Plant biology -

463 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 2001, 4 :463–472 Contents (chosen by) 463 Growth and development (Schneitz and Sablowski) 465 Genome studies and molecular genetics (Lemieux and Grossniklaus) 466 Plant biotechnology (Dunwell) 467 Physiology and metabolism (Hill and Sweetlove) 467 Biotic interactions (Metraux) 470 Cell signalling (Palme and McAinsh) 471 Gene regulation (Weisshaar) 471 Cell biology (Berger) Growth and development Selected by Kay Schneitz Institute of Plant Biology, University of Zürich, Zürich, Switzerland e-mail: [email protected] •• Pollen tube attraction by the synergid cell. Higashiyama T, Yabe S, Sasaki N, Nishimura Y, Miyagishima S-Y, Kuroiwa H, Kuroiwa T: Science 2001, 293 :1480-1483. Significance : This paper provides excellent evidence that the synergids attract the pollen tube during the final step of pollen-tube guidance to the ovule. Findings: In Torenia fournieri, the embryo sac protrudes from the sporophytic tissue allowing the laser-based cell ablation of individual cells in an in vitro culture system. Pollen-tube guidance was only affected when one or both synergids were ablated. Killing one synergid resulted in a reduction of guidance, whereas ablating both synergids led to a complete cessation of guidance. The results indicate that one synergid is sufficient for guidance. The remaining synergid in a fertilized embryo sac was not able to attract a pollen-tube, suggesting that an active mechanism shuts off signaling upon fertilization, thereby preventing polyspermy. •• Control of cell division by a retinoblastoma protein homolog in Chlamydomonas. Umen JG, Goodenough UW : Genes Dev 2001, 15 :1652-1661. Significance : The single-copy locus mat3 was shown to encode a retinoblastoma protein (RB) homolog. The genetic evidence presented in this paper indicates that Mat3p is required for two cell-size-dependent cell cycle decisions: the commitment to the cell cycle and the number of divisions to occur. In contrast to RB from mammals, Mat3p does not appear to be the primary regulator of S-phase entry. Findings: The mat3 gene was cloned and identified by insertional mutagenesis and genomic-rescue experiments. Sequencing revealed that Mat3p is most closely related to plant RBs. A cell-cycle analysis of mat3 and wild-type cells grown under various light/dark regimes revealed several features of the mat3 mutant. The mat3 cells show a reduced cell size, to about 60% of the wild-type size, at commitment to the cell cycle. They also show an increase in the number of cell divisions and a reduction in daughter cell size. Interestingly, the duration from commitment to beginning of mitosis is not significantly different in mat3 and wild-type cells. • Gibberellins signal nuclear import of PHOR1, a photoperiodresponsive protein with homology to Drosophila armadillo. Amador V, Monte E, García-Martínez J-L, Prat S: Cell 2001, 106 :343-354. Significance : The paper identifies PHOR1 as a component of the gibberellic acid (GA)-signaling mechanism. PHOR1 function appears to require its transient, GA-dependent relocation from the cytoplasm to the nucleus. Findings: PHOR1 was identified as an upregulated transcript in a differential display experiment using RNA isolated from leaves of S. demissum grown under either short days or long days. Sequence analysis indicated that PHOR1 encodes a protein with seven ARM repeats and a so-called CPI (Cys-Pro-Ile) domain. Transient and stable transformation experiments using tobacco BY2 cells and various PHOR1::GFP fusion constructs suggested that, in the presence of GA, PHOR1 transiently localizes to the nucleus. The ARM repeats seem to target PHOR1 to the nucleus, whereas the CPI domain appears to mediate the GA-dependent cytoplasmic retention of the protein. Analysis of plants with diminished or excessive PHOR1 expression substantiated the view that PHOR1 constitutes a component of GA signaling. •• Inactivation of AtRac1 by abscisic acid is essential for stomatal closure. Lemichez E, Wu Y, Sanchez J-P, Mettouchi A, Mathur J, Chua N-H: Genes Dev 2001, 15 :1808-1816. Significance : AtRac1, a Rho-type small GTPase, was identified as an important factor in abscisic acid (ABA)-mediated stomatal closure. ABA appears to exert its effect through inactivation of AtRac1, which subsequently results in a disorganization of the actin cytoskeleton. Findings: Using various GFP fusion protein marker lines the authors were able to show that ABA mediates actin, but not tubulin, cytoskeleton disorganization in guard cells during stomatal closure. Analysis of a line harbouring a dominant-negative AtRac1 mutation (AtRac1-T20N) revealed that negative interference with AtRac1 activity also results in actin network disorganization in guard cells and in stomatal closure. Furthermore, constitutive activation of AtRac1 resulted in plants whose stomatal closure was less sensitive than normal to ABA. Biochemical evidence showed that AtRac1 becomes inactivated upon ABA treatment in wild-type cell cultures. In contrast, ABA addition to cell cultures derived from abi1-1 mutants, which are insensitive to ABA, did not result in AtRac1 inactivation. Introducing AtRac1-T20N into an abi1-1 background resulted in stomatal closure. •• Establishment of polarity in lateral organs of plants. Eshed Y, Baum SF, Perea JV, Bowman JL: Curr Biology 2001, 11 :1251-1260. 464 Paper alert Significance : The KANADI (KAN) genes are shown to promote abaxial cell fate in lateral organs. They are distinct from the YABBY genes also implicated in this process. The KAN genes encode putative transcription factors of the GARP family. Findings: Several mutant alleles of KAN2 were identified as enhancers of the kan1 mutant phenotype. The kan1 kan2 double mutants exhibited strong adaxialisation of all lateral organs. The interpretation of the kan1 kan2 mutant phenotype was substantiated by, for example, the findings that the normally adaxially expressed REVOLUTA (REV) gene was ectopically expressed at the abaxial side, whereas the abaxial expression of the YABBY gene FILAMENTOUS FLOW ER (FIL) was either strongly reduced or absent. Cloning of KAN1 and KAN2 revealed the existence of four closely related KAN genes in the Arabidopsis genome, which form a subclass within the much larger GARP family. Ectopic expression of KAN1 , KAN2 , or KAN3 resulted in plants that arrested development at the late embryo or early seedling stage. These plants exhibited severe distortions due to the apparent abaxialisation of the tissues. •• Developmental changes due to long-distance movement of a homeobox fusion transcript in tomato. Kim M, Canio W, Kessler S, Sinha N: Science 2001, 293 :287-289. Significance : Evidence for the long-distance movement of a functional mRNA is presented. The movement overcomes graft borders: the mRNA expression pattern in the scion does not depend on the corresponding promoter activity in the stock. Thus, mRNA expression patterns may sometimes be the result of promoter activity and of transcript movement. Findings: The dominant Mouse ears (Me) mutation of tomato leads to altered leaf morphology. The Me mutation causes a gene fusion between PYROPHOSPHATE-DEPENDENT PHOSPHOFRUCTOKINASE (PFP) and the KNOTTED-1 -like homeobox gene LeT6 . It includes the upstream region of PFP. As a result of this fusion, strong ectopic expression of LeT6 was observed. In grafting experiments, a wild-type scion carrying the semidominant Xanthophyllic (Xa) mutation, which caused yellow normal-shaped leaves, was grafted onto a Me stock. Subsequently, the Xa scion developed leaves resembling the leaves from Me plants. Using reverse transcription (RT)-PCR, a functional PFP::LeT6 fusion transcript was identified only in Me plants and graft stocks, and in the phenotypically altered Xa scion leaves, but not in normal Xa plants. The PFP::LeT6 fusion transcript accumulated in the meristem of Me plants or Xa scions in a pattern that does not correspond to the regular PFP expression pattern. The PFP::LeT6 transcripts were also detected in phloem sieve tubes and companion cells but not in the xylem. Selected by Robert Sablowski John Innes Centre, Norwich, UK e-mail: [email protected] •• Interactions of the COP9 signalosome with the E3 ubiquitin ligase SCFTIR1 in mediating auxin response. Schwechheimer C, Serino G, Callis J, Crosby W L, Lyapina S, Deshaies RJ, Gray W M, Estelle M, Deng X-W : Science 2001, 292 :1379-1382. Significance: The CONSTITUTIVE PHOTOMORPHOGENIC 9 (COP9) complex has been previously implicated in the repression of photomorphogenesis by targeted protein degradation. Here, abundant evidence is presented to show that the same COP9 complex participates in protein degradation in response to auxin. Findings: Mutations in genes that encode components of the COP9 complex are lethal. Viable plants with reduced COP9 levels were obtained by co-suppression of the COP9 subunit CSN5. Adult plants with lower COP9 levels had phenotypes, including loss of apical dominance and resistance to root inhibition by exogenous auxin, similar to those of auxin response mutants. Extracts from these plants had reduced ability to degrade a reporter protein (i.e. a PSIAA6::luciferase fusion) that is targeted for degradation in response to auxin. An ubiquitination complex previously implicated in auxin responses (i.e. SCFTIR1) was associated with the COP9 complex in immunopurification experiments. The interaction between subunits of SCFTIR1 and of COP9 was confirmed in yeast two-hybrid experiments. Reduction of COP9 levels also enhanced the phenotype of axr1 mutants, which are defective in an enzyme cascade that attaches a ubiquitinrelated protein (RUB1) to SCFTIR1 . Plants with lower COP9 levels accumulated the RUB1-conjugated form of a SCFTIR1 subunit, providing additional biochemical evidence that the COP9 complex functions in the AXR1/ SCFTIR1 pathway. • BIG: a calossin-like protein required for polar auxin transport in Arabidopsis. Gil P, Dewey E, Friml J, Zhao Y, Snowden KC, Putterill J, Palme K, Estelle M, Chory J: Genes Dev 2001, 15 :1985-1997. Significance : Auxin is known to mediate growth changes in response to light (e.g. in phototropism). This paper shows an unexpected role of auxin distribution in repressing light-dependent genes in the dark. Findings: doc1 (dark overexpression of CAB ) mutants had been isolated in a screen for CAB (chlorophyll a/b binding protein) expression in dark-grown seedlings. Here, expression profiling with oligonucleotide arrays confirms the misexpression of light-dependent genes in doc1 . tir3 (transport inhibitor response 3 ) mutants had been independently isolated in a screen for resistance to the auxin transport inhibitor, NPA (N-1-naphthylphtalamic acid). The authors of this paper show that doc1 and tir3 map to the same position and have the same auxin-related defects (e.g. loss of apical dominance and reduced formation of lateral roots). tir3 and doc1 do not complement each other, and positional cloning confirms that both mutations affect the same gene. TIR3/DOC1 is renamed BIG because of the unusual size of the encoded protein (i.e. 5077 amino acids). BIG homologs are found in animals from Drosophila to humans, but their biochemical function is not known. The role of BIG in auxin transport is supported by crosses with other mutants that affect auxin transport or response, and by altered intracellular distribution of the auxin efflux carrier PIN1 in the presence of NPA. Ubiquitous increase in auxin levels (using the yucca mutation) suppresses the misexpression of light-dependent genes in tir3/doc1 , suggesting that the transport of auxin to specific cells is necessary to repress light-dependent genes in dark-grown seedlings. • Control of axillary bud initiation and shoot architecture in Arabidopsis through the SUPERSHOOT gene. Tantikanjana T, Yong JW H, Letham DS, Griffith M, Hussain M, Ljung K, Sandberg G, Sundaresan V: Genes Dev 2001, 15 :1577-1588. Significance : Branching of the angiosperm shoot is controlled at two main steps: establishment of axillary meristems and their release from growth arrest. Auxin regulates the growth of established axillary meristems. The authors of this paper suggest that local modulation of cytokinin levels controls the establishment of axillary meristems. Paper alert Findings: The supershoot (sps) Arabidopsis mutant has vastly increased branching, caused by supernumerary axillary meristems at the base of leaves and by failure to inhibit further growth of these meristems. sps plants also have defects in vein pattern and delayed senescence, suggesting increased cytokinin levels, which are confirmed. The gene is cloned by transposon tagging and found to encode a cytochrome P450. SPS is expressed in the vasculature and at the base of leaves, where axillary meristems develop. Together, the results suggest that SPS prevents excessive proliferation of axillary meristems by locally reducing the levels of cytokinin. Genome studies and molecular genetics Selected by Bertrand Lemieux* and Ueli Grossniklaus† *University of Delaware, Newark, Delaware, USA e-mail: [email protected] †Institute of Plant Biology, University of Zürich, Zürich, Switzerland e-mail: [email protected] • Predicting splice variant from DNA chip expression data. Hu G K, Madore SJ, Moldover B, Jatkoe T, Balaban D, Thomas J, Wang Y: Genome Res 2001, 11 :1237-1245. Significance : Alternative splicing of premessenger ribonucleic acids (premRNA) is an important layer of regulation in eukaryotic gene expression. Differences in the messenger RNA (mRNA) population attributed to splice variation have been implicated in various cell growth and differentiation processes. This report indicates that oligonucleotide-probe-based DNA chip assays could be used to detect splice variants on a genomewide scale. Findings: The authors developed two algorithms to normalize and compare the hybridization signals at individual oligonucleotide probe pairs used in GeneChip arrays. Gene expression data from 11 rat tissues were acquired using a high-density oligonucleotide array. On this array, 1600 rat genes were each represented by 20 pairs of oligonucleotide probes. The pairs of oligonucleotides used for these measurements consisted of a perfect match [PM] probe, complementary to the gene coding region, and a mismatch [MM] probe of each probe pair, which contained a mismatch with the known gene coding sequence. The PM probes predicted to detect potential tissue-specific splice variants were identified by the algorithms. The identified candidate splice variants were compared to the alternatively spliced transcripts predicted by expressed sequence tag (EST) clustering. Half of these putative alternative splice variants were confirmed by reverse transcription (RT)-PCR experiments. • An invasive cleavage assay for direct quantitation of specific RNAs. Eis PS, Olson MC, Takova T, Curtis ML, Olson SM, Vener TI, Ip HS, Vedvik KL, Bartholomay CT, Allawi HT et al.: Nat Biotechnol 2001, 19 :673-676. Significance : A flexible ribonucleic acid (RNA) analysis technology that can quantitatively monitor multiple forms of alternatively transcribed and/or processed RNAs is essential in elucidating the role of alternative splicing in the regulation of eukaryotic gene expression. Findings: The authors report the application of the invasive cleavage assay for detecting ribonucleic acid (RNA) in both total-RNA and cell-lysate samples. This method uses an improved 5 ′-nuclease and an isothermal fluorescence resonance energy transfer (FRET)-based signal amplification 465 strategy. The detection format used, termed the RNA invasive cleavage assay, obviates the need for target amplification or additional signal enhancement. In this report, the assay was used to detect low copy number RNA (fewer than 100 copies per reaction). The assay was able to discriminate between sequences that are 95% homologous over a 20 000-fold range, and quantitative differences in the 1.2-fold range were reproducibly detected. •• The composite genome of the legume symbiont Sinorhizobium meliloti. Galibert F, Finan TM, Long SR, Puhler A, Abola P, Ampe F, Barloy-Hubler F, Barnett MJ, Becker A, Boistard P et al.: Science 2001, 293 :668-672. •• Nucleotide sequence and predicted functions of the entire Sinorhizobium meliloti pSymA megaplasmid. Barnett MJ, Fisher RF, Jones T, Komp C, Abola AP, Barloy-Hubler F, Bowser L, Capela D, Galibert F, Gouzy J et al.: Proc Natl Acad Sci USA 2001 98 :9883-9888. •• Analysis of the chromosome sequence of the legume symbiont Sinorhizobium meliloti strain 1021. Capela D, Barloy-Hubler F, Gouzy J, Bothe G, Ampe F, Batut J, Boistard P, Becker A, Boutry M, Cadieu E et al.: Proc Natl Acad Sci USA 2001, 98 :9877-9882. •• The complete sequence of the 1,683-kb pSymB megaplasmid from the N 2 -fixing endosymbiont Sinorhizobium meliloti. Finan TM, Weidner S, Wong K, Buhrmester J, Chain P, Vorholter FJ, Hernandez-Lucas I, Becker A, Cowie A, Gouzy J: Proc Natl Acad Sci USA 2001, 98 :9889-9894. Significance : The observations reported by the authors suggest that nitrogen-fixing bacteria differ significantly in gene content and the organization of their genomes. Findings: Nitrogen fixation is accomplished through a tight metabolic association between legumes and rhizobial bacteria that reduces nitrogen (N2 ) to ammonium (NH 4 + ). The authors report the annotated DNA sequence of Sinorhizobium meliloti, the nitrogen-fixing symbiont of alfalfa. This bacterium has a tripartite genome of 6.7-megabases (Mb), composed of a 3.65-Mb chromosome as well as two plasmids: the 1.35-Mb pSymA and the 1.68-Mb pSymB. Analysis of the distribution of protein-coding regions within this genome indicates that all three elements contribute to symbiosis. The genetic information carried by pSymA or pSymB in S. meliloti is dispersed in the Mesorhizobium loti genome. Comparison of the S. meliloti genome sequence with that of the larger M. loti genome indicates that the latter contains 548 more proteins than the former. Thirty-five percent of M. loti genes have no ortholog in S. meliloti. Comparison of the S. meliloti genome to the sequence of the 536-kb Rhizobium sp. NG R234 symbiotic plasmid indicates that a high proportion (54%) of the genes encoded by this plasmid have no ortholog in S. meliloti. • Resistance of RNA-mediated TGS to Hc-Pro, a viral suppressor of PTGS, suggests alternative pathways for dsRNA processing. Mette MF, Matzke AJM, Matzke MA: Curr Biology 2001, 11 :1119-1123. Significance : Gene silencing in plants has traditionally been separated into transcriptional (TGS) and posttranscriptional gene silencing (PTGS), the latter being similar to RNA interference (RNAi) in animals. Lately, this distinction has become less clear. For instance, small interfering (si) RNAs (Hamilton AJ, Baulcombe DC: Science 1999, 286 :950-952), which were thought to be specific to PTGS, were also found to be involved 466 Paper alert in TGS. During TGS, double-stranded (ds)-RNA that contains promoter sequences can lead to RNA-directed methylation of homologous promoter sequences via a mechanism involving promoter siRNAs (Mette MF et al.: EMBO J 1999, 18 :241-248). This paper reports that HC-Pro, a viral suppressor of PTGS that prevents the accumulation of siRNAs, does not affect TGS induced by promoter dsRNA. These differential effects suggest that the processing of dsRNA into siRNAs can occur by alternative pathways. Findings: The effect of HC-Pro on RNA-mediated TGS was studied using a transgenic TGS system involving the H 9NP silencing locus, which produces dsRNA by transcription from a nopaline synthase promoter (NOSpro) inverted repeat, and an unlinked target NOS locus. Introduction of HC-Pro into this system did not release TGS of the NOS target locus nor did it alleviate H 9NP-induced methylation of the target promoter, as evidenced by Southern blots using methylation-sensitive restriction enzymes. In fact, promoter siRNAs were found to accumulate to approximately five-fold greater levels and methylation of the target promoter was slightly increased in the presence of HC-Pro. •• Requirement of CHROMOM ETHYLASE3 for maintenance of CpXpG methylation. Lindroth AM, Cao X, Jackson JP, Zilberman D, McCallum C, Henikoff S, Jacobson SE: Science 2001, 292 :2077-2080. •• Arabidopsis cmt3 chromomethylase mutations block non-CG methylation and silencing of an endogenous gene. Bartee L, Malagnac F, Bender J: Genes Dev 2001, 15 :1753-1758. •• Maize chromomethylase Zea methyltransferase2 is required for CpNpG methylation. Papa CM, Springer NM, Muszynski MG, Meeley R, Kaeppler SM: Plant Cell 2001, 13 :1919-1928. Significance : Cytosine methylation plays an important role in the regulation of gene expression in both plants and mammals. Many insights into the function of methylation have come from the study of mutants with reduced cytosine methylation in plants. Recently, a new plant-specific class of methylases was reported. In addition to the signature domains of methyltransferases, members of this class also contain a chromodomain (Henikoff S, Comai L: Genetics 1999, 149 :307-318). These reports demonstrate that CHROMOMETHYLASE3 (CMT3 ) and its maize homolog are required for the methylation of CpXpG sequences and for maintaining epigenetic gene silencing. Findings: Mutations in the Arabidopsis CMT3 gene were identified in genetic screens for suppressors of an epigenetically silenced SUPERMAN (SUP) locus (clark kent alleles) and a mutant variant PAI (phosphoribosylanthranilate isomerase) locus, which contains a crippled PAI1 gene and the densely methylated PAI2 gene. One of the second site suppressor loci that relieve silencing of the clark kent alleles and PAI2 was found to affect the CMT3 locus. Either DNA digested with methylationsensitive enzymes for Southern blots or direct sequencing of bisulfite-treated DNA was used to investigate the effect of cmt3 mutations on methylation at the SUP and PAI loci, as well as in other methylated regions (including centromeric repeats and retrotransposons). This analysis clearly showed that CMT3 is specifically responsible for CpXpG methylation. A reverse genetic approach was taken to identify mutants affected in a homolog of CMT3 in maize, Zmet2 . Investigation of the methylation profiles at various repeats showed reduced methylation restricted to CpXpG sites in this mutant. • SWITCH1 ( SWI1 ): a novel protein required for the establishment of sister chromatid cohesion and for bivalent formation at meiosis. Mercier R, Vezon D, Bullier E, Motamayor JC, Sellier A, Lefevre F, Pelletier G, Horlow C: Genes Dev 2001, 15 :1859-1871. Significance : Meiosis is almost universal among eukaryotes, and its genetic control is well characterized in both budding and fission yeast. Although many meiotic mutants have been described in plants, only few of the affected genes have been cloned and studied at the molecular level. Some insights into the control of plant meiosis have come from studying the homologs of yeast genes in Arabidopsis. In contrast, the authors of this paper report the isolation of SW ITCH1 (SW I1 ), a novel gene involved in the establishment of meiotic sister chromatid cohesion, which was identified on the basis of its sterile phenotype. Findings: A second allele of swi1 (swi1-2 ) was isolated from a chemically mutagenized population. Unlike the original swi1-1 allele, which was female specific, swi1-2 affects both male and female meiosis. Cell biological experiments showed that in swi1-2 male meiocytes, a progressive loss of cohesion during prophase I leads to the formation of 20 instead of 5 chromatids at metaphase I and to aberrant chromosome segregation. In contrast, swi1-2 female meiocytes undergo a mitosis-like division, suggesting a different effect or a distinct role of SW I1 in male and female meiosis. Cloning of SW I1 did not reveal homology to known proteins with the exception of limited similarity to a conserved region of SMC (structural maintenance of chromosomes) family proteins. A SW I1::G FP fusion protein was found to be expressed prior to meiosis and during the early stages of prophase I in meiocytes, consistent with the role of SW I1 in the establishment of chromatid cohesion. Plant biotechnology Selected by Jim Dunwell University of Reading, Berkshire, UK e-mail: [email protected] •• Transgenic salt-tolerant tomato plants accumulate salt in foliage but not in fruit. Zhang HX, Blumwald E: Nat Biotechnol 2001, 19 :765-768. Significance : Many strategies have been developed in an attempt to improve the performance of crops in saline soils. This report describes success with a novel method that might have general applicability. Findings: Transgenic tomato plants overexpressing a vacuolar Na+ / H + antiport protein were able to grow, flower, and produce fruit in the presence of 200 mM sodium chloride. Although the leaves accumulated sodium at high concentrations, importantly, the fruit displayed a very low sodium content. These positive results from the use of a single introduced gene demonstrate that it might be possible to produce salt-tolerant crops by modifying far fewer target traits than had been suggested previously. • Feeding value of corn silage estimated with sheep and dairy cows is not altered by genetic incorporation of Bt1376 resistance to Ostrinia nubilalis. Barriere Y, Verite R, Brunschwig P, Surault F, Emile JC: J Dairy Sci 2001, 84 :1863-1871. Significance : Although transgenic field crops are grown over large areas, there have been relatively few published reports on Paper alert the results of feeding of such crops to farm animals. This report provides such data. Findings: Silage from a genetically modified maize hybrid, which is resistant to the European corn borer, and from the conventional isogenic hybrid were evaluated in three separate feeding trials with sheep and cattle. On the basis of data on weight gain, milk yield and milk quality, it was concluded that there was no significant difference in the feeding quality of conventional and genetically modified silage. • Towards development of an edible vaccine against bovine pneumonic pasteurellosis using transgenic white clover expressing a Mannheimia haemolytica A1 leukotoxin 50 fusion protein. Lee RW, Strommer J, Hodgins D, Shewen PE, Niu Y, Lo RY: Infect Immun 2001, 69 :5786-5793. Significance : These results provide further evidence that transgenic plants can be used successfully as a source of edible vaccines. In this example, the vaccine is expressed in a forage crop and could be used to protect cattle against an important veterinary disease, bovine pneumonia pasteurellosis or shipping fever. Findings: A transformation construct that employs the cauliflower mosaic virus 35S promoter linked to a derivative of the Mannheimia haemolytica A1 leukotoxin gene (Lkt) was selected and introduced into white clover by Agrobacterium-tumefaciensmediated transformation. Transgenic lines were recovered, and expression of Lkt was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. An extract from transgenic plants that contained Lkt was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. Physiology and metabolism Selected by Steven Hill* and Lee Sweetlove† University of Oxford, Oxford, UK *e-mail: [email protected] †e-mail: [email protected] • Rerouting the plant phenylpropanoid pathway by expression of a novel bacterial enoyl-CoA hydratase/ lyase enzyme function. Mayer MJ, Narbad A, Parr AJ, Parker ML, Walton NJ, Mellon FA, Michael AJ: Plant Cell 2001, 13 :1669-1682. Significance : The importance of phenylpropanoids in plant growth and development is demonstrated, and a method for generating novel metabolites in plants is described. Findings: Transgenic tobacco plants were produced that overexpress a bacterial enzyme that is capable of modifying intermediates of the plant phenylpropanoid synthesis pathway. The transgenic plants show dramatic phenotypic changes when compared with wildtype plants, including stunting, premature senescence and male sterility. There were also changes in the lignin composition of the xylem and in phloem development. Furthermore, most of the phenylpropanoids that accumulate in the wildtype were present only at very low levels in these transgenics. Significant amounts of novel phenylpropanoids accumulated in the transgenics, however, mostly as glucosides and glucose esters. •• Transpiration, a prerequisite for long-distance transport of minerals in plants? Tanner W, Beevers H: Proc Nat Acad Sci USA 2001, 98 :9443-9447. 467 Significance : This paper challenges the accepted view that transpiration is required for mineral uptake. Findings: Sunflower plants growing hydroponically were supplied with mineral nutrients either during the day (when transpiration rates are high) or during the night (when transpiration rates are much lower). The night-supplied plants grew at the same rate and acquired similar quantities of nutrients as did the day-supplied plants, even though their rate of transpiration during nutrient acquisition was much lower. Measurement of root ion content excludes the possibility that the night-supplied plants were storing ions for translocation during the day. These experiments suggest that transpiration is not required for ion movement, and the authors suggest that root pressure, and water recycling due to phloem transport, may be sufficient to account for ion flow. •• The Arabidopsis sex1 mutant is defective in the R1 protein, a general regulator of starch degradation in plants, and not in the chloroplast hexose transporter. Yu T-S, Kofler H, Häusler RE, Hille D, Flügge U-I, Zeeman SC, Smith AM, Kossmann J, Lloyd J, Ritte G et al.: Plant C ell 2001, 13 :1907-1918. Significance : The authors of this paper identify a mutation responsible for reduced leaf starch degradation in Arabidopsis, and propose a putative role for starch phosphorylation in the regulation of leaf starch degradation. Findings: The sex1 mutation of Arabidopsis has previously been described and proposed to be caused by absence of the chloroplastic glucose transporter. In this work, the sex1 locus was cloned by positional cloning and shown to be homologous to the R1 protein of potato. In a number of alleles of sex1 , the mutation is found in the R1 gene. The starch of sex1 plants has reduced phosphate content, which is also consistent with the proposed role of R1 in starch phosphorylation. It is concluded that starch phosphorylation is required for starch degradation. • A T-DNA insertion knockout of the bifunctional lysineketoglutarate reductase/ saccharopine dehydrogenase gene elevates lysine levels in Arabidopsis seeds. Zhu X, Tang G, Granier F, Bouchez D, Galili G: Plant Physiol 2001, 126 :1539-1545. Significance: The importance of lysine breakdown in determining the lysine content of seeds is shown. Findings: A mutant is isolated that lacks the bifunctional protein that catalyses the first two steps of lysine breakdown. The mutant plants grow at a similar rate to the wild type, and there are no obvious phenotypic differences. However, the lysine content of the seed of the mutant is significantly increased, providing evidence that there is substantial lysine catabolism during normal seed development. Biotic interactions Selected by Jean-Pierre Metraux University of Fribourg, Fribourg, Switzerland e-mail: [email protected] •• NIM1 overexpression in Arabidopsis potentiates plant disease resistance and results in enhanced effectiveness of fungicides. Friedrich L, Lawton K, Dietrich R, Willits M, Cade R, Ryals J: Mol Plant Microbe Interact 2001, 14 :1114-1124. 468 Paper alert Significance : Overexpression of a key regulatory gene in the signal transduction of salicylic acid (SA) improves plant health and leads to improved fungicide effectiveness. Findings: The NIM1 (NON-INDUCIBLE IMMUNITY1 , also known as NPR1 ) gene, which encodes a nuclear-localized regulator of the expression of pathogenesis-related proteins PR1, is an important activator of systemic acquired resistance (SAR) in Arabidopsis. Overexpression of NIM1 in wild-type plants leads to an increase in resistance to different pathogens. NIM1 lines also exhibit an increase in the levels of NIM1 and a rapid induction of PR1 gene expression. However, levels of SA, an endogenous signal required for SAR induction, are not significantly increased in NIM1 lines compared with wild-type plants. Enhanced resistance in NIM1 lines depends on SA, suggesting that overexpression of NIM1 increases the responsiveness to SA or a SA-dependent signal. Further support for this hypothesis was obtained from NIM1 lines that exhibit increased responsiveness to the SAR-inducing compound benzo(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester. Furthermore, the increased efficacy of three fungicides was observed in the NIM1 lines. Thus, a novel disease-control strategy might involve a combination of both transgenic and chemical approaches. •• Transgenic plants producing the bacterial pheromone N-acyl-homoserine lactone exhibit enhanced resistance to the bacterial phytopathogen Erwinia carotovora . Mäe A, Montesano M, Koiv V, Palva TE: Mol Plant Microbe Interact 2001, 14 :1035-1042. Significance : Overexpression of a bacterial pheromone that induces the production of plant cell wall degrading enzymes by the bacteria, and leads to activation of plant defense, protects plants against a phytopathogenic bacterium. Findings: Previous work by these authors demonstrated that the bacterial pheromone N-oxoacyl-homoserine lactone (OHL) is essential for quorum sensing in Erwinia carotovora, the causal agent of soft rot. OHL operates a cell-density-dependent control of the activation of the main virulence genes and the extracellular plant cell wall degrading enzymes (PCW DEs). This quorum sensing might be important for E. carotovora to avoid the premature production of PCW DEs and the subsequent activation of plant defense responses. To test this hypothesis, transgenic tobacco producing OHL were generated by ectopic expression of the E. carotovora gene expI, which is responsible for OHL biosynthesis. Tobacco lines overexpressing expI produced the active pheromone and partially complemented the avirulent phenotype of bacterial expI mutants. The OHL-producing tobacco lines exhibited enhanced resistance to infection by wild-type E. carotovora. The results were confirmed by ectopic treatment of wild-type plants with OHL, which also resulted in increased resistance to E. carotovora. These interesting results might provide an interesting approach for the protection of plants to bacterial diseases. • HSR203 antisense suppression in tobacco accelerates development of hypersensitive cell death. Tronchet M, Ranty B, Marco Y, Roby D: Plant J 2001, 27 :115-127. Significance : A new serine hydrolase with esterase activity controls hypersensitive cell death, defense gene expression and resistance to different pathogens. Findings: The activation of the tobacco gene hsr203 was shown to strongly correlate with programmed cell death occurring in response to diverse pathogens. The authors characterize the gene product and show that it is a serine hydrolase that displays esterase activity. Antisense suppression of hsr203 protein leads to an accelerated hypersensitive response (HR) when the plants are inoculated with an avirulent strain of Ralstonia solanacearum. This was accompanied by a drastic inhibition of in planta bacterial growth. Transgenic plants deficient in HSR203 were also found to show increased resistance to the avirulent bacterium Pseudomonas syringae pv. pisi, and to virulent and avirulent races of Phytophthora parasitica (a fungal pathogen of tobacco), but not to other virulent bacteria. Unexpectedly, the expression of hsr51 , a gene that also correlates with HR, and of the pathogenesis-related defense genes PR1-a and PR5, was strongly reduced in the transgenic lines. Thus, antisense suppression of hsr203 in tobacco can have pleiotropic effects on HR and defense mechanisms, and can induce pathogen resistance. hsr203 might be involved in detoxification or lipid signaling. • Evidence for a disease-resistance pathway in rice similar to the NPR1-mediated signaling pathway in Arabidopsis. Chern MS, Fitzgerald HA, Yadav RC, Canlas PE, Dong X, Ronald P: Plant J 2001, 27 :101-113. Significance : Monocots and dicots share a conserved NPR1 / NIM1 -mediated signal transduction pathway. Findings: The NPR1/NIM1 gene is a key regulator of systemic acquired resistance (SAR) in Arabidopsis. Here, the role of NPR1 was investigated in monocots. The authors overexpressed the Arabidopsis NPR1 gene in rice and challenged the transgenic plants with the rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo). The NPR1 -overexpressing rice plants displayed enhanced resistance to Xoo, and this required an expression of the NPR1 mRNA above a threshold level. The transgenic rice plants were used to identify components mediating the resistance controlled by NPR1 . Four cDNA clones, encoding NPR1 interactors (named rTGA2.1, rTGA2.2, rTGA2.3 and rLG2) belonging to the bZIP family, were identified by yeast two-hybrid screening. Three of these interactors share between 75% and 78% sequence identities with Arabidopsis TGA2. A fourth interactor shares 81% sequence identity to the maize LIG ULELESS2 (LG2) gene product, which is involved in establishing the leaf blade–sheath boundary. The interaction of the rice bZIP proteins was impaired by mutations in the nim gene. In vitro pull-down experiments confirmed NPR1–rTGA2.1 binding, and in gel mobility shift assays showed that rTGA2.1 binds to the rice RCH10 promoter and to a cis-element required for salicylic-acid responsiveness. This is an interesting study as it shows for the first time that monocot and dicot plants share a conserved signal transduction pathway controlling NPR1-mediated resistance. •• A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF: Proc Natl Acad Sci USA 2001, 98 :9448-9453. Significance: A defect in fatty-acid saturation can affect salicylic acid (SA)- and jasmonic acid (JA)-dependent disease resistance. Findings: The NPR1 gene is a key regulator of the SA signaling pathway for systemic acquired resistance (SAR) in Arabidopsis. Other defense signaling pathways have also been observed to operate in addition to that regulated by NPR1 . A screening for suppressors of the npr1-5 mutation identified the ssi2 mutant, which constitutively expresses the PR gene, forms spontaneous lesions, and displays enhanced resistance to Peronospora parasitica. In contrast, certain defense responses regulated by Paper alert the JA signaling pathway, including expression of the defensin gene PDF1.2 and resistance to Botrytis cinerea, are impaired in ssi2 plants. The authors cloned the SSI2 gene and showed that it encodes a stearoyl-ACP desaturase (S-ACP DES). S-ACP DES is a member of a family of soluble fatty acid (FA) desaturases that play an important role in regulating the overall level of desaturated FAs in the cell. Consistent with these findings, the ssi2 mutant exhibits a 10-fold reduction in S-ACP DES enzyme activity, resulting in elevated levels of the 18:0 FA content. These results strongly support a role for FA-derived signals in certain defense responses, and indicate the occurrence of crosstalk among different defense signaling pathways. The Arabidopsis aberrant growth and death2 mutant shows resistance to Pseudomonas syringae and reveals a role for NPR1 in suppressing hypersensitive cell death. Rate DN, Greenberg JT: Plant J 2001, 27 :203-211. •• Significance : A novel Arabidopsis mutant was found that is affected in its resistance to both virulent and avirulent forms of a bacterial pathogen. This mutant reveals a hitherto unknown function of NPR1, an important regulator of systemic induced resistance, in the regulation of the hypersensitive response. Findings: A novel recessive Arabidopsis mutant, called agd2 (aberrant growth and death2 ), was found in a screening of plants with a pathogen-responsive reporter transgene that contained the β-glucuronidase (G US) coding region. The agd2 mutants show overexpression of G US and display the following features: an increase in salicylic acid (SA)-dependent resistance to virulent and avirulent strains of the bacterial pathogen P. syringae, elevated SA levels, a low level of hypersensitivetype spontaneous cell death (HR), callose deposition, and enlarged leaf cells. The SA signaling regulator NPR1 (NONEXPRESSO R O F PR1) is required only for enhanced resistance of agd2 to virulent P. syringae, whereas resistance to P. syringae carrying avrRpt2 is NPR1 -independent. Inactivation of NPR1 is also required for the formation of a HR in agd2 plants. Interestingly, npr1 mutants show a stronger HR than wild-type plants, whereas NPR1 -overproducing plants show a weaker HR when infected with P. syringae carrying the avrRpm1 gene. HR in agd2 is partially suppressed by npr1 , indicating that HR can be modulated positively or negatively by NPR1 , depending on its cellular context. When agd2 plants are depleted of SA, cell growth and callose deposition are strongly enhanced, suggesting a role for SA in regulating growth and certain cell-wall modifications. AG D2 may be involved in the regulation of cell death and/or growth and pathogen defense, making its function comparable to that of NFkB in animal cells. In summary, agd2 affects a SA- and an NPR1-dependent defense pathway that responds to both virulent and avirulent forms of P. syringae, and a SA-dependent, NPR1-independent pathway directed at avirulent races of P. syringae. • Activation of salicylic acid-induced protein kinase, a mitogen-activated protein kinase, induces multiple defense responses in tobacco. Zhang S, Liu Y: Plant Cell 2001, 13 :1877-1889. Significance : Salicylic-acid-induced protein kinase (SIPK) alone is sufficient to activate the salicylic-acid-induced signaling cascade for defense responses. Findings: Previous work by these authors has shown that constitutive overexpression in tobacco of a MAPK kinase, NtMEK2DD, induces the expression of defense genes and hypersensitive response (HR)-like cell death. The activation of 469 two endogenous MAPKs, a SIPK and a wounding-induced protein kinase (W IPK) precede this. This kinase cascade is further explored to characterize the roles of SIPK and W IPK in the induction of defense responses. Both kinases are known to be rapidly activated in plants that are challenged by avirulent pathogens or in cells treated with pathogen-derived elicitors. Expression of SIPK under an inducible promoter in tobacco leaves results in a transient increase of SIPK activity that is sufficient to induce defense gene expression and HR-like cell death. In contrast, expression of W IPK under the same conditions does not lead to an increase in W IPK activity, although W IPK is expressed at a similar level as SIPK. Using chimeras, the authors show that the information for the differential regulation of SIPK and W IPK resides at their carboxyl termini. This gain-of-function study supports a direct role for SIPK in the signaling cascade of multiple defense responses in tobacco. • A gene related to yeast HOS2 histone deacetylase affects extracellular depolymerase expression and virulence in a plant pathogenic fungus. Baidyaroya D, Brosch G, Ahna J, Graessle S, Wegenera S, Tonukaria NJ, Caballero O, Loidl P, Walton JD: Plant Cell 2001, 13 :1609-1624. Significance : A histone deacetylase from a fungal pathogen of maize was found that is required for virulence and the activation of several genes. Findings: Cochliobolus carbonum is a pathogen of maize that produces HC-toxin, an inhibitor of histone deacetylase (HDAC). The authors examined the function of a gene hos1 related to the Saccharomyces cerevisiae HDAC gene, HOS2, by targeted gene replacement in C. carbonum. Mutants have smaller and less septate conidia than wild-type pathogens, exhibit a 50% reduction in total HDAC activity and show a decreased penetration efficiency associated with strongly reduced virulence. Growth of hdc1 mutants in vitro is normal on glucose, slightly decreased on sucrose, and reduced by 30–73% on other simple and complex carbohydrates. The hdc1 mutants exhibit downregulated activities and gene expression levels. Except for abnormal conidial morphology, the phenotypes of hdc1 match those of the C. carbonum mutant ccSNF1 , which is altered in a protein kinase necessary for the expression of glucose-repressed genes. These results show that HDC1 has multiple functions and is required for full virulence of C. carbonum on maize. They demonstrate that HDC1 does not act like a corepressor, as observed for many HDACs, but seems to be required for the induction of a number of fungal genes. It may be possible that the induction of maize defense genes has similar requirements for HDAC activity. • Introduction of the phzH gene of Pseudomonas chlororaphis PCL1391 extends the range of biocontrol ability of phenazine-1-carboxylic acid-producing Pseudomonas spp. strains. Chin-A-Woeng TFC, Thomas-Oates JE, Lugtenberg BJJ, Bloemberg GV: Mol Plant Microbe Interact 2001, 14 :1006-1015. Significance : A single gene was found to extend the biocontrol activity of a soil-borne biocontrol agent. Findings: The biocontrol agent Pseudomonas chlororaphis PCL1391 protects against tomato foot and root rot caused by Fusarium oxysporum f. sp. radicis-lycopersici. Its activity depends on the production of phenazine-1-carboxamide (PCN). In contrast, P. fluorescens 2-79 and P. aureofaciens 30-84, two biocontrol strains that are effective against take all in wheat, 470 Paper alert which produce phenazine-1-carboxylic acid (PCA), do not control F. oxysporum f. sp. radicis-lycopersici. This raises the question of the importance of the amide group in the biocontrol of soft rot. A novel phenazine biosynthetic gene phzH was shown to be required for the presence of the amide group of PCN. The deduced PhzH protein shares homology with asparagine synthetase, a class II glutamine amidotransferase. This indicates that the conversion of PCA to PCN occurs via transamidation that is catalyzed by PhzH. The importance of a functional phzH gene for PCN synthesis was demonstrated in a phzH mutant strain that accumulated PCA. Mutation of phzH caused a loss of biocontrol activity against tomato foot and root rot. PCN production and biocontrol activity were, however, restored by complementing the mutant with an intact phzH gene. Moreover, transformation of biocontrol strains P. fluorescens 2-79 and P. aureofaciens 30-84 with the phzH gene, enables these strains to produce PCN instead of PCA and to suppress tomato foot and root rot. This demonstrates nicely how the introduction of a single gene can efficiently extend the range of the biocontrol activity of bacterial strains. Cell signalling Selected by Klaus Palme Max-Delbrück Laboratory, Köln, Germany e-mail: [email protected] •• Reciprocal regulation between TOC1 and LHY/ CCA1 within the Arabidopsis circadian clock. Alabadí D, Oyama T, Yanovsky MJ, Harmon FG, Más P, Kay SA: Science 2001, 293:834-838. Significance : Interactions form a loop that is critical for clock function in Arabidopsis. Findings: Interactive regulation of the Arabidopsis clock genes LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1), and TIMING OF CAB EXPRESSION 1 (TOC1 ) is central to oscillator function. Moreover, the MYB transcription factors LHY and CCA1 negatively regulate TOC1 expression, and both proteins bind to a region in the TOC1 promoter that is critical for its clock regulation. •• Developmental abnormalities and reduced fruit softening in tomato plants expressing an antisense RAB11 GTPase gene. Lu C, Zainai Z, Tucker GA, Lycett GW : Plant Cell 2001, 13 :1819-1833. Significance : The antisense phenotypes of RAB11 GTPase provide insights into exocytosis/endocytosis in plants. Findings: A tomato cDNA encoding a protein with homology to the rab11/YPT3 class of small GTPases is characterized. Its mRNA is developmentally regulated during fruit ripening. Its expression was inhibited in several ripening mutants (e.g. Never-ripe), indicating that it may be induced by ethylene in fruit. Antisense fruit changed color as expected but failed to soften normally and showed reduced levels of cell-wall pectinesterase and polygalacturonase. Other phenotypic effects observed in antisense plants were determinate growth, reduced apical dominance, branching of inflorescences, abnormal floral structure, and the formation of ectopic shoots on the leaves. In antisense plants, ethylene production was reduced. The data suggest an alternative or additional role for this RAB11 GTPase gene in the exocytosis or endocytosis of homeotic proteins, in hormone carriers, or in receptors. •• The TRANSPARENT TESTA12 gene of Arabidopsis encodes a multidrug secondary transporter-like protein required for flavonoid sequestration in vacuoles of the seed coat endothelium. Debeaujon I, Peeters AJM, Léon-Kloosterziel KM, Koornneef M: Plant C ell 2000, 100 :469-478. •• Arabidopsis ALF5 , a multidrug efflux transporter gene family member, confers resistance to toxins. Diener AC, Gaxiola RA, Fink G R: Plant Cell 2001, 13 :1625-1637. •• The Arabidopsis thaliana ABC transporter AtMRP5 controls root development and stomata movement. Gaedeke N, Klein M, Kolukisaoglu U, Forestier C, Muller A, Ansorge M, Becker D, Mamnun Y, Kuchler K, Schulz B et al.: EMBO J 2001, 20 :1875-1887. Significance : Analysis of several multidrug transporters from Arabidopsis reveals important functions. Findings: Multidrug transporters represent a large group of genes in the Arabidopsis genome. Five superfamilies were found: the AB C transporter family, the major facilitator family, the small drug resistance family, the resistance–nodulation–cell-division family and the multidrug and toxic compound extrusion (MATE) family. These papers present significant advances in our understanding of different members of these groups. Debeaujon et al. reported that the TRANSPARENT TESTA12 (TT12 ) gene encodes a member of the MATE family in Arabidopsis. This gene controls the vacuolar sequestration of flavonoids in the seed coat endothelium. Mutant seeds lacking TT12 are pale and less dormant, supporting an important role for flavonoids in seed biology. Diener et al. characterized an Arabidopsis root mutant (alf5 ) that has greatly reduced lateral roots. ALF5 was cloned and shown to be another member of the MATE family that, when heterologously expressed in yeast, conferred resistance to the toxic cation tetramethylammonium. Finally, Gaedeke and coworkers characterized a member of the AB C transporter family (AtMRP5 ) that showed cystic fibrosis transmembrane conductance regulator (CFTR)-like properties. The data suggest that this gene may play a role in coordinating the efflux of cations during stomatal closure. Interestingly, altered hormone (i.e. auxin) levels were found in mutants, probably accounting for some of the phenotypic changes observed. Selected by Martin R McAinsh Lancaster University, Lancaster, UK e-mail: [email protected] • Reciprocal regulation between TOC1 and LHY/ CCA1 within the Arabidopsis circadian clock. Alabadi D, Oyama T, Yanovsky MJ, Harmon FG, Más P, Kay SA: Science 2001, 293 :880-883. Significance : A critical role for the interactive regulation among clock genes in the function of the Arabidopsis circadian clock. Findings: The authors examined the interaction between three genes LATE ELONGATED HYPOCOTYL (LHY), CIRCADIAN CLOCK ASSOCIATED 1 (CCA1 ), and TIMING OF CAB EXPRESSION 1 (TOC1 ), which are components of the Arabidopsis circadian clock. They show that the MYB transcription factors LHY and CCA1 act as negative elements that repress TOC1 expression by binding to a region in the TOC1 promoter (the evening element [EE]) that is central to its clock regulation. Conversely, TOC1 appears to be a positive element for LHY and CCA1 expression. A model showing the interaction among TOC1 , LHY and CCA1 within the Arabidopsis circadian clock is presented. Paper alert •• An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis. Hugouvieux V, Kwak JM, Schroeder JI: C ell 2001, 106 :477-487. Significance : Demonstration of a novel functional link between mRNA processing and modulation of early ABA signal transduction. Findings: The authors isolated a recessive ABA-hypersensitive mutant, abh1 , which has enhanced ABA sensitivity rather than biosynthesis and shows no significant effects in response to other known hormones. ABH1 encodes an 848-amino-acid protein that is a subunit of an Arabidopsis heterodimeric nuclear mRNA cap-binding complex. Gene-chip analysis shows that the abh1 mutation modulates only a limited number of genes, several of which are implicated in ABA signalling. The abh1 mutation enhances the probability of ABA-induced increases in guard cell cytosolic calcium, and stomata of abh1 plants exhibit ABA-hypersensitive closure. The authors conclude that abh1 amplifies early ABA signalling and that ABH1 modulates ABA signalling by altering transcript levels of early signalling components. • Ethylene inhibits the Nod factor signal transduction pathway of Medicago truncatula . Oldroyd GED, Engstrom EM, Long SR: Plant Cell 2001, 13 :1835-1849. Significance : Interaction between the ethylene and Nod factor signal transduction pathways is demonstrated. Findings: The authors used direct and indirect ethylene application, inhibition of ethylene biosynthesis and the ethylene-insensitive mutant sickle (skl) to study the effects of ethylene on nodulation responses in Medicago truncatula. They show that ethylene can modulate many aspects of the plant’s response to Nod factors including root-hair deformation, the induction of RIP1 and ENOD11 gene expression, and the initiation of calcium spiking. The authors conclude that ethylene acts at or upstream of the point of calcium spiking in the Nodfactor signal transduction pathway, either directly or through feedback from ethylene effects on downstream events. A model for the interaction between the ethylene-perception pathway and the Nod-factor-perception pathway is presented. • A fatty acid desaturase modulates the activation of defense signaling pathways in plants. Kachroo P, Shanklin J, Shah J, Whittle EJ, Klessig DF: Proc Natl Acad Sci USA 2001, 98 :9448-9453. Significance : A role for fatty-acid-derived signals in modulating crosstalk between defence signalling pathways. Findings: The authors used a map-based approach to clone the SSI2 gene from Arabidopsis. They show that ssi2 mutants exhibit constitutive activation of a NPR1 -independent defence signalling pathway, leading to spontaneous lesion formation, pathogenesis-related gene expression, and enhanced resistance to Peronospora parasitica. In contrast, the activation of jasmonic-acid-inducible defence responses, including the expression of the PDF1.2 gene and resistance to Botrytis cinerea, is impaired in ssi2 plants. The SSI2 gene encodes a stearoyl-ACP desaturase (S-ACP DES), which is a member of a family of soluble fatty acid desaturases. ssi2 mutants exhibit a 10-fold reduction in S-ACP DES activity, resulting in an elevation of the 18:0 fatty-acid content of plants. Therefore, the authors suggest that a fatty-acidderived signal modulates crosstalk between different defence signalling pathways. 471 Gene regulation Selected by Bernd Weisshaar Max-Planck-Institut für Züchtungsforschung, Köln, Germany e-mail: [email protected] • The Arabidopsis TT2 gene encodes an R2R3 MYB domain protein that acts as a key determinant for proanthocyanidin accumulation in developing seed. Nesi N, Jond C, Debeaujon I, Caboche M, Lepiniec L: Plant Cell 2001, 13:2099-2114. Significance : The authors present data that give new insights into the regulation of seed coat pigmentation in A. thaliana. Following on from the identification of the ‘structural’ transparent testa (tt) loci, which encode enzymes of phenylpropanoid metabolism, a picture of the regulation of these genes is now emerging from studies addressing the ‘regulatory’ tt loci. Findings: Isolation and functional characterisation of the gene encoding TT2 (or AtMYB123). The regulation of seed coat pigmentation in A. thaliana resembles that of kernel pigmentation in Zea mays: together, the R2R3-MYB factor C1 and bHLH factors, such as R, control the latter, whereas TT2 (a R2R3-MYB factor) and TT8 (a bHLH factor) are both required for the former. TT2 and TT8 function in a combinatorial way to activate the genes encoding enzymes for late steps of proanthocyanidin biosynthesis. The BANYULS gene, which is believed to encode the late enzyme leucoanthocyanidin reductase (LAR), has been identified as a potential target gene for TT2. • Developmentally distinct MYB genes encode functionally equivalent proteins in Arabidopsis. Lee MM, Schiefelbein J: Development 2001, 128 :1539-1546. • Ectopic expression of the Arabidopsis AtMYB23 gene induces differentiation of trichome cells. Kirik V, Schnittger A, Radchuk V, Adler K, Hülskamp M, Baumlein H: Devel Biol 2001, 235 :366-377. Significance : G L1, W ER, and AtMYB23 are shown to encode functionally equivalent proteins or at least factors with strongly overlapping function at the level of the protein. Findings: The authors studied the action of two members of the large family of R2R3-MYB factors that show significant amino-acid sequence similarity to G L1 (G LABRA1, AtMYB0), which is required for trichome formation: W EREWOLF (W ER, AtMYB66), which is involved in root hair formation, and AtMYB23. Without detailed analysis, these three genes might have been considered ‘redundant’. However, although these genes seem to perform related functions in gene regulation, their different temporal and/or spatial expression characteristics result in their involvement in different biological processes. Cell biology Selected by Frederic Berger Reproduction et Development des Plantes, Lyon, France e-mail: [email protected] • Male meiotic spindle lengths in normal and mutant Arabidopsis cells. Yang M, Ma H: Plant Physiol 2001, 126 :622-630. Significance : Careful observations of the meiotic spindle in the wildtype and a mutant lead to provocative speculations on the mechanism of spindle elongation. 472 Paper alert Findings: Meiosis is studied in microspore mother cells of both the wildtype and the mutant ask1-1 . Careful measurements show that spindles elongate to a greater extent during meiosis I than during meiosis II. In the ask1-1 mutant, the spindle assembles in meiosis I as in the wildtype, but homologous chromosomes do not separate in the mutant at anaphase. Several spindle lengths are observed in ask1-1 mutants, which encompass the lengths observed in the wildtype. This suggests that the mechanisms that govern spindle elongation are not affected in the mutant and that the extent of elongation is affected by the absence of chromosome separation. The fact that spindle length distribution is not continuous but discrete suggests that the spindle elongates in steps. These steps measure 0.7 µm. The striking observation that the spindle also elongates in multiples of 0.7 µm in other organisms may underlie a conserved mechanism. Significance : A combination of in vivo observations and biochemical approaches shows that the Cyclin-dependent kinase 2 (Cdc2) associates with microtubules involved in the mitotic apparatus. Findings: A fusion protein Cdc2::G FP is expressed in cell lines obtained from transgenic plants in which the coding sequence is placed under a constitutive promoter. During preprophase, the fusion protein accumulates in a pattern similar to that of the preprophase band. During mitosis, Cdc2::G FP relocates from chromatin to the spindle. This accumulation is enhanced by treatment with drugs that block the transition to anaphase. The association with the mitotic spindle depends on Cdc2 activity: it is prevented by the Cdc2 inhibitor roscovitin. Eventually, Cdc2::GFP accumulates at the phragmoplast. Biochemical copurification of Cdc2 with tubulin correlates with in vivo observations. • Pollen germinates precociously in the anthers of raringto-go, an Arabidopsis gametophytic mutant. Johnson SA, McCormick S: Plant Physiol 2001, 126 :685-695. Significance : A series of novel mutants for pollen-tube growth and pollination signal is presented. The phenotypes of these mutants are likely to lead to essential genes involved in tipgrowth and polarity in pollen. Findings: The wall of the pollen tube is enriched in callose in comparison to most cell walls in the sporophyte. An elegant screen based on callose production in pollen grains in the absence of pollination identified the mutant raring-to-go in which pollen-tube growth is initiated in the absence of pollination. Two other mutants in which callose is produced at random at the surface of pollen grains might have a defect in polarity. Another class of mutants of special interest is constituted of the two gift-wrapped pollen mutants in which the pollen-tube germinates inwardly and gradually fills the pollen grain. This phenotype is reminiscent of the shift in direction of tip-growth that is observed in root hairs of Medicago in response to Rhizobium. • A new dynamin-like protein, ADL6, is involved in trafficking from the trans-Golgi network to the central vacuole in Arabidopsis. Jin JB, Kim YA, Lee SH, Kim DH, Cheong GW, Hwang I: Plant Cell 2001, 13 :1511-1525. Significance : A careful study using G FP markers identifies a key player in trafficking from the trans-Golgi network (TG N) to the vacuole. Findings: The dynamin-like protein ADL6 is isolated on the basis of sequence identity. ADL6 is immunolocalised to the Golgi apparatus, as identified by the JIM84 epitope. In agreement with this result, treatment with Brefeldin A, which disrupts the Golgi apparatus, causes diffuse staining for antibodies against JIM84 and ADL6. In protoplasts, in vivo labelling of the Golgi apparatus by fusion of the red fluorescent protein with the rat sialyltransferase (ST::RFP) co-localises with the distribution of the fusion ADL6::G FP. The function of ADL6 in trafficking from the TG N to the vacuole is tested using the dominant negative version ADL6 [K51E]. The transport of the G FP fusion with the storage protein sporamin from the endoplasmic reticulum to the vacuole is detected. Co-transformation of protoplasts with ADL6 [K51E] leads to a decrease in vacuole targeting and to a pattern of accumulation that corresponds to the Golgi apparatus. No effect is detected on trafficking to the plasma membrane, as show by the correct targeting of the integral plasma membrane protein H + -ATPase::G FP. • Dynamic recruitment of Cdc2 to specific microtubule structures during mitosis. Weingartner M, Binarova P, Drykova D, Schweighofer A, David J-P, Heberle-Bors E, Doonan J, Bögre L: Plant Cell 2001, 13 :1929-1943.