Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

Characterization of plant immunity-activating mechanism by a pyrazole derivative.

A newly identified chemical, 4-{3-[(3,5-dichloro-2-hydroxybenzylidene)amino]propyl}-4,5-dihydro-1H-pyrazol-5-one (BAPP) was characterized as a plant immunity activator. BAPP enhanced disease resistance in rice against rice blast disease and expression of a defense-related gene without growth inhibition. Moreover, BAPP was able to enhance disease resistance in dicotyledonous tomato and Arabidopsis plants against bacterial pathogen without growth inhibition, suggesting that BAPP could be a candidate as an effective plant activator. Analysis using Arabidopsis sid2-1 and npr1-2 mutants suggested that BAPP induced systemic acquired resistance (SAR) by stimulating between salicylic acid biosynthesis and NPR1, the SA receptor protein, in the SAR signaling pathway.

Suppression of phospholipase D genes improves chalky grain production by high temperature during the grain-filling stage in rice.

High temperature (HT) during the grain developing stage causes deleterious effects on rice quality resulting in mature grains with a chalky appearance. Phospholipase D (PLD) plays an important role in plants, including responses to environmental stresses. OsPLDα1, α3 and ß2-knockdown (KD) plants showed decreased production of chalky grains at HT. HT ripening increased H2O2 accumulated in the developing grains. However, the increase was canceled by the knockdown of OsPLDß2. Expression levels of OsCATA which is one of three rice catalase genes, in developing grains of OsPLDß2-KD plants at 10 DAF were increased compared with that in vector-controls in HT growth conditions. Overexpression of OsCATA markedly suppressed the production of chalky grains in HT growth conditions. These results suggested that OsPLDß2 functions as a negative regulator of the induction of OsCATA and is involved in the production of chalky grains in HT growth conditions.

Laser Microdissection-Based Tissue-Specific Transcriptome Analysis Reveals a Novel Regulatory Network of Genes Involved in Heat-Induced Grain Chalk in Rice Endosperm.

Heat stress occurrence during seed filling leads to the formation of a chalky portion in the limited zone of the starchy endosperm of rice grains. In this study, isolation of aleurone, dorsal, central and lateral tissues of developing endosperm by laser-microdissection (LM) coupled with gene expression analysis of a 44 K microarray was performed to identify key regulatory genes involved in the formation of milky-white (MW) and white-back (WB) grains during heat stress. Gene regulatory network analysis classified the genes changed under heat stress into five modules. The most distinct expression pattern was observed in modules where most of the small heat shock proteins and cellular organization genes were changed under heat stress in dorsal aleurone cells and dorsal starchy endosperm zones. The histological observation supported the significant increase in cell number and size of dorsal aleurone cells in WB grains. With regard to the central starchy endosperm zone, preferential down-regulation of high molecular weight heat shock proteins (HMW HSPs), including a prominent member encoding endoplasmic reticulum (ER) chaperones, by heat stress was observed, while changes in expression of starch biosynthesis genes were minimal. Characterization of transgenic plants suppressing endosperm lumenal binding protein gene (BiP1), an ER chaperone preferentially down-regulated at the MW zone under heat stress, showed evidence of forming the chalky grains without disturbing the expression of starch biosynthesis genes. The present LM-based comprehensive expression analysis provides novel inferences that HMW HSPs play an important role in controlling redox, nitrogen and amino acid metabolism in endosperm leading to the formation of MW and WB chalky grains under heat stress.

Involvement of ethylene signaling in Azospirillum sp. B510-induced disease resistance in rice.

A bacterial endophyte Azospirillum sp. B510 induces systemic disease resistance in the host without accompanying defense-related gene expression. To elucidate molecular mechanism of this induced systemic resistance (ISR), involvement of ethylene (ET) was examined using OsEIN2-knockdown mutant rice. Rice blast inoculation assay and gene expression analysis indicated that ET signaling is required for endophyte-mediated ISR in rice. ABBREVIATIONS: ACC: 1-aminocyclopropane-1-carboxylic acid; EIN2: ethylene-insensitive protein 2; ET: ethylene; ISR: induced systemic resistance; JA: jasmonic acid; RNAi: RNA interference; SA: salicylic acid; SAR: systemic acquired resistance.

MutMapPlus identified novel mutant alleles of a rice starch branching enzyme IIb gene for fine-tuning of cooked rice texture.

Physicochemical properties of storage starch largely determine rice grain quality and food characteristics. Therefore, modification of starch property is effective to fine-tune cooked rice textures. To obtain new resources with modified starch property as breeding materials, we screened a mutant population of a japonica cultivar Nipponbare and found two independent mutant lines, altered gelatinization (age)1 and age2, with moderate changes in starch gelatinization property. A combination of conventional genetic analyses and the latest mapping method, MutMapPlus, revealed that both of these lines harbour novel independent mutant alleles of starch branching enzyme IIb (BEIIb) gene. In age1, amino acid substitution of Met-723 to Lys completely abolished BEIIb enzyme activity without significant reduction in its protein level. A transposon insertion in an intron of BEIIb gene reduced BEIIb protein level and activity in age2. Production of a series of the mutant lines by combining age alleles and indica-type starch synthase IIa allele established stepwise alteration of the physicochemical properties of starch including apparent amylose content, thermal property, digestibility by α-amylase and branched structures of amylopectin. Consistent with the alteration of starch properties, the results of a sensory evaluation test demonstrated that warm cooked rice of the mutants showed a variety of textures without marked reduction in overall palatability. These results suggest that a series of the mutant lines are capable of manipulation of cooked rice textures.

High Temperature-Induced Expression of Rice α-Amylases in Developing Endosperm Produces Chalky Grains.

Global warming impairs grain filling in rice and reduces starch accumulation in the endosperm, leading to chalky-appearing grains, which damages their market value. We found previously that high temperature-induced expression of starch-lytic α-amylases during ripening is crucial for grain chalkiness. Because the rice genome carries at least eight functional α-amylase genes, identification of the α-amylase(s) that contribute most strongly to the production of chalky grains could accelerate efficient breeding. To identify α-amylase genes responsible for the production of chalky grains, we characterized the histological expression pattern of eight α-amylase genes and the influences of their overexpression on grain appearance and carbohydrate components through a series of experiments with transgenic rice plants. The promoter activity of most α-amylase genes was elevated to various extents at high temperature. Among them, the expression of Amy1A and Amy3C was induced in the internal, especially basal to dorsal, region of developing endosperm, whereas that of Amy3D was confined near the ventral aleurone. These regions coincided with the site of occurrence of chalkiness, which was in clear contrast to conventionally known expression patterns of the enzyme in the scutellum and aleurone during seed germination. Furthermore, overexpression of α-amylase genes, except for Amy3E, in developing endosperm produced various degrees of chalky grains without heat exposure, whereas that of Amy3E yielded normal translucent grains, as was the case in the vector control, even though Amy3E-overexpressing grains contained enhanced α-amylase activities. The weight of the chalky grains was decreased due to reduced amounts of starch, and microscopic observation of the chalky part of these grains revealed that their endosperm consisted of loosely packed round starch granules that had numerous pits on their surface, confirming the hydrolysis of the starch reserve by α-amylases. Moreover, the chalky grains contained increased amounts of soluble sugars including maltooligosaccharides at the expense of starch. The integrated analyses proposed that expression of Amy1A, Amy3C, and Amy3D at the specific regions of the developing endosperm could generate the chalkiness. This finding provides the fundamental knowledge to narrow down the targets for the development of high temperature-tolerant premium rice.

An Activity-Staining Method on Filtration Paper Enables High-Throughput Screening of Temperature-Sensitive and Inactive Mutations of Rice α-Amylase for Improvement of Rice Grain Quality.

α-Amylase is a starch-hydrolyzing enzyme (EC 3.2.1.1) indispensable for germination of cereal seeds, but it is also expressed during the ripening stage. Previous studies demonstrated that the enzyme is activated in developing rice seeds under extremely hot weather and triggers a loss of grain quality by hindering the accumulation of storage starch in the endosperm. Since inactive or, preferably, heat-labile α-amylases are preferable for breeding premium rice, we developed a method for rapid screening of inactive and temperature-sensitive mutants of the enzyme by combining the random mutagenesis by error-prone PCR and an on-filter activity test of the recombinant enzyme expressed by Escherichia coli. This technique was applied to a major α-amylase in the developing seed, Amy3D, and the activity of the isolated mutant enzymes was verified with both the bacteria-expressed recombinant proteins and the extract from the endosperm overexpressing each of them. Then, we identified several substitutions leading to loss of the activity of amino acid residues (Leu28, Asp112, Cys149, Trp201, Asp204, Gly295, Leu300 and Cys342), as well as a variety of heat-sensitive substitutions of Asp83, Asp187 and Glu252. Furthermore, variations of the heat-labile enzymes were created by combining these heat-sensitive mutations. The effects of the respective mutations and their relationship to the structure of the enzyme molecule are discussed.

Overexpression of TIFY genes promotes plant growth in rice through jasmonate signaling.

Because environmental stress can reduce crop growth and yield, the identification of genes that enhance agronomic traits is increasingly important. Previous screening of full-length cDNA overexpressing (FOX) rice lines revealed that OsTIFY11b, one of 20 TIFY proteins in rice, affects plant size, grain weight, and grain size. Therefore, we analyzed the effect of OsTIFY11b and nine other TIFY genes on the growth and yield of corresponding TIFY-FOX lines. Regardless of temperature, grain weight and culm length were enhanced in lines overexpressing TIFY11 subfamily genes, except OsTIFY11e. The TIFY-FOX plants exhibited increased floret number and reduced days to flowering, as well as reduced spikelet fertility, and OsTIFY10b, in particular, enhanced grain yield by minimizing decreases in fertility. We suggest that the enhanced growth of TIFY-transgenic rice is related to regulation of the jasmonate signaling pathway, as in Arabidopsis. Moreover, we discuss the potential application of TIFY overexpression for improving crop yield.

Overexpression of a rice TIFY gene increases grain size through enhanced accumulation of carbohydrates in the stem.

Screening of rice full-length cDNA overexpressing (FOX) lines allowed the identification of a TIFY gene, TIFY11b, as a growth-promoting gene whose overexpression increased plant height and seed size. The grains of TIFY11b-overexpressing plants exceeded those of non-transformants in length, width and thickness, resulting in 9-21% increases in grain weight. The increase was achieved by overexpressing the gene in the whole plant body, but not by seed-restricted expression, indicating that seed enlargement is attributable to overexpression in vegetative organs such as the leaf. The whole-body overexpressing plants developed longer leaves along with higher levels of starch and sucrose in the leaf sheath and culm at the heading stage than the non-transformants. Although overexpression of TIFY11b did not alter the photosynthetic rate per leaf area before and after heading, it caused an accumulation of higher levels of the carbohydrate assimilate, probably due to increased photosynthesis per plant, suggesting that the increase in grain size and weight is attained by enhanced accumulation and translocation of the carbohydrate in the culms and leaf sheaths of the transgenic plants. Thus, TIFY11b is a novel grain-size increasing gene.

Suppression of α-amylase genes improves quality of rice grain ripened under high temperature.

High temperature impairs rice (Oryza sativa) grain filling by inhibiting the deposition of storage materials such as starch, resulting in mature grains with a chalky appearance, currently a major problem for rice farming in Asian countries. Such deterioration of grain quality is accompanied by the altered expression of starch metabolism-related genes. Here we report the involvement of a starch-hydrolyzing enzyme, α-amylase, in high temperature-triggered grain chalkiness. In developing seeds, high temperature induced the expression of α-amylase genes, namely Amy1A, Amy1C, Amy3A, Amy3D and Amy3E, as well as α-amylase activity, while it decreased an α-amylase-repressing plant hormone, ABA, suggesting starch to be degraded by α-amylase in developing grains under elevated temperature. Furthermore, RNAi-mediated suppression of α-amylase genes in ripening seeds resulted in fewer chalky grains under high-temperature conditions. As the extent of the decrease in chalky grains was highly correlated to decreases in the expression of Amy1A, Amy1C, Amy3A and Amy3B, these genes would be involved in the chalkiness through degradation of starch accumulating in the developing grains. The results show that activation of α-amylase by high temperature is a crucial trigger for grain chalkiness and that its suppression is a potential strategy for ameliorating grain damage from global warming.

The biotron breeding system: a rapid and reliable procedure for genetic studies and breeding in rice.

Oryza sativa is widely used as a model organism for many aspects of research in monocots and cereals. However, it has certain disadvantages as a model species compared with Arabidopsis thaliana, the eudicot species most widely used in plant sciences: first, it has a long cultivation time; and second, it requires considerably more space for growth. Here, we introduce a biotron breeding system, which allows rapid and reliable rice cultivation using a well-equipped artificial environmental chamber. This system involves use of regulation of CO2 levels, removal of tillers and embryo rescue to overcome the disadvantages of rice cultivation. The rice cultivars Nipponbare, Koshihikari, Taichung 65 and Kasalath all showed vigorous growth and sufficient seed production in the biotron breeding system with accelerated flowering time. Nipponbare, which was the earliest among these cultivars, flowered at about 50 d after sowing. The life cycle of these plants could be further shortened using an embryo rescue technique on immature seeds at 7 d after pollination, thereby avoiding the lengthy process of seed maturation. Overall, it was possible to shorten the life cycle of Nipponbare to about 2 months under the controlled conditions. Furthermore, controlled crosses, which can be difficult with conventional cultivation methods, were easy to perform as we could control the exact timing of anther dehiscence. Thus, our biotron breeding system offers a valuable new approach to genetic and breeding studies in rice.

A novel factor FLOURY ENDOSPERM2 is involved in regulation of rice grain size and starch quality.

Rice (Oryza sativa) endosperm accumulates a massive amount of storage starch and storage proteins during seed development. However, little is known about the regulatory system involved in the production of storage substances. The rice flo2 mutation resulted in reduced grain size and starch quality. Map-based cloning identified FLOURY ENDOSPERM2 (FLO2), a member of a novel gene family conserved in plants, as the gene responsible for the rice flo2 mutation. FLO2 harbors a tetratricopeptide repeat motif, considered to mediate a protein-protein interactions. FLO2 was abundantly expressed in developing seeds coincident with production of storage starch and protein, as well as in leaves, while abundant expression of its homologs was observed only in leaves. The flo2 mutation decreased expression of genes involved in production of storage starch and storage proteins in the endosperm. Differences between cultivars in their responsiveness of FLO2 expression during high-temperature stress indicated that FLO2 may be involved in heat tolerance during seed development. Overexpression of FLO2 enlarged the size of grains significantly. These results suggest that FLO2 plays a pivotal regulatory role in rice grain size and starch quality by affecting storage substance accumulation in the endosperm.

Atlas of rice grain filling-related metabolism under high temperature: joint analysis of metabolome and transcriptome demonstrated inhibition of starch accumulation and induction of amino acid accumulation.

High temperature impairs grain filling by inhibiting the deposition of storage materials such as starch and protein. To comprehend its impact on grain filling metabolism in rice (Oryza sativa), levels of metabolites and transcripts related to central pathways of metabolism were simultaneously determined in developing caryopses exposed to high temperature (33 degrees C/28 degrees C) and a control temperature (25 degrees C/20 degrees C) during the milky stage. A capillary electrophoresis-based metabolomic analysis revealed that high temperature increased the accumulation of sucrose and pyruvate/ oxaloacetate-derived amino acids and decreased levels of sugar phosphates and organic acids involved in glycolysis/gluconeogenesis and the tricarboxylic acid (TCA) cycle, respectively. A transcriptomic analysis using a whole genome-covering microarray unraveled the possible metabolic steps causing the shortage of storage materials under the elevated temperature. Starch deposition might be impaired by down-regulation of sucrose import/degradation and starch biosynthesis, and/or up-regulation of starch degradation as well as inefficient ATP production by an inhibited cytochrome respiration chain, as indicated by the response of gene expression to high temperature. Amino acid accumulation might be attributed to the heat-stable import of amino acids into the caryopsis and/or repression of protein synthesis especially the tRNA charging step under high temperature. An atlas showing the effect of high temperature on levels of metabolites and gene expression in the central metabolic pathways is presented.

Suppression of a phospholipase D gene, OsPLDbeta1, activates defense responses and increases disease resistance in rice.

Phospholipase D (PLD) plays an important role in plants, including responses to abiotic as well as biotic stresses. A survey of the rice (Oryza sativa) genome database indicated the presence of 17 PLD genes in the genome, among which OsPLDalpha1, OsPLDalpha5, and OsPLDbeta1 were highly expressed in most tissues studied. To examine the physiological function of PLD in rice, we made knockdown plants for each PLD isoform by introducing gene-specific RNA interference constructs. One of them, OsPLDbeta1-knockdown plants, showed the accumulation of reactive oxygen species in the absence of pathogen infection. Reverse transcription-polymerase chain reaction and DNA microarray analyses revealed that the knockdown of OsPLDbeta1 resulted in the up-/down-regulation of more than 1,400 genes, including the induction of defense-related genes such as pathogenesis-related protein genes and WRKY/ERF family transcription factor genes. Hypersensitive response-like cell death and phytoalexin production were also observed at a later phase of growth in the OsPLDbeta1-knockdown plants. These results indicated that the OsPLDbeta1-knockdown plants spontaneously activated the defense responses in the absence of pathogen infection. Furthermore, the OsPLDbeta1-knockdown plants exhibited increased resistance to the infection of major pathogens of rice, Pyricularia grisea and Xanthomonas oryzae pv oryzae. These results suggested that OsPLDbeta1 functions as a negative regulator of defense responses and disease resistance in rice.

Expressed nifH Genes of Endophytic Bacteria Detected in Field-Grown Sweet Potatoes (Ipomoea batatas L.).

We examined the nitrogenase reductase (nifH) genes of endophytic diazotrophic bacteria expressed in field-grown sweet potatoes (Ipomoea batatas L.) by reverse transcription (RT)-PCR. Gene fragments corresponding to nifH were amplified from mRNA obtained from the stems and storage roots of field-grown sweet potatoes several months after planting. Sequence analysis revealed that these clones were homologous to the nifH sequences of Bradyrhizobium, Pelomonas, and Bacillus sp. in the DNA database. Investigation of the nifH genes amplified from the genomic DNA extracted from these sweet potatoes also showed high similarity to various α-proteobacteria including Bradyrhizobium, ß-proteobacteria, and cyanobacteria. These results suggest that bradyrhizobia colonize and express nifH genes not only in the root nodules of leguminous plants but also in sweet potatoes as diazotrophic endophytes.

Comprehensive expression profiling of rice grain filling-related genes under high temperature using DNA microarray.

To elucidate the effect of high temperature on grain-filling metabolism, developing rice (Oryza sativa) "Nipponbare" caryopses were exposed to high temperature (33 degrees C/28 degrees C) or control temperature (25 degrees C/20 degrees C) during the milky stage. Comprehensive gene screening by a 22-K DNA microarray and differential hybridization, followed by expression analysis by semiquantitative reverse transcription-PCR, revealed that several starch synthesis-related genes, such as granule-bound starch synthase I (GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate dikinase gene were down-regulated by high temperature, whereas those for starch-consuming alpha-amylases and heat shock proteins were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with the diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted in the occurrence of grains with various degrees of chalky appearance and decreased weight. Among them, severely chalky grains contained amylopectin enriched particularly with long chains compared to slightly chalky grains, suggesting that such alterations of amylopectin structure might be involved in grain chalkiness. However, among high temperature-tolerant and sensitive cultivars, alterations of neither amylopectin chain-length distribution nor amylose content were correlated to the degree of grain chalkiness, but rather seemed to be correlated to grain weight decrease, implying different underlying mechanisms for the varietal difference in grain chalkiness. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are discussed.

Catalytic activation of the plant MAPK phosphatase NtMKP1 by its physiological substrate salicylic acid-induced protein kinase but not by calmodulins.

MAPK phosphatases (MKPs) are negative regulators of MAPKs. Previously, we identified NtMKP1 as a novel calmodulin (CaM)-binding protein (Yamakawa, H., Katou, S., Seo, S., Mitsuhara, I., Kamada, H., and Ohashi, Y. (2004) J. Biol. Chem. 279, 928-936). In this study, we characterized the interaction of NtMKP1 with substrate MAPKs and CaM. NtMKP1 (produced by in vitro transcription/translation) inactivated salicylic acid-induced protein kinase (SIPK) through dephosphorylation of the TEY motif of SIPK. CaM bound but unexpectedly did not activate the phosphatase activity of NtMKP1. NtMKP1 has four characteristic domains, viz. a dual-specificity phosphatase catalytic domain, a gelsolin homology domain, a CaM-binding domain, and C-terminal domain. Deletion analysis revealed that the N-terminal non-catalytic region of NtMKP1 bound SIPK and was essential for inactivating SIPK, whereas the CaM-binding and C-terminal domains were dispensable. Moreover, the phosphatase activity of NtMKP1 was increased strongly by the binding of SIPK, but weakly by another MAPK, wound-induced protein kinase. Swapping and site-directed mutagenesis of SIPK and wound-induced protein kinase revealed that the strong activation of NtMKP1 phosphatase activity by SIPK partially depended on the putative common docking domain of SIPK. On the other hand, conversion of Lys(41) and Arg(43) of NtMKP1 to Ala (K41A/R43A) abolished the interaction with SIPK. Expression of constitutively active MAPK kinase in Nicotiana benthamiana induced activation of SIPK and cell death. Simultaneous expression of either NtMKP1 or NtMKP1 L443R, which was unable to bind CaM, compromised the constitutively active MAPK kinase-induced responses, whereas that of NtMKP1 K41A/R43A did not. These results indicate that the regulation of NtMKP1 activity by SIPK binding, but not by CaM binding, is important for the function of NtMKP1.

Three types of tobacco calmodulins characteristically activate plant NAD kinase at different Ca2+ concentrations and pHs.

We previously reported that three types of tobacco calmodulin (CaM) isoforms originated from 13 genes are differently regulated at the transcript and protein levels in response to wounding and tobacco mosaic virus-induced hypersensitive reaction (HR); wound-inducible type I and HR-inducible type III levels increased after wounding and HR, respectively, while type II, whose expression is constitutive and wound responsible, remained unchanged. Here, we show that these CaMs differentially activate target enzymes; rat NO synthase was activated most effectively by type III, moderately by type I and weakly by type II, and plant NAD kinase (NADK) was activated in the inverse order. Furthermore, we found that a suitable Ca2+ concentration differs by type; type II activated NADK at lower Ca2+ of around 0.1 microM, which is the cytosolic concentration in unstimulated cells, type I did so at 1-5 microM, which is the increased Ca2+ concentration in stimulated cells, while type III did not at any Ca2+ level. NADK activation was highest over a pH range of 7.1-6.8 for which the cytosolic pH reportedly changed from 7.5 after being stimulated. Thus, tobacco CaMs, especially type I, effectively activate NADK in stimuli-induced conditions.

Plant MAPK phosphatase interacts with calmodulins.

A mitogen-activated protein kinase (MAPK) phosphatase gene, designated NtMKP1, was isolated as a candidate gene for a calmodulin (CaM)-binding protein from tobacco. NtMKP1 protein has four characteristic domains conserved among plant MAPK phosphatases reported so far, namely a dual specificity protein phosphatase catalytic domain, gelsolin-like domain, putative CaM-binding domain (CaMBD), and serine-rich region, indicating that NtMKP1 is the ortholog of Arabidopsis MKP1. The bacterially expressed NtMKP1 protein physically interacted with three plant-specific types of CaM in an overlay assay with labeled CaMs, showing high affinity to NtCaM1 and NtCaM3 but lower affinity to NtCaM13. The peptide for the putative CaMBD bound both NtCaM1 and NtCaM3 significantly but bound NtCaM13 only slightly. Moreover, CaM overlay assays with mutated CaMBDs revealed that Trp440 and Leu443 in the middle of the basic amphiphilic alpha-helix motif (amino acids 436-453) are critical for binding CaM. In comparison with the transient accumulation of a wound-induced MAPK, WIPK transcript, a prolonged activation of NtMKP1 expression was found in response to wounding and tobacco mosaic virus-induced hypersensitive reaction. In transgenic tobacco plants overexpressing NtMKP1, wound-induced activation of SIPK, salicylic acid-induced MAPK, and WIPK was inhibited. These results suggest that plant CaMs are involved in these stress-activated MAPK cascades via NtMKP1.