Heterologous expression of wheat WRKY transcription factor genes transcriptionally activated in hybrid necrosis strains alters abiotic and biotic stress tolerance in transgenic Arabidopsis.

Hybrid necrosis and hybrid chlorosis are sometimes observed in interspecific hybrids between the tetraploid wheat cultivar Langdon and diploid wild wheat Aegilops tauschii. Many WRKY transcription factor genes are dramatically upregulated in necrosis and chlorosis wheat hybrids. Here, we isolated cDNA clones for four wheat WRKY transcription factor genes, TaWRKY49, TaWRKY92, TaWRKY112, and TaWRKY142, that were commonly upregulated in the hybrid necrosis and hybrid chlorosis and belonged to the same clade of the WRKY gene family. Expression patterns of the four TaWRKY genes in response to several stress conditions were similar in wheat seeding leaves. The four TaWRKY-GFP fusion proteins were targeted to the nucleus in onion epidermal cells. The TaWRKY gene expression levels were increased by high salt, dehydration, darkness, and blast fungus treatment in common wheat. Expression of either of the TaWRKY genes increased salinity and osmotic stress tolerance accompanied with overexpression of STZ/Zat10, and induced overexpression of the salicylic acid-signal pathway marker gene AtPR1 in transgenic Arabidopsis. TaWRKY142 expression also induced the jasmonic acid-pathway marker gene AtPDF1.2 and enhanced resistance against the fungal pathogen Colletotrichum higginsianum in transgenic Arabidopsis. These results suggest that the four TaWRKY genes act as integrated hubs of multiple stress signaling pathways in wheat and play important roles in autoimmune response-inducing hybrid necrosis and hybrid chlorosis.

Genotypic effects on sugar and by-products of liquid hydrolysates and on saccharification of acid-insoluble residues from wheat straw.

Wheat straw is one of the major attractive resources for low-cost raw materials for renewable energy, biofuels and biochemicals. However, like other sources of lignocellulosic biomass, straw is a heterogeneous material due to its mixed origin from different tissue and cell types. Here, to examine the genotypic effects on biorefinery usage of wheat straw, straw obtained from different wheat cultivars and experimental lines was pretreated with dilute acid. Significant differences between cultivars were observed in the concentrations of glucose and toxic by-products of the liquid hydrolysates. A higher content of xylose than glucose was found in liquid hydrolysates from wheat straw, and the xylose content appeared to be affected by both environmental and genetic factors. Analysis using chromosome substitution lines of the common wheat cultivar Chinese Spring showed that chromosomes 2A and 3A from other wheat cultivars, Hope and Timstein, significantly increased the xylose content. However, no significant relationship was observed between the liquid hydrolysate xylose content and the glucose content obtained from enzymatic saccharification of the acid-insoluble residue. These results highlight the potential of wheat breeding to improve biomass-related traits in wheat straw.

Global gene expression profiling related to temperature-sensitive growth abnormalities in interspecific crosses between tetraploid wheat and Aegilops tauschii.

Triploid wheat hybrids between tetraploid wheat and Aegilops tauschii sometimes show abnormal growth phenotypes, and the growth abnormalities inhibit generation of wheat synthetic hexaploids. In type II necrosis, one of the growth abnormalities, necrotic cell death accompanied by marked growth repression occurs only under low temperature conditions. At normal temperature, the type II necrosis lines show grass-clump dwarfism with no necrotic symptoms, excess tillers, severe dwarfism and delayed flowering. Here, we report comparative expression analyses to elucidate the molecular mechanisms of the temperature-dependent phenotypic plasticity in the triploid wheat hybrids. We compared gene and small RNA expression profiles in crown tissues to characterize the temperature-dependent phenotypic plasticity. No up-regulation of defense-related genes was observed under the normal temperature, and down-regulation of wheat APETALA1-like MADS-box genes, considered to act as flowering promoters, was found in the grass-clump dwarf lines. Some microRNAs, including miR156, were up-regulated, whereas the levels of transcripts of the miR156 target genes SPLs, known to inhibit tiller and branch number, were reduced in crown tissues of the grass-clump dwarf lines at the normal temperature. Unusual expression of the miR156/SPLs module could explain the grass-clump dwarf phenotype. Dramatic alteration of gene expression profiles, including miRNA levels, in crown tissues is associated with the temperature-dependent phenotypic plasticity in type II necrosis/grass-clump dwarf wheat hybrids.

Three dominant awnless genes in common wheat: Fine mapping, interaction and contribution to diversity in awn shape and length.

The awn is a long needle-like structure formed at the tip of the lemma in the florets of some grass species. It plays a role in seed dispersal and protection against animals, and can contribute to the photosynthetic activity of spikes. Three main dominant inhibitors of awn development (Hd, B1 and B2) are known in hexaploid wheat, but the causal genes have not been cloned yet and a genetic association with awn length diversity has been found only for the B1 allele. To analyze the prevalence of these three awning inhibitors, we attempted to predict the genotypes of 189 hexaploid wheat varieties collected worldwide using markers tightly linked to these loci. Using recombinant inbred lines derived from two common wheat cultivars, Chinese Spring and Mironovskaya 808, both with short awns, and a high-density linkage map, we performed quantitative trait locus analysis to identify tightly linked markers. Because this linkage map was constructed with abundant array-based markers, we converted the linked markers to PCR-based markers and determined the genotypes of 189 hexaploids. A significant genotype-phenotype correlation was observed at the Hd and B1 regions. We also found that interaction among these three awning inhibitors is involved in development of a membranous outgrowth at the base of awn resembling the Hooded mutants of barley. For the hooded awn phenotype, presence of the Hd dominant allele was essential but not sufficient, so B2 and other factors appear to act epistatically to produce the ectopic tissue. On the other hand, the dominant B1 allele acted as a suppressor of the hooded phenotype. These three awning inhibitors largely contribute to the genetic variation in awn length and shape of common wheat.

A high-resolution physical map integrating an anchored chromosome with the BAC physical maps of wheat chromosome 6B.

BACKGROUND: A complete genome sequence is an essential tool for the genetic improvement of wheat. Because the wheat genome is large, highly repetitive and complex due to its allohexaploid nature, the International Wheat Genome Sequencing Consortium (IWGSC) chose a strategy that involves constructing bacterial artificial chromosome (BAC)-based physical maps of individual chromosomes and performing BAC-by-BAC sequencing. Here, we report the construction of a physical map of chromosome 6B with the goal of revealing the structural features of the third largest chromosome in wheat. RESULTS: We assembled 689 informative BAC contigs (hereafter reffered to as contigs) representing 91% of the entire physical length of wheat chromosome 6B. The contigs were integrated into a radiation hybrid (RH) map of chromosome 6B, with one linkage group consisting of 448 loci with 653 markers. The order and direction of 480 contigs, corresponding to 87% of the total length of 6B, were determined. We also characterized the contigs that contained a part of the nucleolus organizer region or centromere based on their positions on the RH map and the assembled BAC clone sequences. Analysis of the virtual gene order along 6B using the information collected for the integrated map revealed the presence of several chromosomal rearrangements, indicating evolutionary events that occurred on chromosome 6B. CONCLUSIONS: We constructed a reliable physical map of chromosome 6B, enabling us to analyze its genomic structure and evolutionary progression. More importantly, the physical map should provide a high-quality and map-based reference sequence that will serve as a resource for wheat chromosome 6B.

Extracellular trafficking of a wheat cold-responsive protein, WLT10.

A cold-responsive wheat gene, WLT10, encodes a member of the cereal-specific low temperature-responsive/cold-responsive protein family, which contains a hydrophobic N-terminal 20 amino acid sequence that corresponds to signal peptides associated with extracellular trafficking. To verify the subcellular localization of WLT10 and the function of its putative signal peptide, we constructed three chimeric genes in which either the WLT10 signal peptide, a signal peptide with only 6 additional amino acids, or the full-length WLT10 polypeptide was fused to the N-terminus of green fluorescent protein (GFP). These fusion constructs were transiently introduced into onion epidermal cells by particle bombardment. GFP signals were observed not only in the extracellular space (ECS) but also in the endoplasmic reticulum (ER) and Golgi apparatus. The time course of GFP signal localization suggests the movement of WLT10 through the ER/Golgi pathway and into the ECS. Thus, WLT10 is a cold-responsive secreted protein, and its N-terminal 20 amino acid region is important for transport to the ECS.

A high-density genetic map with array-based markers facilitates structural and quantitative trait locus analyses of the common wheat genome.

The large genome and allohexaploidy of common wheat have complicated construction of a high-density genetic map. Although improvements in the throughput of next-generation sequencing (NGS) technologies have made it possible to obtain a large amount of genotyping data for an entire mapping population by direct sequencing, including hexaploid wheat, a significant number of missing data points are often apparent due to the low coverage of sequencing. In the present study, a microarray-based polymorphism detection system was developed using NGS data obtained from complexity-reduced genomic DNA of two common wheat cultivars, Chinese Spring (CS) and Mironovskaya 808. After design and selection of polymorphic probes, 13,056 new markers were added to the linkage map of a recombinant inbred mapping population between CS and Mironovskaya 808. On average, 2.49 missing data points per marker were observed in the 201 recombinant inbred lines, with a maximum of 42. Around 40% of the new markers were derived from genic regions and 11% from repetitive regions. The low number of retroelements indicated that the new polymorphic markers were mainly derived from the less repetitive region of the wheat genome. Around 25% of the mapped sequences were useful for alignment with the physical map of barley. Quantitative trait locus (QTL) analyses of 14 agronomically important traits related to flowering, spikes, and seeds demonstrated that the new high-density map showed improved QTL detection, resolution, and accuracy over the original simple sequence repeat map.

Short report: Identification of virulence-associated plasmids in Rhodococcus equi in humans with and without acquired immunodeficiency syndrome in Brazil.

Virulence of Rhodococcus equi strains from 20 humans in Brazil was investigated by using a polymerase chain reaction to characterize isolates as virulent (VapA), intermediately virulent (VapB), and avirulent. Nine isolates were obtained from human immunodeficiency virus (HIV)-positive patients, six from HIV-negative patients, and five from patients of unknown status. Five isolates were VapB positive, four were VapA positive, and eleven were avirulent. Among the nine isolates from HIV-positive patients, five contained VapB plasmids and two contained VapA plasmids. Five VapB-positive isolates had the type 8 virulence plasmid. Eleven of the patients had a history of contact with livestock and/or a farm environment, and none had contact with pigs.

Cryptogein-induced cell cycle arrest at G2 phase is associated with inhibition of cyclin-dependent kinases, suppression of expression of cell cycle-related genes and protein degradation in synchronized tobacco BY-2 cells.

Induction of defense responses by pathogens or elicitors is often accompanied by growth inhibition in planta, but its molecular mechanisms are poorly understood. In this report, we characterized the molecular events that occur during cryptogein-induced cell cycle arrest at G(2) phase in synchronously cultured tobacco Bright Yellow-2 (BY-2) cells. Concomitant with the proteinaceous elicitor-induced G(2) arrest, we observed inhibition of the histone H1 kinase activity of cyclin-dependent kinases (CDKs), which correlated with a decrease in mRNA and protein levels of CDKB1. In contrast, the amount of CDKA was almost unaffected by cryptogein even at M phase. Cryptogein rapidly inhibited the expression not only of positive, e.g. A- and B-type cyclins and NtCAK, but also of negative cell cycle regulators such as WEE1, suggesting that cryptogein affects multiple targets to inactivate CDKA to induce G(2) arrest by mechanisms distinct from known checkpoint regulation. Moreover, we show that CDKB1 and cyclin proteins are also rapidly degraded by cryptogein and that the proteasome-dependent protein degradation has a crucial role in the control of cryptogein-induced hypersensitive cell death.

Regulation of microbe-associated molecular pattern-induced hypersensitive cell death, phytoalexin production, and defense gene expression by calcineurin B-like protein-interacting protein kinases, OsCIPK14/15, in rice cultured cells.

Although cytosolic free Ca(2+) mobilization induced by microbe/pathogen-associated molecular patterns is postulated to play a pivotal role in innate immunity in plants, the molecular links between Ca(2+) and downstream defense responses still remain largely unknown. Calcineurin B-like proteins (CBLs) act as Ca(2+) sensors to activate specific protein kinases, CBL-interacting protein kinases (CIPKs). We here identified two CIPKs, OsCIPK14 and OsCIPK15, rapidly induced by microbe-associated molecular patterns, including chitooligosaccharides and xylanase (Trichoderma viride/ethylene-inducing xylanase [TvX/EIX]), in rice (Oryza sativa). Although they are located on different chromosomes, they have over 95% nucleotide sequence identity, including the surrounding genomic region, suggesting that they are duplicated genes. OsCIPK14/15 interacted with several OsCBLs through the FISL/NAF motif in yeast cells and showed the strongest interaction with OsCBL4. The recombinant OsCIPK14/15 proteins showed Mn(2+)-dependent protein kinase activity, which was enhanced both by deletion of their FISL/NAF motifs and by combination with OsCBL4. OsCIPK14/15-RNAi transgenic cell lines showed reduced sensitivity to TvX/EIX for the induction of a wide range of defense responses, including hypersensitive cell death, mitochondrial dysfunction, phytoalexin biosynthesis, and pathogenesis-related gene expression. On the other hand, TvX/EIX-induced cell death was enhanced in OsCIPK15-overexpressing lines. Our results suggest that OsCIPK14/15 play a crucial role in the microbe-associated molecular pattern-induced defense signaling pathway in rice cultured cells.

The Arabidopsis cyclin-dependent kinase-activating kinase CDKF;1 is a major regulator of cell proliferation and cell expansion but is dispensable for CDKA activation.

Cyclin-dependent kinases (CDKs) play an essential role in cell cycle regulation during the embryonic and post-embryonic development of various organisms. Full activation of CDKs requires not only binding to cyclins but also phosphorylation of the T-loop domain. This phosphorylation is catalysed by CDK-activating kinases (CAKs). Plants have two distinct types of CAKs, namely CDKD and CDKF; in Arabidopsis, CDKF;1 exhibits the highest CDK kinase activity in vitro. We have previously shown that CDKF;1 also functions in the activation of CDKD;2 and CDKD;3 by T-loop phosphorylation. Here, we isolated the knockout mutants of CDKF;1 and showed that they had severe defects in cell division, cell elongation and endoreduplication. No defect was observed during embryogenesis, suggesting that CDKF;1 function is primarily required for post-embryonic development. In the cdkf;1 mutants, T-loop phosphorylation of CDKA;1, an orthologue of yeast Cdc2/Cdc28p, was comparable to that in wild-type plants, and its kinase activity did not decrease. In contrast, the protein level and kinase activity of CDKD;2 were significantly reduced in the mutants. Substitution of threonine-168 with a non-phosphorylatable alanine residue made CDKD;2 unstable in Arabidopsis tissues. These results indicate that CDKF;1 is dispensable for CDKA;1 activation but is essential for maintaining a steady-state level of CDKD;2, thereby suggesting the quantitative regulation of a vertebrate-type CAK in a plant-specific manner.

Role of endogenous nitric oxide (NO) and NO synthases in healing of indomethacin-induced intestinal ulcers in rats.

We examined the roles of nitric oxide (NO) and NO synthase (NOS) isozymes in the healing of indomethacin-induced small intestinal ulcers in rats. Animals were given indomethacin (10 mg/kg, s.c.) and killed 1, 4 and 7 days after the administration. Indomethacin (2 mg/kg), N(G)-nitro-L-arginine methyl ester (L-NAME: a nonselective NOS inhibitor: 10 mg/kg) and aminoguanine (a relatively selective iNOS inhibitor: 20 mg/kg) were given s.c. once daily for 6 days, the first 3 days or the last 3 days during a 7-day experimental period. Both indomethacin and L-NAME significantly impaired healing of these lesions, irrespective of whether they were given for 6 days, first 3 days or last 3 days. The healing was also impaired by aminoguanine given for the first 3 days but not for the last 3 days. Expression of iNOS mRNA in the intestine was up-regulated after ulceration, persisting for 2 days thereafter, and the Ca(2+)-independent iNOS activity also markedly increased with a peak response during 1-2 days after ulceration. Vascular content in the ulcerated mucosa as measured by carmine incorporation was decreased when the healing was impaired by indomethacin and L-NAME given for either the first or last 3 days as well as aminoguanidine given for the first 3 days. These results suggest that endogenous NO plays a role in healing of intestinal lesions, in addition to prostaglandins, yet the NOS isozyme mainly responsible for NO production differs depending on the stage of healing: iNOS in the early stage and cNOS in the late stage.

Diverse phosphoregulatory mechanisms controlling cyclin-dependent kinase-activating kinases in Arabidopsis.

For the full activation of cyclin-dependent kinases (CDKs), not only cyclin binding but also phosphorylation of a threonine (Thr) residue within the T-loop is required. This phosphorylation is catalyzed by CDK-activating kinases (CAKs). In Arabidopsis three D-type CDK genes (CDKD;1-CDKD;3) encode vertebrate-type CAK orthologues, of which CDKD;2 exhibits high phosphorylation activity towards the carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II. Here, we show that CDKD;2 forms a stable complex with cyclin H and is downregulated by the phosphorylation of the ATP-binding site by WEE1 kinase. A knockout mutant of CDKD;3, which has a higher CDK kinase activity, displayed no defect in plant development. Instead, another type of CAK - CDKF;1 - exhibited significant activity towards CDKA;1 in Arabidopsis root protoplasts, and the activity was dependent on the T-loop phosphorylation of CDKF;1. We propose that two distinct types of CAK, namely CDKF;1 and CDKD;2, play a major role in CDK and CTD phosphorylation, respectively, in Arabidopsis.

Roles of endogenous prostaglandins and cyclooxygenase isozymes in healing of indomethacin-induced small intestinal lesions in rats.

The role of prostaglandins (PGs)/cyclooxygenase (COX) in the healing of indomethacin-induced small intestinal ulcers was examined in rats. Animals were given indomethacin (10 mg/kg s.c.) and killed 1, 2, 3, 5, and 7 days later. Indomethacin (2 mg/kg), 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560; COX-1 inhibitor; 3 mg/kg), and rofecoxib (COX-2 inhibitor; 3 mg/kg) were given p.o. once daily for 6 days, during the first 3 days or last 3 days of the experimental period. All COX inhibitors given for 6 days significantly impaired the healing of these ulcers. Healing was also impaired by rofecoxib given for the first 3 days or by SC560 given for the last 3 days. The expression of COX-2 mRNA in the intestine was up-regulated after ulceration, persisting for 3 days and dissipating thereafter. Mucosal PGE2 contents decreased within 3 h after ulceration, recovered 24 h later, and increased above normal 1 approximately 3 days later. The PGE2 content at 4 days after ulceration was decreased by rofecoxib but not SC560, whereas that at 7 days was suppressed by SC560 but not rofecoxib. Vascular content in the ulcerated mucosa decreased when the healing was impaired by COX inhibitors. The deleterious effect of indomethacin on healing was mimicked by a prostacyclin E receptor (EP) 4 antagonist and reversed by coadministration of PGE2 as well as an EP4 agonist. In conclusion, endogenous PGs play a role in the healing of intestinal ulcers through EP4 receptors, yet the COX isozyme involved differs depending on the stage of healing; COX-2 in the early stage and COX-1 in the late stage.

Improvement of freezing tolerance in tobacco plants expressing a cold-responsive and chloroplast-targeting protein WCOR15 of wheat.

Cold acclimation, an adaptive process for developing freezing tolerance in over-wintering plants, is associated with increased expression levels of a series of cold-responsive (Cor)/late embryogenesis abundant (Lea) genes. To investigate the function of Wcor15, a member of the wheat Cor/Lea gene family, for improvement of freezing tolerance, two types of transgenic tobacco lines expressing Wcor15-containing chimeric genes were produced and characterized. Immunoblot and gene expression analyses of a transgenic tobacco line expressing the Wcor15-GFP fusion gene under control of the CaMV35S promoter showed transport and abundant accumulation of the WCOR15 protein in the stromal compartment of the chloroplasts. The 5' upstream region of Wcor15 induced expression of the GFP reporter gene under low-temperature conditions in the transgenic tobacco. Both transgenic lines expressing the Wcor15-GFP fusion gene showed a similar and significantly improved level of freezing tolerance compared with the wild-type tobacco plants. Our results demonstrate that the induced expression of the wheat Wcor15 gene positively contributes to the development of freezing tolerance in the heterologous tobacco plants.

A distinct type of cyclin D, CYCD4;2, involved in the activation of cell division in Arabidopsis.

The Arabidopsis genome encodes 10 D-type cyclins (CYCD); however, their differential role in cell cycle control is not well known. Among them, CYCD4;2 is unique in the amino acid sequence; namely, it lacks the Rb-binding motif and the PEST sequence that are conserved in CYCDs. Here, we have shown that CYCD4;2 suppressed G1 cyclin mutations in yeast and formed a kinase complex with CDKA;1, an ortholog of yeast Cdc28, in insect cells. Hypocotyl explants of CYCD4;2 over-expressing plants showed faster induction of calli than wild-type explants on a medium containing lower concentration of auxin. These results suggest that CYCD4;2 has a promotive function in cell division by interacting with CDKA;1 regardless of the unusual primary sequence.

Differential and coordinated expression of Cbf and Cor/Lea genes during long-term cold acclimation in two wheat cultivars showing distinct levels of freezing tolerance.

The cold acclimation process in plants is primarily regulated through the signal transduction pathways that lead to the induction and enhancement of expression of different sets of Cor/Lea genes. Winter wheat 'Mironovskaya 808' (M808) exhibited a much higher level of freezing tolerance than spring wheat 'Chinese Spring' (CS), and the difference became clearer after the long-term cold acclimation. To understand the molecular basis of this cultivar difference, we isolated two CBF/DREB1 homologs, Wcbf2, which are the candidate gene for a transcription factor of the Cor/Lea genes. Expression of the Wcbf2 gene was induced rapidly by low temperature (LT) and drought but not by abscisic acid (ABA). The gene expression was temporal and at least twice up-regulated by LT. The first up-regulation occurred within 1-4 h, which might correspond to the rapid response to LT, while the second up-regulation occurred during 2-3 weeks of cold acclimation. After the second up-regulation, the amount of Wcbf2 transcript greatly decreased in CS, while it increased again in M808 after 4 weeks until 9 weeks (end of the test period). The maintenance of this high level of the Wcbf2 transcript might represent the long-term effect of cold acclimation. The activation of Cor/Lea genes followed the accumulation of Wcbf2 transcript suggested direct involvement of the Wcbf2 gene in the induction and enhancement of the Cor/Lea gene expression. The cultivar difference in freezing tolerance developed during different stages of cold acclimation can be at least partly explained by the differential and coordinated regulation of the predicted Cor/Lea gene signal transduction pathway that is mediated by the CBF/DREB1 transcription factors in common wheat.

Regulation by Vrn-1/Fr-1 chromosomal intervals of CBF-mediated Cor/Lea gene expression and freezing tolerance in common wheat.

Vrn-1/Fr-1 chromosomal regions of common wheat possess major QTLs for both winter hardiness (Fr) and vernalization requirement (Vrn). The Vrn-1/Fr-1 intervals are assigned to long arms of the homologous group 5 chromosomes. To investigate the role of the Vrn-1/Fr-1 intervals on the low-temperature (LT) inducibility of wheat Cor/Lea genes and its putative transcription factor gene Wcbf2, LT response of these genes was monitored using near-isogenic lines (NILs) for the Vrn-1 loci. The Wcbf2 transcript accumulated rapidly after LT treatment and remained at a high level in lines without any dominant Vrn-1 alleles. By contrast, the Wcbf2 transcript level was greatly reduced in lines carrying the Vrn-1 alleles. The Vrn-1 NILs accumulated much lower amounts of Cor/Lea transcripts and COR/LEA proteins than the non-carrier line. The observed patterns and levels of gene expression, particularly in the Vrn-A1 NIL, agreed with the higher sensitivity to freezing damage in this line than in the non-carrier line. Up-regulation of the expression of the WAP1 gene, a candidate of the Vrn-1 loci, was much delayed in the non-carrier line than all the NILs carrying the Vrn-1 loci. Neither positive nor negative relationships were found between the WAP1 expression and the Cbf2/Cor/Lea expression. These results support the intimate relationship between the Cbf2/Cor/Lea expression and the level of freezing tolerance, and suggest that a functional Fr-A1 allele linked to the vrn-A1 allele, instead of the vernalization gene itself, plays a major role in regulating the CBF-mediated Cor/lea gene expression in wheat.

Cell cycle dependence of elicitor-induced signal transduction in tobacco BY-2 cells.

The molecular links between the cell cycle and defense responses in plants are largely unknown. Using synchronized tobacco BY-2 cells, we analyzed the cell cycle dependence of elicitor-induced defense responses. In synchronized cultured apoaequorin-expressing cells, the increase in cytosolic free Ca2+ induced by a proteinaceous elicitor, cryptogein, was greatly suppressed during the G2 and M phases in comparison with G1 or S phases. Treatment with cryptogein during the G1 or S phases also induced biphasic (rapid/transient and slow/prolonged) responses in activation of mitogen-activated protein kinases (MAPKs) and production of reactive oxygen species (ROS). In contrast, elicitor treatment during the G2 or M phases induced only a rapid and transient phase of MAPK activation and ROS production. Their slow and prolonged phases as well as expression of defense-related genes, cell cycle arrest and cell death were induced only after the cell cycle progressed to the G1 phase; removal of the elicitor before the start of the G1 phase inhibited these responses. These results suggest that although cryptogein recognition occurred at all phases of the cell cycle, the recognition during the S or G1 phases, but not at the G2 or M phases, induces the prolonged activation of MAPKs and the prolonged production of ROS, followed by cell cycle arrest, accumulation of defense-related gene transcripts and cell death. Elicitor signal transduction depends on the cell cycle and is regulated differently at each phase.