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, 5:81–88 Contents (chosen by) 81 Growth and development (Schneitz and Sablowski) 83 Genome studies and molecular genetics (Lemieux and

Grossniklaus) 84 84 86 87

Plant biotechnology (Dunwell) Biotic interactions (Metraux) Cell signalling (Palme and McAinsh) Cell biology (Berger)

Growth and development Selected by Kay Schneitz Institute of Plant Biology, University of Zürich, Zürich, Switzerland e-mail: [email protected]

•• Two-component circuitry in Arabidopsis cytokinin signal transduction. Hwang I, Sheen J: Nature 2001, 413:383-389. Significance: This paper provides evidence of a two-component cytokinin signaling mechanism. The phosphorelay involves a plasma-membrane-based histidine kinase, histidine phosphotransmitters that translocate from the cytoplasm to the nucleus, and response regulators acting as transcriptional activators that become liberated by the cytokinin-derived signal. Some response regulators are cytokinin-inducible transcriptional repressors that may provide a negative feedback loop in cytokinin signaling. Findings: The authors made use of a transient expression system that involved leaf mesophyll protoplasts carrying a cytokinin-inducible reporter gene construct. CKI1, a hybrid histidine kinase, was shown to localise to the plasma membrane and to confer hypersensitivity to biologically active cytokinins. The histidine phosphotransmitters AHP1 and AHP2, but not AHP5, were shown to translocate transiently from the cytoplasm to the nucleus in the presence of exogenous cytokinin. The B-type response regulators ARR1, ARR2, and ARR10 activated the transcription of the reporter gene, whereas the A-type response regulators ARR4, ARR5, ARR6 and ARR7 repressed it. Both types of response regulators were shown to localise to the nucleus. Altering the receiver domain of both types of response regulators did not change their behavior, indicating that cytokinin signaling relieves the repression of ARR1, ARR2 and ARR10. Corresponding studies with transgenic plants corroborated the results obtained with this cell culture system. •• ARR1, a transcription factor for genes immediately responsive to cytokinins. Sakai H, Honma T, Aoyama T, Sato S, Kato T, Tabata S, Oka A: Science 2001, 294:1519-1521.

Significance: Further elucidation of the phosphorelay mechanism underlying cytokinin signal transduction in planta. The B-type response regulator ARR1 mediates cytokinin-induced signal transduction through its receiver domain and directly activates the ARR6 gene, which encodes a cytokinin-inducible A-type response regulator, at the transcriptional level. The data indicate that cytokinin signalling relieves ARR1 from repression that exists in the absence of cytokinin. Findings: The authors analysed the phenotypes of Arabidopsis plants ectopically expressing variants of ARR1 in the presence or absence of exogenous cytokinin. The expression of an ARR1 variant lacking the receiver domain, ARR1∆DDK, caused severe distortions in the absence of cytokinin whereas a wildtype variant caused only mild phenotypic effects. In the presence of cytokinin, however, the wildtype variant caused a phenotype resembling the strong phenotype caused by ARR1∆DDK. The authors also compared root length, callus formation, and ARR6 transcription levels in arr1-1 mutants, wildtype plants, and plants carrying a 35S::ARR1 construct. The results indicated that arr1-1 mutants and 35S::ARR1 plants are less or more sensitive, respectively, to cytokinin application than are wildtype plants. Results obtained with plants carrying a steroid-inducible ARR1∆DDK variant showed that ARR6 transcription could be increased upon addition of dexamethasone, even in the presence of the protein synthesis inhibitor cycloheximide. •• Polycomb repression of flowering during early plant development. Kinoshita T, Harada JJ, Goldberg RB, Fischer RL: Proc Natl Acad Sci USA 2001, 98:14156-14616. Significance: The polycomb group gene FERTILIZATION INDEPENDENT ENDOSPERM (FIE) is important for the repression of reproductive development in the seedling shoot apical meristem (SAM) and for the repression of shoot and floral organ formation outside of the seedling’s SAM. Findings: Introducing a FIE::GFP construct, under the control of a partial FIE promoter, rescued the seed abortion phenotype usually observed in homozygous Arabidopsis fie mutants. The resulting seedlings showed ectopic shoot and floral organ formation in the hypocotyls and roots. In addition, these seedlings did not form rosettes; instead, flowers originated directly from the seedling apex. The transcript levels of genes such as LEAFY (LFY), APETALA1 (AP1), AGAMOUS (AG), and PISTILLATA (PI) were ectopically elevated in the FIE::GFP seedlings in a temporal and spatial manner. Semiquantitative PCR analysis using RNA obtained from dissected walking-stick-stage embryos indicated that FIE-mediated repression of floral meristem identity and floral organ identity genes occurs before this stage of embryogenesis. • FHY1: a phytochrome A-specific signal transducer. Desnos T, Puente P, Whitelam GC, Harberd NP: Genes Dev 2001, 15:2980-2990. Significance: FHY1 is a novel light-regulated protein that specifically mediates the PHYTOCHROME A (PHYA)-based far-red (FR) light response. Interestingly, FHY1 transcription is subject to regulation by a negative feedback loop that is also mediated through PHYA.

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Findings: The FHY1 locus was cloned by chromosome walking. It encodes a novel protein that is rich in serines and glutamic acids. Analysis of plants carrying functional constructs of FHY1::GFP fusion proteins revealed that FHY1 accumulates in the nucleus and the cytoplasm of plants grown in the dark. GFP fluorescence was not detected in the cells of plants raised in constant FR light. A GFP signal could still be detected, however, after dark-grown seedlings were exposed to FR light for two hours. Phenotypic analysis of fhy1 mutants and plants ectopically expressing FHY1 revealed that FHY1 is a specific positive regulator of the PHYA-dependent FR response. Investigations of the FHY1 transcript levels of wildtype plants and various mutants both grown under different light regimes indicated that PHYA mediates a negative feedback loop that regulates FHY1 transcription. Furthermore, FHY3, another gene implicated in FR signalling, is a light-independent positive regulator of FHY1 transcription. Selected by Robert Sablowski John Innes Centre, Norwich, UK e-mail: [email protected]

• The Arabidopsis nectary is an ABC-independent floral structure. Baum SF, Esched Y, Bowman JL: Development 2001, 128:4657-4667. Significance: The classical ABC model of floral development implies separate mechanisms for establishing the concentric domains (whorls) occupied by floral organs and for determining the identity of the organs in each whorl. This paper shows that nectary development is a marker for the third whorl that is independent of organ identity. Two genes that pattern the expression of ABC genes, LEAFY (LFY) and UNUSUAL FLORAL ORGANS (UFO), also seem to pattern the number of whorls in the flower. Findings: Nectaries form at the base of stamens in the third whorl of wildtype Arabidopsis flowers. In this work, nectary development was monitored in many genetic backgrounds with altered organ identity. The overall conclusion was that nectaries form in the third whorl, regardless of the identity of the organs in that whorl. The organ identity (i.e. ABC) genes are controlled by LFY and UFO. Combined ufo and lfy mutations eliminated nectaries, whereas gain of UFO function caused the formation of ectopic nectaries. Given that nectary development is whorl-specific, this implied that LFY and UFO establish the third whorl. Previous work had shown that CRABS CLAW (CRC) is expressed throughout nectary development, and is required for nectary development but not for establishing the third whorl. The results described in this paper show that CRC is necessary but not sufficient for nectary development: ubiquitous CRC expression did not induce ectopic nectaries. The authors also discuss their findings from the evolutionary point of view. The independence of nectary and floral-organ development may have allowed the highly variable position of nectary glands across plants. •• The VERNALIZATION2 gene mediates the epigenetic regulation of vernalisation in Arabidopsis. Gendall AR, Levy YY, Wilson A, Dean C: Cell 2001, 107:525-535. Significance: Flowering in Arabidopsis is accelerated by a period of cold treatment that mimics overwintering. Cold perception and flowering response can be separated by weeks, and the cellular ‘memory’ of the stimulus is stable through several rounds of mitosis but not through meiosis. The work described in this paper provides molecular evidence that this ‘memory’ has an epigenetic basis.

Findings: vernalisation2-1 (vrn2-1) was initially isolated as a mutation that reduced the vernalisation response of the lateflowering Arabidopsis mutant fca-1. In this work, vrn2-1 was shown to reduce the vernalisation response of other late-flowering mutants (i.e. FRI and fve-1), confirming that vrn2-1 has a general role in the vernalisation response and is not simply a modifier of fca-1. Positional cloning revealed that the sequence of VRN2 is similar to EMBRYONIC FLOWER 2 and FERTILIZATION-INDEPENDENT SEED 2 from Arabidopsis, and to Suppressor of zeste 12 (Su[z]12) from Drosophila. Structural similarity to Su[z]12 suggested that VRN2 could function in maintaining transcriptional repression by controlling chromatin structure. This hypothesis was supported by results relating to the interaction between VRN2 and a gene that represses flowering, FLOWERING LOCUS C (FLC). VRN2 was required to maintain low levels of FLC expression after vernalization, but not for the initial repression of FLC by cold treatment. In addition, the FLC gene was more accessible to DNAse I digestion in nuclei purified from vrn2-1 mutants. A GFP::VRN2 fusion protein was localised in discrete regions of onion cell nuclei, in accordance with a role for VRN2 in transcriptional control. VRN2 expression was not affected by vernalisation. •• Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M: Nature 2001, 414:271-276. Significance: This paper reveals a direct molecular link between auxin-regulated protein degradation and the downstream transcriptional responses to auxin. Findings: Fusions to β-glucuronidase (GUS) were used to monitor the stability of the AUX2 and AUX3 proteins. The fusion proteins had a short half-life, which was further shortened by auxin treatment; this effect was blocked by the proteasome inhibitor MG132. Reporter proteins corresponding to the products of the dominant auxin-resistant mutant alleles axr2-1 and axr3-1 were more stable and resisted auxin-mediated degradation. The wildtype reporter fusions were also stabilised by mutations affecting the function of the ubiquitin ligase SCFTIR1 (i.e. by transport inhibitor resistant1 [tir1] and axr1). Immunoprecipitation and glutathione S-transferase (GST) pulldown assays showed that AXR2 and AXR3, but not axr2-1 and axr3-1, bound to SCFTIR1 in plant extracts. That binding was increased in extracts from auxin-treated plants. Deletions showed that binding to SCFTIR1 occurred through the highly conserved domain II of INDOLE ACETIC ACID [IAA]/AUX proteins, the same domain that is mutated in axr2-1 and axr3-1. The data indicate that auxin targets IAA/AUX proteins for destruction by promoting the association of these transcriptional regulators to the SCFTIR1 ubiquitin ligase complex. EMBRYONIC FLOWER2, a novel polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis. Yoshida N, Yanai Y, Chen L, Kato Y, Hiratsuka J, Miwa T, Sing ZR, Takahashi S: Plant Cell 2001, 13:2471-2481. Significance: EMBRYONIC FLOWER (EMF) prevents the premature initiation of reproductive development in Arabidopsis. This paper reports that EMF2 encodes a polycomb-group protein, suggesting that it may modulate chromatin structure to repress genes that are required for reproductive development. Findings: Positional cloning revealed that EMF2 is similar to FERTILIZATION-INDEPENDENT SEED 2 and VERNALIZATION2 from Arabidopsis (see summary of paper by Gendall

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et al. above), and to Suppressor of zeste 12 (Su[z]12) from Drosophila. Similarity to (Su[z]12) suggests a role for EMF2 in controlling chromatin structure. Antisense inhibition of EMF2 caused various degrees of premature flowering, the most severe inhibition producing extremely early flowering like that of emf mutants. This finding suggests that EMF2 has a quantitative effect on flowering time. During the transition to flowering, however, the levels of EMF2 mRNA did not change, implying that transcriptional control of EMF2 is not involved in the transition to flowering. In situ hybridisation showed that EMF2 mRNA is present in most actively proliferating tissues, including embryos, root tips, the vegetative meristem, leaf primordia, inflorescences and floral meristems.

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]

•• Detection of simple mutations and polymorphisms in large genomic regions. Sokurenko EV, Tchesnokova V, Yeung AT, Oleykowski CA, Trintchina E, Hughes KT, Rashid RA, Brint JM, Moseley SL, Lory S: Nucleic Acids Res 2001, 29:E111. Significance: A method that can detect single nucleotide polymorphisms (SNPs) that does not require PCR amplification of the target DNA regions and that has a number of advantages over existing methods. Findings: The authors report the development of a novel technology that can detect SNPs directly within a sample of total genomic DNA. This method, designated Genomic Identity Review by Annealing of Fractionated Fragments (GIRAFF), exploits preparative size-fractionation of restriction-digested genomic DNA and a newly discovered property of the mismatch-specific endonuclease CEL I to cleave heteroduplex DNA. This technology was applied to the discovery of allelic variants in the genomic DNA of isogenic pairs of recombinant Pseudomonas aeruginosa, Escherichia coli and Salmonella isolates. Total genomic DNA from two bacterial strains was purified and combined. After a complete endonuclease restriction of the combined genomic DNA and size-fractionation of the fragments, heteroduplex DNA was formed by heat-denaturation and re-annealing of the fractionated DNA fragments. These fragments were treated with the CEL I mismatch-specific endonuclease and the CEL1 digestion products separated by agarose gel electrophoresis. A Southern blot was then used to determine the sequence identity of genomic regions that contained SNPs. These mutation scans correctly detected a GA insertion in the cell-density-dependent, quorumsensing regulatory gene. • Detection of single base alterations in genomic DNA by solid phase polymerase chain reaction on oligonucleotide microarrays. Huber M, Losert D, Hiller R, Harwanegg C, Mueller MW, Schmidt WM: Anal Biochem 2001, 299:24-30. Significance: The method described in this paper provides an alternative to hybridisation-based SNP detection and traditional single-base extension methods. Findings: The authors describe a method for the simultaneous solid- and liquid-phase amplification of DNA using the

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polymerase chain reaction (PCR) on polyethyleneimine-coated glass slides. This method is applied to the ‘on-chip’ amplification of specific alleles of the P53 gene using a gene-specific oligonucleotide tiling array that interrogates single nucleotide positions within exons 7 and 8 of this gene in combination with genespecific unbound primers. Genomic DNA is used as template for the simultaneous amplification in the liquid phase and the allele-specific amplification in the solid phase. These amplifications take place in a single reaction on a glass microarray sealed with a coverslip. As the amplification products generated by the immobilised oligonucleotide primers remain covalently bound to the solid support, they are visualised by the incorporation of fluorescent dyes into DNA during the PCR. The authors report signal intensity ratios of more than 10 between the perfect match and the mismatch oligonucleotides. The authors also present data that suggest that these assays can discriminate between homozygous and heterozygous alleles. •• The VERNALIZATION2 gene mediates the epigenetic regulation of vernalization in Arabidopsis. Gendall AR, Levy YY, Wilson A, Dean C: Cell 2001, 107:525-535. • EMBRYONIC FLOWER2, a novel Polycomb group protein homolog, mediates shoot development and flowering in Arabidopsis. Yoshida N, Yanai Y, Chen L, Kato Y, Hiratsuka J, Miwa T, Sung ZR, Takahashi S: Plant Cell 2001, 13:2471-2481. Significance: Polycomb group (PcG) genes play an important role in the control of gene expression in invertebrates, vertebrates and plants. They are thought to regulate the transcription of target genes by modifying their higher order chromatin structure. In plants, it had been previously shown that PcG proteins are involved in the repression of floral homeotic genes (e.g. CURLY LEAF) and the regulation of cell proliferation during seed development (mediated by the FIS-class genes MEDEA, FERTILIZATION-INDEPENDENT SEED2 (FIS2), and FERTILIZATION-INDEPENDENT ENDOSPERM). These two papers report the molecular characterisation of two further Arabidopsis PcG proteins that are similar to the Drosophila PcG protein Suppressor of zeste 12 and the Arabidopsis protein FIS2. EMBRYONIC FLOWER2 (EMF2) plays a role in repressing the reproductive program and VERNALIZATION2 (VRN2) mediates vernalization, the exposure to a period of low temperature that accelerates flowering. As the flowering response occurs several weeks after the cold treatment, an epigenetic ‘memory’ is thought to be involved in vernalization. The molecular characterisation of VRN2 shows that a PcG protein plays a role in maintaining this memory. Findings: The EMF2 and VRN2 genes were isolated through map-based cloning procedures. EMF2 is expressed in the embryo, as well as in vegetative and reproductive meristems. The expression of VRN2 is unaltered by vernalization but the vrn2 mutation affects the expression of FLC, a central regulator of flowering. It has been shown previously that vernalization promotes flowering by downregulating FLC, a repressor of genes that promote flowering. Because the vrn2 mutation alone has no effect on flowering, its role is only revealed when vrn2 is combined with mutations that upregulate FLC expression, such as fca. In a fca vrn2 double mutant, FLC is initially downregulated upon vernalization. However, unlike in the fca single mutant where FLC is stably repressed, FLC expression increases again after the initial downregulation in this double mutant. This loss of repression correlates with an increased DNAse sensitivity of the FLC locus in the fca vrn2 double mutant, suggesting a

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difference in chromatin organisation between the vrn2 and fca vrn2 mutants. These findings suggest that the PcG protein VRN2 is required for the stable maintenance of a repressed state of FLC and provides a molecular mechanism for the epigenetic memory involved in vernalization. •• A QTL for flowering time in Arabidopsis reveals a novel allele of CRY2. El-Din El-Assal S, Alonso-Blanco C, Peeters AJM, Raz V, Koornneef M: Nat Genetics 2001, 29:435-440. •• Natural variation in light sensitivity of Arabidopsis. Maloof JN, Borevitz JO, Dabi T, Lutes J, Nehring RB, Redfern JL, Trainer GT, Wilson JM, Asami T, Berry CC, Weigel D, Chory J: Nat Genetics 2001, 29:441-446. Significance: The work described in these two papers illustrates the power of studying natural genetic variation, an area that is rapidly gaining in popularity among Arabidopsis researchers. It clearly demonstrates how the plasticity in plant growth and development seen among different Arabidopsis accessions can be used advantageously to dissect plant biology and will ultimately lead to a better understanding of natural selection. Using two alternative directed approaches, natural variation in flowering time and light sensitivity was identified and linked to molecular changes in the photoreceptor genes CRYPTOCHROME2 (CRY2) and PHYTOCHROME A (PHYA), respectively. Findings: Using recombinant inbred lines generated from crosses between the Arabidopsis laboratory strain Landsberg erecta (Ler) and an accession from the Cape Verde Islands (Cvi), several quantitative trait loci (QTL) were identified that contribute to differences in the flowering response to photoperiod. Positional cloning of one major QTL showed it to be a novel allele of CRY2, the blue-light photoreceptor gene that had previously been shown to promote flowering under a long-day photoperiod. The QTL could be mapped to one of the 12 base changes between the Ler and Cvi CRY2 alleles. Although CRY2 is an abundant protein throughout the day under long-day conditions, it was found to be rapidly degraded after exposure to light under short-day conditions. This unexpected and rapid decay was dramatically slowed down if the CRY2 protein contained the relevant amino-acid change found in the Cvi accession. This amino-acid change has thus provided a molecular basis for the early-flowering phenotype of the Cvi accession. In the second paper, 141 Arabidopsis accessions were examined for their response to four different light conditions, two hormone conditions and darkness. Cluster analysis showed that some accessions had response patterns that were similar to those of known photoreceptor mutants, which allowed the authors to associate the far-red response of the Lm-2 accession to a single amino-acid change in PHYA. This change alters the photochemical properties and auto-phosphorylation capacity of the PHYA protein and stabilises the light-labile protein. The Lm-2 PHYA protein is 100-fold less sensitive to far-red light than are laboratory strains.

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

• The fate of antibiotic resistance marker genes in transgenic plant feed material fed to chickens. Chambers PA, Duggan PS, Heritage J, Forbes JM: J Antimicrob Chemother 2001, 49:161-164.

Significance: There has been widespread debate about the possibility of antibiotic resistance marker genes being transferred from transgenic foodstuffs to bacteria in the digestive systems of animals. This study examines this possibility. Findings: The authors examined the fate of the ampicillin antibiotic resistance marker when incorporated into transgenic maize and fed to chicks. Although plant-derived markers were found in the crops of five birds that had been fed transgenic maize and in the stomach contents of two birds, the marker gene was not found in the intestines. Furthermore, the survival of the marker gene was similar to that of plant DNA targets, demonstrating that it survives no better than other DNA. It was concluded that it is very unlikely that bacteria in the gut of chickens will be transformed to ampicillin resistance when the birds are fed transgenic maize. • Unhydroxylated triple helical collagen I produced in transgenic plants provides new clues on the role of hydroxyproline in collagen folding and fibril formation. Perret S, Merle C, Bernocco S, Berland P, Garrone R, Hulmes DJ, Theisen M, Ruggiero F: J Biol Chem 2001, 276:43693-43698. Significance: There is much interest in the possibility of using plant-derived collagen, a constituent of gelatine, as a substitute for animal-derived gelatine. This study provides valuable experimental data concerning the formation of functional collagen in transgenic plants. Findings: Human unhydroxylated homotrimeric triple-helical collagen I produced in transgenic plants was used to investigate the role of hydroxyproline in molecular folding and fibril formation. The absence of hydroxyproline residues does not prevent the correct folding of the recombinant collagen, although it markedly slows down its propagation rate compared with that of bovine fully hydroxylated homotrimeric collagen I. Because of the lack of hydroxylation, recombinant collagen molecules showed increased flexibility and a reduced melting temperature compared with those of native homotrimers and heterotrimers. These data confirm the predicted importance of hydroxyproline in collagen self-assembly.

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

•• Complementation of the Magnaporthe grisea deltacpkA mutation by the Blumeria graminis PKA-c gene: functional genetic analysis of an obligate plant pathogen. Bindslev L, Kershaw MJ, Talbot NJ, Oliver RP: Mol Plant Microbe Interact 2001, 14:1368-1375. Significance: The signalling components for pathogenicity among various phytopathogens are remarkably conserved. Findings: The authors had previously described a gene similar to that encoding the catalytic subunit of cyclic AMP-dependent protein kinase A (named Bka1) in the obligate pathogenic fungus Blumeria graminis (syn. Erysiphe graminis) f. sp. hordei. PKA-c, a close homolog of Bka1, had already been characterized functionally by expression in a mutant (deltacpkA ) of the non-obligate rice pathogen Magnaporthe grisea. DeltacpkA mutants displayed delayed and incomplete appressorium development, suggesting a role for PKA-c in the signal

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transduction for the development of infection structures. Transformation of the deltacpkA mutant with Bka1 controlled by the M. grisea MPG1 promoter, restored its pathogenicity and appressorium maturation kinetics to those of wildtype M. grisea. The results demonstrate a surprising conservation of signalling components regulating infection-related development in pathogenic fungi. • Chemically induced virus resistance in Arabidopsis thaliana is independent of pathogenesis-related protein expression and the NPR1gene. Wong CE, Carson RAJ, Carr JP: Mol Plant Microbe Interact 2002, 15:75-81. Significance: The authors discover a novel defence signalling pathway operating in virus resistance. Findings: In Arabidopsis thaliana, salicylic acid (SA)-induced resistance to turnip vein clearing virus (TVCV) is shown to be induced by non-lethal concentrations of cyanide and antimycin (AA) without concomitant induction of pathogenesis-related PR-1 gene expression. Induced resistance to TVCV is characterized by an inhibition of virus replication at the site of inoculation. Cyanide and AA induce resistance to viruses as well as the expression of a mitochondrial alternative oxidase (a potential target for cyanide and AA) independently of NPR1, a regulatory gene located downstream of SA that is involved in the expression of the PR genes. These findings show that resistance to RNA viruses can be induced via a distinct branch of the defensive signal transduction pathway in plants. •• Isochorismate synthase is required to synthesize salicylic acid for plant defence. Wildermuth MC, Dewdney J, Wu G, Ausubel FM: Nature 2001, 414:562-565. Significance: Pathogen-induced salicylic acid (SA) is synthesised from chorismate. Findings: The activation of local and systemic defences of plants against pathogens requires SA. SA accumulates both locally and systemically after pathogen infection, and its bio synthesis has been subject to great interest. Previous work has suggested that plants can synthesise SA from phenylalanine. However, it has never been shown unambiguously that this pathway is unique and limits the establishment of systemic acquired resistance (SAR). The authors used sid2, a Arabidopsis thaliana mutant known to be impaired in SAR and SA biosynthesis. They showed that Sid2 encodes an isochorismate synthase that complements the sid2 mutation. This surprising finding indicates that SA required for SAR is synthesised from chorismate using a similar biosynthetic pathway to that found in many bacteria. •• A phosphate transporter expressed in arbusculecontaining cells in potato. Rausch C, Daram P, Brunner S, Jansa J, Laloi M, Leggewie G, Amrhein N, Bucher M: Nature 2001, 414:462-470. Significance: The expression of a phosphate-transporter gene was identified exclusively in roots colonised by mycorrhiza. Findings: Arbuscular mycorrhiza are common symbiotic fungi in the roots of many plants. The arbuscular mycorrhizal symbiosis is based on nutrient exchange: for example, the fungus acts as a source of phosphate for the plant while the plants provides glucose to the fungus. The authors identified a phosphate-transporter gene, StPT3, in potato. The functionality of this gene was confirmed by yeast complementation. StPT3 is expressed only in root sectors where mycorrhizal structures are formed. A motif in the sequence of the StPT3 promoter is similar to transposon-like

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elements. Thus, mutualistic symbiosis might have evolved by genetic rearrangements in the StPT3 promoter. • Caspase inhibitors reduce symptom development and limit bacterial proliferation in susceptible plant tissues. Richael C, Lincoln JE, Bostock RM, Gilchrist DG: Physiol Mol Plant Pathol 2001, 59:213-221. Significance: Necrotrophic bacteria use programmed plant cell death in a susceptible host to achieve successful infection. Findings: The co-injection of various caspase inhibitors and pathogenic bacteria into a susceptible plant host resulted in reduced plant cell death, cessation of bacterial growth and a decrease in the expression level of all of the pathogenesisrelated transcripts that were assayed. No deleterious effects of the caspase inhibitors on bacterial or plant cells were observed. These results suggest that virulent bacteria can activate programmed plant cell death as a means to successfully initiate and maintain an infection of their host. • Sensitivity of different ecotypes and mutants of Arabidopsis thaliana toward the bacterial elicitor flagellin correlates with the presence of receptor-binding sites. Bauer Z, Gómez-Gómez L, Boller T, Felix G: J Biol Chem 2001, 276:45669-45676. Significance: This paper describes a membranous high affinitybinding site that acts as a physiological receptor for flagellin. Findings: A radio-labelled derivative of flg22, a synthetic peptide representing the elicitor-active domain of flagellin, was used to probe the interaction of flagellin with its receptor in A. thaliana. Binding detected in intact cells and membrane preparations showed specificity for flagellin-derived peptides that were either agonists or antagonists of the biologically responses. Binding was measurable in all ecotypes of A. thaliana that show sensitivity to flagellin. However, binding was barely detectable in several flagellin-insensitive mutants or in the flagellin-insensitive ecotype Ws-0, which is affected in FLS1, a locus previously identified as essential for flagellin perception. In addition, no binding was found in plants carrying a mutation in the leucine-rich domain of FLS2, a transmembrane receptor-like kinase. The tight correspondence between the presence of this binding site and the biological response indicate that a functional receptor-binding site depends on FLS1 and FLS2, and acts as the flagellin receptor. •• Characterization of an Arabidopsis–Phytophthora pathosystem: resistance requires a functional PAD2 gene and is independent of salicylic acid, ethylene and jasmonic acid signalling. Roetschi A, Si-Ammour A, Belbahri L, Mauch F, Mauch-Mani B: Plant J 2001, 28:293-305. Significance: A novel pathway for resistance operates against an oomycete in Arabidopsis. Findings: The authors have established a pathosystem to study the interaction between Arabidopsis thaliana and Phytophthora porri, a relative of the devastating Phytophthora infestans, the causal agent of potato late blight. Extensive colonisation and sporulation characterise compatible interactions. In contrast, incompatible interactions are typified by the build up of a hypersensitive reaction and the formation of papillae in resistant Arabidopsis accessions. Mutants such as jar1 (jasmonicacid-resistant), etr1 (ethylene receptor) and ein1 (ethyleneinsensitive) that are impaired in various steps of the defence pathway retain wildtype levels of resistance toward P. porri. Mutants that lack salicylic acid, such as npr1 (non-expresser of

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PR1) or sid2 (salicylic acid-induction deficient), are only slightly less resistant to P. porri than wildtype plants. The phytoalexindeficient mutant pad2 is hypersusceptible to infection by P. porri. Low levels of the phytoalexin camalexin are unlikely to be the cause of hypersusceptibility as pad3 mutants blocked in camalexin biosynthesis are resistant. Hence, this work highlights a novel signalling pathway for disease resistance to a fungal pathogen.

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

•• Auxin regulates SCFTIR1-dependent degradation of AUX/IAA proteins. Ramos JA, Zenser N, Leyser O, Callis J: Plant Cell 2001, 13:2349-2360. •• AUX/IAA proteins are active repressors, and their stability and activity are modulated by auxin. Tiwari SB, Wang X-J, Hagen G, Guilfoyle T: Plant Cell 2001, 13:2809-2822. •• Rapid degradation of auxin/indoleacetic acid proteins requires conserved amino acids of domain II and Is proteasome dependent. Gray WM, Kepinski S, Rouse D, Leyser O, Estelle M: Nature 2001, 414:271-276. •• Antisense expression of an Arabidopsis Ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress. Kim S-H, Arnold D, Lloyd A, Roux SJ: Plant Cell 2001, 13:2619-2630. •• Multidrug resistance-like genes of Arabidopsis required for auxin transport and auxin-mediated development. Noh B, Murphy AS, Spalding EP: Plant Cell 2001, 13:2441-2454. •• Localization of the auxin permease AUX1 suggests two functionally distinct hormone transport pathways operate in the Arabidopsis root apex. Swarup R, Friml J, Marchant A, Ljung K, Sandberg G, Palme K, Bennett M: Genes Dev 2001, 15:2648-2653. •• The role of auxin-binding protein 1 in the expansion of tobacco leaf cells. Chen J-G, Shimomura S, Sitbon F, Sandberg G, Jones AM: Plant J 2001, 28:607-617. Significance: A rich harvest of papers reflecting progress towards understanding the molecular mechanisms of auxin action. Findings: Auxin plays an important role in numerous plant processes. Among auxin-responsive genes, the AUX/IAA genes are particularly well characterized. They are rapidly induced by auxin and their products have very short half-lives (6–8 min), suggesting that protein degradation has a primary role in the regulation of their activity. Ramos et al. show that domain II of the AUX/IAA proteins is required for their rapid proteasome-mediated degradation in response to auxin. Protein abundance and half-life of AUX/IAA proteins were measured in tobacco protoplasts and in transgenic Arabidopsis seedlings by monitoring luciferase fused to various portions of AUX/IAA proteins. Tiwari et al. observed that mutations in domain II resulted in increased repression of auxin-induced responses, whereas mutations in domains I and III partially relieved this repression. Their data suggest that AUX/IAAs function as active repressors by dimerising with auxin-response factors that are bound to auxin-response elements, and that early auxin-response genes are regulated by the auxin-modulated stabilities of AUX/IAA proteins. Using inhibitors, the

proteasome pathway was shown by Gray et al. to have a specific involvement in auxin response. Gray and coworkers further demonstrated that auxin stimulates and promotes SCFTIR1-dependent degradation of AUX/IAA proteins. By stabilising the SCFTIR1 complex using either proteasome inhibitors or genetic mutations, this complex was shown to physically interact with AUX/IAAs, an interaction that is mediated by domain II. Nevertheless, the important question of how auxin regulation is mediated remains unanswered. Does it involve perception or transport or both? Kim et al. report surprising data showing that antisense expression of Arabidopsis RanBP1, a protein modulating GTP/GDP-bound states of Ran GTPases, renders plants hypersensitive to auxin; primary root growth is enhanced but growth of lateral roots is supressed. Given the established role of RanBPs in the transport of proteins into the nucleus, these findings are consistent with a key role for AtRanBP in the delivery to the nucleus of proteins that suppress auxin action and regulate mitotic progress in root tips. Two other papers by Noh et al. and Swarup et al. provide new insights into key aspects of auxin transport. Localisation of the auxin permease AUX1 in the Arabidopsis root apex revealed a novel phloem-based auxin transport pathway. AUX1 was found to be asymmetrically localised opposite to the putative auxin efflux carrier PIN1 in the plasma membrane of root protophloem cells. These data suggest a key role for AUX1 in root gravitropism in facilitating basipetal auxin transport to distal elongation zones. Members of the multidrug resistance (MDR) genes of the ATP-binding cassette transporters are novel components of the auxin transport machinery. Double mutants that have mutations in both AtMDR1 and AtPGP1 have are greatly impaired in auxin transport activity, and have epinastic cotyledons and reduced apical dominance. Most interestingly, 1-naphthylphthalamic acid (NPA; an inhibitor of auxin efflux) bound tightly to these proteins, suggesting that they may be the elusive NPA receptor(s). Finally, the paper by Chen et al. describes work on ABP1, an unusual putative receptor for auxin that resides in the endomembrane lumen. ABP1 acts at a relatively low auxin concentration to mediate cell expansion, whereas at higher auxin levels, cell division is stimulated by an unknown mechanism. Together, these papers present major advances and add information to the patchwork of data that is slowly filling the many gaps in our picture of how auxin acts. •• Overexpression of Arabidopsis ESR1 induces initiation of shoot regeneration. Banno H, Keda Y, Niu Q-W, Chua N-H: Plant Cell 2001, 13:2609-2618. Significance: ESR1 is described as a gene that may regulate the induction of shoot regeneration after the acquisition of competence for organogenesis. Findings: ESR1, for Enhancer of Shoot Regeneration1, was isolated by functional screening of an Arabidopsis cDNA library. It confers cytokinin-independent shoot formation when overexpressed in Arabidopsis root explants. ESR1 encodes a putative transcription factor with an AP2/EREBP domain. ESR1 overexpression greatly increased the efficiency of shoot regeneration from root explants in the presence of cytokinin, with a shift in the optimal cytokinin concentration required for this process. The effect of ESR1 overexpression on shoot regeneration was synergistic with the effects of cytokinin. In wildtype Arabidopsis plants, ESR1 expression was induced by cytokinin, and ESR1 transcript levels increased transiently during shoot regeneration from root explants, most probably in response to cytokinin in the shoot-inducing medium. Transient increases in

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ESR1 transcription occurred after acquisition of competence for shoot regeneration and before shoot formation, which is consistent with the physiological effects of ESR1 overexpression. Selected by Martin R McAinsh Lancaster University, Lancaster, UK e-mail: [email protected]

•• Phot1 and phot2 mediate blue light regulation of stomatal opening. Kinoshita T, Doi M, Suetsugu N, Kagawa T, Wada M, Shimazaki K-I: Nature 2001, 414:656-660. Significance: phot1 and phot2 are identified as blue-light receptors mediating stomatal opening. Findings: The authors used phot1 phot2 double mutant Arabidopsis plants to study the role of the phot1 and phot2 blue-light receptors in blue-light-mediated stomatal opening. They show that phot1 phot2 plants do not express PHOT1 mRNA or produce active phot2 protein. Single mutations in the PHOT1 and PHOT2 genes decrease the sensitivity of stomata to a blue-light stimulus, whereas stomata of the phot1 phot2 double mutant do not open in response to blue light. Importantly, these effects are not due to changes in the activity of the plasma membrane H+-ATPase, which drives stomatal opening. The authors conclude that phot1 and phot2 work simultaneously and redundantly as blue-light receptors mediating stomatal opening. • ARR1, a transcription factor for genes immediately responsive to cytokinins. Sakai H, Honma T, Aoyama T, Sato S, Kato T, Tabata S, Oka A: Science 2001, 294:1519-1521. Significance: The Arabidopsis response regulator (ARR)1 is positioned immediately after cytokinin perception in the cytokinin signal transduction pathway. Findings: The morphological characteristics of ARR1-overexpressing plants (i.e. transgenic plants carrying either full-length ARR1 genes or truncated versions missing the NH2-terminal signal receiver domain) and arr1 mutants plants were analysed in the presence and absence of cytokinin in order to examine the role of ARR1 in the cytokinin signalling pathway. The signal receiver domain suppresses the function of ARR1 in the absence of cytokinins. A cytokinin signal releases this suppression, and the level of ARR1 expression correlates with sensitivity to cytokinin. In addition, ARR1 directly activates ARR6, a gene that responds to cytokinins without de novo protein synthesis. The authors conclude that ARR1 is a principal transcription-factor-type response regulator that is involved in an early step of cytokinin signal transduction. • Differential contribution of cytoplasmic Ca2+ and Ca2+ influx to gamete fusion and egg activation in maize. Antoine A-F, Faure J-E, Dumas C, Feijo JA: Nat Cell Biol 2001, 3:1120-1123. Significance: Two Ca2+-dependent signalling cascades are shown to be triggered by gamete fusion in higher plants such as maize. Findings: The authors simultaneously monitored extracellular Ca2+ fluxes with a Ca2+-vibrating probe and cytosolic Ca2+ ([Ca2+]cyt) by wide-field imaging in order to investigate Ca2+-dependent events following gamete fusion in higher plants. They show that fusion triggers a Ca2+ influx followed by a long-lasting increase in [Ca2+]cyt after a lag of 40–120 s. Studies using the Ca2+-channel blocker Gd3+ indicate that there are two independent Ca2+ influx mechanisms: a Gd3+-insensitive

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Ca2+ influx localised to the site of sperm fusion and a global Gd3+-sensitive Ca2+ influx. The authors suggest that the increase in [Ca2+]cyt may be both necessary and sufficient to launch egg activation, whereas the initial Ca2+ influx may be involved in upstream events such as sperm incorporation. • 14-3-3 proteins regulate intracellular localization of the bZIP transcriptional activator RSG. Igarashi D, Ishida S, Fukazawa J, Takahashi Y: Plant Cell 2001, 13:2483-2497. Significance: 14-3-3 proteins are identified as regulators of the intracellular localisation of the REPRESSION OF SHOOT GROWTH (RSG) transcription factor in tobacco. Findings: The authors used yeast two-hybrid screening to identify the 14-3-3 signalling proteins as RSG-binding partners. They show that the serine-114 residue of RSG is critical to this interaction. A RSG mutant in which serine-114 had been changed to alanine was no longer able to bind 14-3-3s, was localised exclusively to the nucleus rather than throughout the nucleus and cytoplasm, and exhibited greater transcriptional activation capability than wildtype RSG. In addition, leptomycin B (an inhibitor of nuclear export) caused a rapid relocation of RSG from the cytosol to the nucleus. The authors conclude that 14-3-3 proteins may negatively modulate RSG, which is involved in the regulation of endogenous gibberellin concentrations, by controlling its intracellular localisation.

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

• A novel plant kinesin-related protein specifically associates with the phragmoplast organelles. Lee YRJ, Giang HM, Liu B: Plant Cell 2001, 13: 2427-2439. Significance: A new protein that is associated with the phragmoplast is identified. Findings: In silico analysis has identified the sequence of a kinesin-related gene (AtPAKRP2) in the Arabidopsis genome. The predicted protein shows a tripartite organisation that is distinct from the organisation of other plant kinesins. Phylogenetic analysis shows that AtPAKRP2 is distantly related to other kinesins, and it is not clear whether the protein behaves like a motor protein. Immunolocalisation shows that the protein associates with the phragmoplast with a pattern distinct from those of the other kinesin AtPAKRP1 and the syntaxin-related KNOLLE. Pharmacological data suggest that the protein associates with microtubules and Golgi vesicles, and might be involved in vesicular transport during cytokinesis. • Ara6, a plant-unique novel type Rab GTPase, functions in the endocytic pathway of Arabidopsis thaliana. Ueda T, Yamaguchi M, Uchimiya H, Nakano A: EMBO J 2001, 20:4730-4741. Significance: A novel Rab GTPase identifies a plant-specific endocytic pathway. Findings: The genes ARA6 and ARA7 have been cloned in Arabidopsis on the basis of their sequence identity to Rab5 GTPases. ARA7 appears to encode an ortholog of Rab5, whereas ARA6 encodes a GTPase that lacks the typical isoprenylation site and contains an amino-terminal myristoylation site and a palmitoylation site. Similarities between the dynamics

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of the fluorescence pattern of the GFP::ARA6 fusion protein and of the pattern of the dye FM4-64 suggest that ARA6 is involved in the endocytic pathway. This function requires amino-terminal myristoylation and palmitoylation coordinately with a carboxy-terminal domain. These requirements are unique to ARA6 amongst RabGTPASes. • Mammalian Bax-induced plant cell death can be downregulated by overexpression of Arabidopsis Bax Inhibitor-1 (AtBI-1). Kawai-Yamada M, Jin L, Yoshinaga K, Hirata H, Uchimiya H: Proc Natl Acad Sci USA 2001, 21:12295-12300. Significance: A plant endogenous pathway inhibits the induction of cell death by the mammalian proapoptotic protein BAX in Arabidopsis. Findings: When expressed under a dexamethasone-inducible promoter, the mammalian protein BAX causes cell death in Arabidopsis. In yeast, the Bax Inhibitor-1 protein prevents BAX-induced cell death. The ortholog of Bax Inhibitor-1 in Arabidopsis, AtBI-1, is isolated and a functional fusion protein

AtBI-1::GFP is produced and localised to the endosplasmic reticulum. Overexpression of AtBI-1 under the CaMV 35S promoter suppresses cell-death induction by BAX. • A cell cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex. Boniotti MB, Guttierrez C: Plant J 2001, 28:341-350. Significance: This study provides a valuable biochemical approach to studying the function of the retinoblastoma protein (RB) in the regulation of the G1→S transition in plants. Findings: RB from maize is used as a phosphorylation target to test the presence of a cell-cycle-related kinase (CDK) activity. A correlation between RB phosphorylation and proliferation is shown using extracts from various parts of wheat leaves. Synchronised BY-2 tobacco cultures are used to demonstrate the phosphorylation of RB by a CDK activity at the G1→S transition. Pharmacological data suggest that RB is phosphorylated by a PSTAIRE-type CDK.