Effects of formulation on transport of pyrethroids in residential settings.

Washoff of 17 pyrethroid products resulting from a 1-h, 25.4-mm rainfall occurring 24 h after application was measured in indoor studies with concrete slabs. These products included different pyrethroid active ingredients and a range of formulation types. Based on this replicated study, 5 product pairs with contrasting washoff behaviors were chosen for an outdoor study using 6 full-scale house fronts in central California. Products in 4 of these pairs were applied once to different rectangular areas on the driveway (1 product in each pair to 3 house lots and the other to the remaining 3 house lots). The products in the fifth pair were applied 3 times at 2-mo intervals to vertical stucco walls above the driveway. All house lots received natural and simulated rainfall over 7 mo. Indoor studies showed differences up to 170-fold between paired products, whereas the maximum difference between paired products in the field was only 5-fold. In the pair applied to the wall, 1 product had 91 times the washoff of the other in the indoor study, whereas in the field the same product had 15% lower washoff. These results show that, although the formulation may influence washoff under actual use conditions, its influence is complex and not always as predicted by indoor experiments. Because the formulation also affects insect control, washoff research needs to be conducted together with efficacy testing.

Major transport mechanisms of pyrethroids in residential settings and effects of mitigation measures.

The major pathways for transport of pyrethroids were determined in runoff studies conducted at a full-scale test facility in central California, USA. The 6 replicate house lots were typical of front lawns and house fronts of California residential developments and consisted of stucco walls, garage doors, driveways, and residential lawn irrigation sprinkler systems. Each of the 6 lots also included a rainfall simulator to generate artificial rainfall events. Different pyrethroids were applied to 5 surfaces­driveway, garage door and adjacent walls, lawn, lawn perimeter (grass near the house walls), and house walls above grass. The volume of runoff water from each house lot was measured, sampled, and analyzed to determine the amount of pyrethroid mass lost from each surface. Applications to 3 of the house lots were made using the application practices typically used prior to recent label changes, and applications were made to the other 3 house lots according to the revised application procedures. Results from the house lots using the historic application procedures showed that losses of the compounds applied to the driveway and garage door (including the adjacent walls) were 99.75% of total measured runoff losses. The greatest losses were associated with significant rainfall events rather than lawn irrigation events. However, runoff losses were 40 times less using the revised application procedures recently specified on pyrethroid labels.

Calling dynamics and call synchronization in a local group of unison bout callers.

In many species of chorusing frogs, callers can rapidly adjust their call timing with reference to neighboring callers so as to maintain call rate while minimizing acoustic interference. The rules governing the interactions, in particular, who is listening to whom are largely unknown, presumably influenced by distance between callers, caller density, and intensities of interfering calls. We report vocal interactions in a unison bout caller, the green tree frog (Hyla cinerea). Using a microphone array, we monitored bouts from a local group of six callers embedded in a larger chorus. Data were analyzed in a 21-min segment at the peak of the chorus. Callers within this group were localized and their voices were separated for analysis of spatio-temporal interactions. We show that callers in this group: (1) synchronize with one another, (2) prefer to time their calls antiphonally, almost exactly at one-third and two-thirds of the call intervals of their neighbors, (3) tolerate call collision when antiphonal calling is not possible, and (4) perform discrete phase-hopping between three preferred phases when tracking other callers. Further, call collision increases and phase-locking decreases, with increasing inter-caller spacing. We conclude that the precise phase-positioning, phase-tracking, and phase-hopping minimizes acoustic jamming while maintaining chorus synchrony.

Laboratory degradation rates of 11 pyrethroids under aerobic and anaerobic conditions.

Degradation of 11 pyrethroids was measured over approximately 100 days in three sediment/water systems under aerobic and anaerobic conditions at 25 °C in the dark. The three California sediments represented a range of textures and organic matter. Test compounds were bifenthrin, cypermethrin, ζ-cypermethrin, cyfluthrin, ß-cyfluthrin, deltamethrin, esfenvalerate, fenpropathrin, γ-cyhalothrin, λ-cyhalothrin, and permethrin. A non-standard design was employed to keep conditions essentially the same for all compounds. The test compounds were applied as two test mixtures (six active ingredients per mixture, with bifenthrin common to both) at approximately 50 µg of test compound/kg of sediment (dry weight). Extracts of sediment/water were cleaned up by solid-phase extraction, concentrated, and analyzed by gas chromatography/mass spectrometry (except deltamethrin) against matrix-matched standards, with cyfluthrin-d6 as an internal standard. Deltamethrin was analyzed by liquid chromatography/tandem mass spectrometry using deltamethrin-phenoxy-(13)C6 as an internal standard. Similar degradation rates of bifenthrin and for related isomeric compounds (e.g., cyfluthrin and ß-cyfluthrin) were generally measured in both mixtures for each sediment. First-order half-lives under aerobic conditions ranged from 2.9 to greater than 200 days, with a median value of 18 days. Under anaerobic conditions, the range was from 20 to greater than 200 days, with a median value of 70 days.

Nitric oxide methods in seed biology.

The ubiquitous signaling molecule nitric oxide (NO) plays an important role in seed biology. Experiments with this biologically important gas require special provisions because NO in aerobic environments is readily converted into other oxides of nitrogen. In this chapter, we describe methods for the application of NO as a gas, and through the use of NO-donor compounds. We included information on the removal or reduction of NO with NO scavengers. Methods for detecting NO using NO-reactive fluorescent probes, and an apparatus incorporating an oxidizer column are also described.

Runoff transport of pyrethroids from a residential lawn in central California.

An irrigation runoff study on a residential lawn was conducted in California, northeast of Sacramento, during the summer and fall of 2008 to investigate the contribution of turf uses of pyrethroids to residues in Californian urban creek sediments. This study examined how over irrigation (i.e., irrigation that produces runoff) in the summer season may transport recently applied pyrethroids. The study included liquid and granular applications of both bifenthrin [(2-methyl-3-phenyl-phenyl) methyl 3-(2-chloro-3,3,3-trifluoro-prop-1-enyl)-2,2-dimethyl-cyclopropane-1-carboxylate] and beta-cyfluthrin [Cyano(4-fluoro-3-phenoxyphenyl)methyl 3-(2,2-dichloroethenyl)-2,2-dimethyl-cyclopropanecarboxylate]. Generally, runoff did not occur at irrigation rates of 2.03 cm/h (0.8 in/h) but did occur when the irrigation rates were increased to about 3.81 cm/h (1.5 in/h), generating chemical losses in the first runoff event of up to 0.58 and 0.08% of applied for beta-cyfluthrin and bifenthrin, respectively. Chemical runoff losses dropped significantly between over-irrigation events with the third over-irrigation event chemical runoff losses representing 0.026 and 0.015% of applied for beta-cyfluthrin and bifenthrin, respectively. Runoff losses were generally less for liquid formulations than granular formulations but within a factor of three. Additionally, the study included a simulated winter rainstorm 8 wk after application. The low runoff losses from turf seen in this study suggest that other sources could be contributing to observed residues in urban streams. Other sources could include pyrethroids ending up on impervious surfaces, such as concrete driveways from off-target applications to turf, spills, and other poor handling practices, or pyrechroids applied directly to impervious surfaces for insect control.

Hydrogen sulfide stimulates ß-amylase activity during early stages of wheat grain germination.

We recently reported that H 2S could significantly promote the germination of wheat grains subjected to aluminum (Al(3+)) stress.1 In these experiments seeds were pretreated with the H 2S donor NaHS for 12 h prior to Al(3+) stress. During this pre-incubation period we observed that H2S increased the activity of grain amylase in the absence of Al(3+). Using embryoless half grains of wheat we now show that H2S preferentially affects the activity of endosperm ß-amylase and that α-amylase synthesis and activity is unaffected by this treatment.

Hydrogen sulfide alleviates aluminum toxicity in germinating wheat seedlings.

Protective role of hydrogen sulfide (H(2)S) on seed germination and seedling growth was studied in wheat (Triticum) seeds subjected to aluminum (Al(3+)) stress. We show that germination and seedling growth of wheat is inhibited by high concentrations of AlCl(3). At 30 mmol/L AlCl(3) germination is reduced by about 50% and seedling growth is more dramatically inhibited by this treatment. Pre-incubation of wheat seeds in the H(2)S donor NaHS alleviates AlCl(3)-induced stress in a dose-dependant manner at an optimal concentration of 0.3 mmol/L. We verified that the role of NaHS in alleviating Al(3+) stress could be attributed to H(2)S/HS(-) by showing that the level of endogenous H(2)S increased following NaHS treatment. Furthermore, other sodium salts containing sulfur were ineffective in alleviating Al(3+) stress. NaHS pretreatment significantly increased the activities of amylases and esterases and sustained much lower levels of MDA and H(2)O(2) in germinating seeds under Al(3+) stress. Moreover, NaHS pretreatment increased the activities of guaiacol peroxidase, ascorbate peroxidase, superoxide dismutase and catalase and decreased that of lipoxygenase. NaHS pretreatment also decreased the uptake of Al(3+) in AlCl(3)-treated seed. Taken together these results suggest that H(2)S could increase antioxidant capability in wheat seeds leading to the alleviation of Al(3+) stress.

Measuring NO production by plant tissues and suspension cultured cells.

We describe an inexpensive and reliable detector for measuring NO emitted in the gas phase from plants. The method relies on the use of a strong oxidizer to convert NO to NO2 and subsequent capture of NO2 by a Griess reagent trap. The set-up approaches the sensitivity for NO comparable to that of instruments based on chemiluminescence and photoacoustic detectors. We demonstrate the utility of our set-up by measuring NO produced by a variety of well established plant sources. NO produced by nitrate reductase (NR) in tobacco leaves and barley aleurone was readily detected, as was the production of NO from nitrite by the incubation medium of barley aleurone. Arabidopsis mutants that overproduce NO or lack NO-synthase (AtNOS1) also displayed the expected NO synthesis phenotype when assayed by our set-up. We could also measure NO production from elicitor-treated suspension cultured cells using this set-up. Further, we have focused on the detection of NO by a widely used fluorescent probe 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM). Our work points to the pitfalls that must be avoided when using DAF-FM to detect the production of NO by plant tissues. In addition to the dramatic effects that pH can have on fluorescence from DAF-FM, the widely used NO scavengers 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) can produce anomalous and unexpected results. Perhaps the most serious drawback of DAF-FM is its ability to bind to dead cells and remain NO-sensitive.

Summary of potable well monitoring conducted for aldicarb and its metabolites in the United States in 2005.

United States potable well monitoring in the Pacific Northwest, California, Texas, Mississippi delta, and the Southeast was conducted in 2005 to provide estimates of dietary exposure to aldicarb carbamate residues (aldicarb and its two carbamate metabolites) in potentially vulnerable potable wells. Samples were analyzed from 1,673 drinking water wells in nine major use areas that were within 300 m of fields treated at least once with aldicarb between 2002 and 2005. Analyses were performed with a high-performance liquid chromatography/tandem mass spectrometry analytical method with limits of quantitation of 0.021 microg/L for aldicarb and aldicarb sulfone and 0.027 microg/L for aldicarb sulfoxide. The method detection limits were 0.0070 microg/L for aldicarb and aldicarb sulfone and 0.0090 microg/L for aldicarb sulfoxide. Samples from 1,513 of the 1,673 wells contained no aldicarb carbamate residues. Only 10 wells had total aldicarb carbamate residues above 1 microg/L, the maximum being 2.9 microg/L. All residues were below the U.S. Environmental Protection Agency Health Advisory Limit (HAL) of 10 microg/L. Given the number of samples collected in this study, there is a 95% confidence level that the maximum concentration in this study exceeds the 99.8th percentile of concentrations of all potable wells located within 300 m of fields recently treated with aldicarb. Therefore, the data from this study show that the restrictions and management practices on the current product label continue to be effective in preventing residues in excess of the 10 microg/L HAL in potable wells in these use areas.

The Arabidopsis aleurone layer responds to nitric oxide, gibberellin, and abscisic acid and is sufficient and necessary for seed dormancy.

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.

Sodium nitroprusside, cyanide, nitrite, and nitrate break Arabidopsis seed dormancy in a nitric oxide-dependent manner.

The seeds of many plant species are dormant at maturity and dormancy loss is a prerequisite for germination. Numerous environmental and chemical treatments are known to lessen or remove seed dormancy, but the biochemical changes that occur during this change of state are poorly understood. Several lines of research have implicated nitric oxide (NO) as a participant in this process. Here, we show that dormant seeds of Arabidopsis thaliana (L.) Heynh. will germinate following treatment with the NO donor sodium nitroprusside (SNP), cyanide (CN), nitrite or nitrate. In all cases, the NO scavenger c-PTIO effectively promotes the maintenance of seed dormancy. c-PTIO does not, however, inhibit germination of fully after-ripened seeds, and c-PTIO does not interact directly with nitrite, nitrate or CN. We also show that volatile CN effectively breaks dormancy of Arabidopsis seeds, and that CN is the volatile compound in SNP that promotes dormancy loss. Our data support the hypothesis that NO is a signaling molecule that plays an important role in the loss of seed dormancy.

Global patterns of gene expression in the aleurone of wild-type and dwarf1 mutant rice.

The cereal aleurone layer is a model system for studying the regulation of transcription by gibberellin (GA) and abscisic acid (ABA). GA stimulates and ABA prevents the transcription of genes for alpha-amylases and other secreted hydrolytic enzymes, but how GA and ABA affect the transcription of other genes is largely unknown. We characterized gene expression in rice (Oryza sativa) aleurone using a half-genome rice microarray. Of the 23,000 probe sets on the chip, approximately 11,000 hybridized with RNA from rice aleurone treated with ABA, GA, or no hormone. As expected, GA regulated the expression of many genes, and 3 times as many genes were up-regulated by GA at 8 h than were down-regulated. Changes in gene expression resulting from ABA treatment were not consistent with the hypothesis that the role of ABA in this tissue is primarily to repress gene expression, and 10 times more genes were up-regulated by ABA at 8 h than were down-regulated by ABA. We also measured transcript abundance in aleurone of dwarf1 (d1) mutant rice. The d1 protein is the sole alpha-subunit of heterotrimeric G-proteins in rice. Genes up-regulated by GA or ABA had higher expression in wild type than in d1 aleurone, and genes down-regulated by GA had lower expression in wild type relative to d1 aleurone. The d1 mutation did not result in a decrease in sensitivity to GA at the level of transcription. Rather, changes in transcript abundance were smaller in the d1 mutant than in wild type.

Nitric oxide reduces seed dormancy in Arabidopsis.

Dormancy is a property of many mature seeds, and experimentation over the past century has identified numerous chemical treatments that will reduce seed dormancy. Nitrogen-containing compounds including nitrate, nitrite, and cyanide break seed dormancy in a range of species. Experiments are described here that were carried out to further our understanding of the mechanism whereby these and other compounds, such as the nitric oxide (NO) donor sodium nitroprusside (SNP), bring about a reduction in seed dormancy of Arabidopsis thaliana. A simple method was devised for applying the products of SNP photolysis through the gas phase. Using this approach it was shown that SNP, as well as potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN), reduced dormancy of Arabidopsis seeds by generating cyanide (CN). The effects of potassium cyanide (KCN) on dormant seeds were tested and it was confirmed that cyanide vapours were sufficient to break Arabidopsis seed dormancy. Nitrate and nitrite also reduced Arabidopsis seed dormancy and resulted in substantial rates of germination. The effects of CN, nitrite, and nitrate on dormancy were prevented by the NO scavenger c-PTIO. It was confirmed that NO plays a role in reducing seed dormancy by using purified NO gas, and a model to explain how nitrogen-containing compounds may break dormancy in Arabidopsis is presented.

Nitric oxide gas stimulates germination of dormant Arabidopsis seeds: use of a flow-through apparatus for delivery of nitric oxide.

Nitric oxide (NO) is a gaseous free radical that reacts with O(2) in air and aqueous solution. NO donors have been widely used to circumvent the difficulties inherent in working with a reactive gas, but NO donors do not deliver NO at a constant rate for prolonged periods of time. Furthermore, some of the most commonly used NO donors produce additional, bioactive decomposition products. We designed and built an apparatus that allowed for the precise mixing of gaseous NO with air and the delivery of gas through sample vials at fixed rates. This experimental setup has the added advantage that continuous flow of gas over the sample reduces the buildup of volatile breakdown products. To show that this experimental setup was suitable for studies on the dormancy and germination of Arabidopsis thaliana seeds, we introduced vapors from water or sodium nitroprusside (SNP) into the gas stream. Seeds remained dormant when treated with water vapor, but gases generated by SNP increased germination to 90%. When pure NO was mixed with air and passed over dormant seeds, approximately approximately 30% of the seeds germinated. Because nitrite accumulates in aqueous solutions exposed to NO gas, we measured the accumulation of nitrite under our experimental conditions and found that it did not exceed 100 microM. Nitrite or nitrate at concentrations of up to 500 microM did not increase germination of C24 ecotype Arabidopsis seeds to more than 10%. These data support the hypothesis that NO participates in the loss of Arabidopsis seed dormancy, and they show that for some dormant seeds, exposure to exogenous NO is sufficient to trigger germination.

Comparison of regulatory estimates of drinking water concentrations with monitoring data.

Currently, regulatory practice in the United States is to estimate potential concentrations in drinking water from surface water by using an index reservoir scenario. This approach extrapolates results from the modeling of a single field with maximum application rates to the watershed scale, based on a percent crop area estimate. Since 1998, Bayer CropScience and its predecessor companies have conducted drinking water monitoring studies with a number of different compounds. The results from these studies show that the index reservoir scenario greatly overpredicts residues in surface water. The most important factor is the overestimation of use within a watershed. Other factors contributing to the overestimation of concentrations are the conservative procedures used to obtain the chemical fate related input parameters and the simplified hydrology. A new procedure based on the USGS WARP model, being developed by a group of scientists from the EPA, USGS, USDA, and industry, will provide more realistic estimates of concentrations of pesticides and their metabolites in drinking water.

Hormonal regulation of gluconeogenesis in cereal aleurone is strongly cultivar-dependent and gibberellin action involves SLENDER1 but not GAMYB.

Storage oil is a major constituent in the cereal aleurone layer. The aim of this study was to investigate how gibberellin (GA) and abscisic acid (ABA) regulate conversion of oil to sugar in barley aleurone. The activity of the glyoxylate cycle enzyme isocitrate lyase (ICL) was surveyed in eight barley cultivars. Surprisingly, some cultivars do not require GA for the induction of ICL (e.g. Himalaya), whereas some do (e.g. Golden Promise). Furthermore, in Golden Promise, GA also stimulates triacylglycerol breakdown and enhances the net flux of carbon from acetate to sugar. In contrast, ABA strongly represses ICL activity and the flux of carbon from oil to sugar in both Golden Promise and Himalaya. Biolistics using a promoter reporter showed that GA and ABA regulate ICL at the level of transcription. Studies using barley and rice mutants and pharmacological agents show that GA-dependent induction of ICL activity is mediated by SLENDER1 and requires cGMP, but does not involve the transcription factor GAMYB. Gibberellin and ABA therefore act antagonistically to regulate gluconeogenesis in the aleurone layer as well as controlling the production and secretion of hydrolases into the starchy endosperm. We suggest that the variation between different barley cultivars might be a result of selective breeding to alter seed dormancy.

A gibberellin-regulated calcineurin B in rice localizes to the tonoplast and is implicated in vacuole function.

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Pulsed losses and degradation of aldicarb in a South Florida agricultural watershed.

The objectives of these studies were to characterize patterns of movement of aldicarb, aldicarb sulfoxide, and aldicarb sulfone from a typical canalized South Florida watershed and to evaluate aldicarb dissipation in surface water in situ within a citrus grove. Surface water samples were collected daily or every other day from the discharge point for the watershed beginning May 15, 2001, through August 15, 2002. Of 457 samples collected, aldicarb, aldicarb sulfoxide, and aldicarb sulfone were detected in 6, 1, and 13, respectively. Aldicarb was detected from February through May 2002, corresponding to the legal application season of January 1 through April 30 in Florida. Aldicarb concentrations ranged from <0.16 to 4.97 ng ml(-1). A single detection (0.99 ng ml(-1)) of aldicarb sulfoxide occurred in March 2001. The majority of aldicarb sulfone detections occurred during June and July, 2001, after the application season, and ranged from <0.22 to 0.89 ng ml(-1). The half-life for aldicarb in fortified, native surface water ranged from 1.86 to 3.64 days depending on the source of water and the presence of sediments. These results demonstrated the utility of sampling on a frequent basis (compared with monthly or quarterly) for better characterizing pesticide discharges, especially in flashy systems such as canal-drained watersheds within South Florida.

Characterization of a novel calcium/calmodulin-dependent protein kinase from tobacco.

A cDNA encoding a calcium (Ca2+)/calmodulin (CaM)-dependent protein kinase (CaMK) from tobacco (Nicotiana tabacum), NtCaMK1, was isolated by protein-protein interaction-based screening of a cDNA expression library using 35S-labeled CaM as a probe. The genomic sequence is about 24.6 kb, with 21 exons, and the full-length cDNA is 4.8 kb, with an open reading frame for NtCaMK1 consisting of 1,415 amino acid residues. NtCaMK1 has all 11 subdomains of a kinase catalytic domain, lacks EF hands for Ca2+-binding, and is structurally similar to other CaMKs in mammal systems. Biochemical analyses have identified NtCaMK1 as a Ca2+/CaMK since NtCaMK1 phosphorylated itself and histone IIIs as substrate only in the presence of Ca2+/CaM with a Km of 44.5 microm and a Vmax of 416.2 nm min(-1) mg(-1). Kinetic analysis showed that the kinase not previously autophosphorylated had a Km for the synthetic peptide syntide-2 of 22.1 microm and a Vmax of 644.1 nm min(-1) mg(-1) when assayed in the presence of Ca2+/CaM. Once the autophosphorylation of NtCaMK1 was initiated, the phosphorylated form displayed Ca2+/CaM-independent behavior, as many other CaMKs do. Analysis of the CaM-binding domain (CaMBD) in NtCaMK1 with truncated and site-directed mutated forms defined a stretch of 20 amino acid residues at positions 913 to 932 as the CaMBD with high CaM affinity (Kd = 5 nm). This CaMBD was classified as a 1-8-14 motif. The activation of NtCaMK1 was differentially regulated by three tobacco CaM isoforms (NtCaM1, NtCaM3, and NtCaM13). While NtCaM1 and NtCaM13 activated NtCaMK1 effectively, NtCaM3 did not activate the kinase.