher-than-wildtype levels in fry2. Furthermore, FRY2 encodes a novel protein that has limited homology to yeast and human carboxy-terminal domain phosphatases, which are implicated in gene transcription and pre-mRNA processing. The authors conclude that FRY2 acts upstream of CBF/DREB transcription factors and has important roles in regulating stress and ABA tolerance.

Gene regulation Selected by Bernd Weisshaar Max-Planck-Institut für Züchtungsforschung, Köln, Germany e-mail: [email protected]

• Selective interaction of plant homeodomain proteins mediates high DNA-binding affinity. Smith HMS, Boschke I, Hake S: Proc Natl Acad Sci USA 2002, 99:9579-9584.

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Significance: The paper describes the formation of heterodimers among plant homeodomain proteins and presents evidence that the DNA binding of homeodomain proteins is modulated by heterodimerisation. The data also exemplify that plant homeodomain proteins may act via combinatorial and cooperative interactions to control promoter activity. Findings: A protein that specifically interacts with Zea mays KNOTTED1 (KN1) was identified using the yeast two-hybrid system. Both the new protein, KIP (KNOTTED-INTERACTING PROTEIN), and KN1 are homeodomain factors. The authors determined the DNA-binding site sequence of KN1, and demonstrated that a combination of KN1 and KIP bound DNA more strongly than each of the two proteins alone. • ANTHOCYANIN1 of petunia controls pigment synthesis, vacuolar pH, and seed coat development by genetically distinct mechanisms. Spelt C, Quattrocchio F, Mol JNM, Koes R: Plant Cell 2002, 14:2121-2135. Significance: Identification of multiple downstream effects of the Petunia hybrida basic region/helix-loop-helix (bHLH) transcription factor ANTHOCYANIN1 (AN1). Findings: The authors present detailed analyses of an allelic series of mutations at the AN1 locus. The data indicate that distinct domains of AN1 control vacuolar pH and anthocyanin biosynthesis. Mutations in the bHLH domain, which do not significantly change the level of AN1 protein expression, interfere with AN1 activity and with the control of vacuolar pH and anthocyanin biosynthesis. • Molecular structure of the GARP family of plant Myb-related DNA binding motifs of Arabidopsis response regulators. Hosoda K, Imamura A, Katoh E, Hatta T, Tachiki M, Yamada H, Mizuno T, Yamazaki T: Plant Cell 2002, 14:2015-2029. Significance: Biochemical and structural features of the MYB-related domain of the type-B response regulator ARR10 are described. This domain also occurs in the members of the GARP family of MYB-related transcription factors, which were named according to the ‘family founder members’ Zea mays GOLDEN2, Arabidopsis thaliana response regulators, and A. thaliana PHR1. Findings: The authors show that the GARP domain (of ARR10) contains a nuclear localisation signal and that it binds to DNA in a sequence-specific way. The consensus sequence of the DNA binding site was found to be AGATN(C/T)(G/T). The three-dimensional structure of a GARP domain polypeptide in solution was determined by NMR spectroscopy. In addition, the authors deduced the GARP-domain–DNA interaction map.

Cell biology Selected by Frederic Berger Reproduction et Development des Plantes, Lyon, France e-mail: [email protected]

•• Expansion of the cell plate in plant cytokinesis requires a kinesin-like protein/MAPKKK complex. Nishihama R, Soyano T, Ishikawa M, Araki S, Tanaka H, Asada T, Irie K, Ito M, Terada M, Banno H et al.: Cell 2002, 109:87-99. Significance: The isolation of a complex of protein kinases that are essential for cytokinesis in plant cells. Findings: An original screen in yeast for activators of the previously isolated mitogen-activated protein kinase kinase

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kinase (MAPKKK) NPK1 permitted the identification of two other MAPKKKs, NACK1 and NACK2 from tobacco. NACK1 and NACK2 are expressed during mitosis. NACK1 activates NPK1 through direct interaction. In vivo localisation of green fluorescent protein (GFP) fusions and immunolocalisation show that NACK1 recruits NPK1 to the phragmoplast. Overexpression of a dominant-negative form of NACK1 and loss of function of NACK1 cause incomplete cytokinesis, both in culture cells and in planta. Two genes that encode orthologues of NACK1 are present in Arabidopsis thaliana, and loss of function of either of these genes impairs cytokinesis during the embryogenesis and vegetative development of weak plants that ultimately die. •• Role of formins in actin assembly: nucleation and barbed-end association. Pruyne D, Evangelista M, Yang C, Bi E, Zygmond S, Bretscher A, Boone C: Science 2002, 297:612-615. Significance: Evidence for nucleation of actin filaments by a new mechanism. Findings: Nucleation of branched actin filaments is mediated by the conserved ARP2/3 complex, which acts on the pointed end of the filament. Before this work, no nucleator that is responsible for unbranched filaments had been found. Formins are members of a protein family that is involved in cytokinesis and cell polarity, and whose activity is mediated by actin dynamics. In this report, the yeast formin Bnip1 is shown to nucleate actin filament from the barbed end. The conserved domain FH2, for which no function had been ascribed until now, appears to be responsible for the nucleation whereas the FH1 domain interacts with profilin. Bnip1 is immunolocalised to the barbed end of actin filament and its function in unbranched actin filament assembly in vivo is shown by its overexpression in yeast. • The Arabidopsis TONNEAU2 gene encodes a putative novel protein phosphatase 2A regulatory subunit essential for the control of the cortical cytoskeleton. Camilleri C, Azimzadeh J, Pastuglia M, Bellini C, Grandjean O, Bouchez D: Plant Cell 2002, 14:833-845. Significance: A gene that is involved in the regulation of the enigmatic preprophase band is isolated for the first time. The corresponding protein might regulate the organisation of microtubules into arrays by controlling the phosphorylation of an as yet unknown protein. Findings: The tonneau2 (ton2)/fass mutants are characterised by a stunted embryo with abnormal patterns of division. The preprophase band of microtubules, which precedes the onset of mitosis and indicates the site of cell division in the wildtype, is absent or mislocated in ton2. The mitotic spindle and the phragmoplast are not affected in ton2, whereas the interphase cortical microtubule array is randomised with defects increasing in parallel with the strength of the mutant allele. TON2 encodes a highly conserved protein with homologies to the B’’ regulatory subunit of protein phosphatase type 2A (PP2A). TON2 has a yeast-two-hybrid interaction with the subunit Aα of the AtPP2A. • Differential messenger RNA gradients in the unicellular alga Acetabularia acetabulum. Role of the cytoskeleton. Vogel H, Grieninger GE, Zetsche KH: Plant Physiol 2002, 129:1407-1416.

Significance: This study provides evidence of widespread mRNA gradients in the giant cell of Acetabularia. This finding leads us to re-visit historical experiments that led to the discovery of the role of the nucleus. Findings: The authors performed semi-quantitative PCR analyses for twelve genes of mRNA isolated from different positions along the basal–apical axis of the giant cell of Acetabularia. A group of mRNAs have a uniform distribution, but mRNAs encoded by other genes are distributed along an apical–basal gradient. Interestingly, genes of the same family may have mRNA gradients that have opposite directions or both mRNAs with a gradient and mRNAs with uniform expression. The gradient associated with certain genes can vary according to the developmental stage. The establishment of mRNA gradients depends on the activity of the cytoskeleton. In his classic grafting experiments in the thirties, Haemmerling realised that substances that were produced by the nucleus were responsible for the development of the different parts of the cell. Fragments of Acetabularia could autonomously generate some of the developmental program and retain their identity. This property probably relies on the gradients of mRNAs described in this study. • Centromeric localization and adaptative evolution of an Arabidopsis histone H3 variant. Talbert PB, Masuelli R, Tyagi AP, Comai L, Henikoff S: Plant Cell 2002, 14:1053-1066. Significance: In many organisms, a variant of the histone H3 is specifically located to the centromeres. Here, the authors report the identification of the centromeric histone of Arabidopsis thaliana. Findings: Sequence similarities are used to identify HTR12, a putative centromeric variant of the histone H3, in Arabidopsis. Antibodies against HTR12 show that it is preferentially localised in centromere in mitotically active cells, as well as in pollen during mitosis. In pollen, HTR12 accumulates in unusual bodies whose function remains unknown. The expression of HTR12 diminishes in terminally differentiated cells, as does the expression of the Drosophila centromeric histone CID. Comparisons of the sequences of HTR12 in different ecotypes of Arabidopsis thaliana and the related species Arabis arenosa shows a sharp change in the amino-terminal tail, which suggests a strong adaptative evolution of the Arabidopsis centromeric histone. A similar phenomenon is observed for CID in Drosophila, suggesting that centromeric histones are under the same evolutionary constraints across species. • Microtubule basis for left-handed helical growth in Arabidopsis. Thitamadee S, Tuchihara K, Hashimoto T: Nature 2002, 417:193-196. Significance: Left–right asymmetry and twisting are usual in plant organs. This report shows an essential and direct role of microtubule arrangement in this growth habit. Findings: Wildtype Arabidopsis stem and leaves do not twist but right-handed twisting mutants, spiral1 and spiral2, have been characterised. A suppressor screen for spiral1 led to the identification of the lefty1 and lefty2 mutants, which exhibit left-handed twisting of stems and leaves. The lefty mutations originate from single-base-pair changes in the tubulin genes TUA6 and TUA4. Unlike transverse microtubule cortical arrays in wildtype plants, lefty arrays form right-handed helices and have reduced microtubule stability.