RESUMEN
Pseudovivipary is an environmentally induced flowering abnormality in which vegetative shoots replace seminiferous (sexual) inflorescences. Pseudovivipary is usually retained in transplantation experiments, indicating that the trait is not solely induced by the growing environment. Pseudovivipary is the defining characteristic of Festuca vivipara, and arguably the only feature separating this species from its closest seminiferous relative, Festuca ovina. We performed phylogenetic and population genetic analysis on sympatric F. ovina and F. vivipara samples to establish whether pseudovivipary is an adaptive trait that accurately defines the separation of genetically distinct Festuca species. Chloroplast and nuclear marker-based analyses revealed that variation at a geographical level can exceed that between F. vivipara and F. ovina. We deduced that F. vivipara is a recent species that frequently arises independently within F. ovina populations and has not accumulated significant genetic differentiation from its progenitor. We inferred local gene flow between the species. We identified one amplified fragment length polymorphism marker that may be linked to a pseudovivipary-related region of the genome, and several other markers provide evidence of regional local adaptation in Festuca populations. We conclude that F. vivipara can only be appropriately recognized as a morphologically and ecologically distinct species; it lacks genetic differentiation from its relatives. This is the first report of a 'failure in normal flowering development' that repeatedly appears to be adaptive, such that the trait responsible for species recognition constantly reappears on a local basis.
Asunto(s)
Adaptación Biológica/genética , Festuca/genética , Flores/fisiología , Especiación Genética , Genética de Población , Filogenia , Análisis del Polimorfismo de Longitud de Fragmentos Amplificados , Secuencia de Bases , ADN de Cloroplastos/genética , Festuca/fisiología , Flores/anatomía & histología , Flujo Génico/genética , Datos de Secuencia Molecular , Reproducción/genética , Reproducción/fisiología , Análisis de Secuencia de ADN , Especificidad de la Especie , Reino UnidoRESUMEN
A total of 45 microsatellites (SSRs) were developed for mapping in Fragaria. They included 31 newly isolated codominant genomic SSRs from F. nubicola and a further 14 SSRs, derived from an expressed sequence tagged library (EST-SSRs) of the cultivated strawberry, F. x ananassa. These, and an additional 64 previously characterised but unmapped SSRs and EST-SSRs, were scored in the diploid Fragaria interspecific F2 mapping population (FVxFN) derived from a cross between F. vesca 815 and F. nubicola 601. The cosegregation data of these 109 SSRs, and of 73 previously mapped molecular markers, were used to elaborate an enhanced linkage map. The map is composed of 182 molecular markers (175 microsatellites, six gene specific markers and one sequence-characterised amplified region) and spans 424 cM over seven linkage groups. The average marker spacing is 2.3 cM/marker and the map now contains just eight gaps longer than 10 cM. The transferability of the new SSR markers to the cultivated strawberry was demonstrated using eight cultivars. Because of the transferable nature of these markers, the map produced will provide a useful reference framework for the development of linkage maps of the cultivated strawberry and for the development of other key resources for Fragaria such as a physical map. In addition, the map now provides a framework upon which to place transferable markers, such as genes of known function, for comparative mapping purposes within Rosaceae.
Asunto(s)
Mapeo Cromosómico , Cromosomas de las Plantas , Diploidia , Fragaria/genética , Repeticiones de Microsatélite , ADN de Plantas , Etiquetas de Secuencia Expresada , Biblioteca de Genes , Genes de Plantas , Ligamiento Genético , Marcadores Genéticos , Genoma de Planta , Polimorfismo GenéticoRESUMEN
BACKGROUND AND AIMS: The control of dormancy in yam (Disocorea spp.) tubers is poorly understood and attempts to shorten the long dormant period (i.e. cause tubers to sprout or germinate much earlier) have been unsuccessful. The aim of this study was to identify and define the phases of dormancy in Dioscorea rotundata tubers, and to produce a framework within which dormancy can be more effectively studied. METHODS: Plants of 'TDr 131' derived from tissue culture were grown in a glasshouse simulating temperature and photoperiod at Ibadan (7 degrees N), Nigeria to produce tubers. Tubers were sampled on four occasions: 30 d before shoot senescence (149 days after planting, DAP), at shoot senescence (179 DAP), and twice during storage at a constant 25 degrees C (269 and 326 DAP). The development of the apical shoot bud was described from tissue sections. In addition, the responsiveness of shoot apical bud development to plant growth regulators (gibberellic acid, 2-chloroethanol and thiourea) applied to excised tuber sections was also examined 6 and 12 d after treatment. KEY RESULTS AND CONCLUSIONS: Three phases of tuber dormancy are proposed: Phase I, from tuber initiation to the appearance of the tuber germinating meristem; Phase II, from the tuber germinating meristem to initiation of foliar primordium; and Phase III, from foliar primordium to appearance of the shoot bud on the surface of the tuber. Phase I is the longest phase (approx. 220 d in 'TDr 131'), is not affected by PGRs and is proposed to be an endo-dormant phase. Phases II and III are shorter (<70 d in total), are influenced by PGRs and environmental conditions, and are therefore endo-/eco-dormant phases. To manipulate dormancy to allow off-season planting and more than one generation per year requires that the duration of Phase I is shortened.
Asunto(s)
Dioscorea/fisiología , Tubérculos de la Planta/fisiología , Dioscorea/anatomía & histología , Etilenclorhidrina/farmacología , Giberelinas/fisiología , Organogénesis , Reguladores del Crecimiento de las Plantas/fisiología , Brotes de la Planta/embriología , Tubérculos de la Planta/anatomía & histología , TioureaRESUMEN
BACKGROUND AND AIMS: The aims of this investigation were to highlight the qualitative and quantitative diversity apparent between nine diploid Fragaria species and produce interspecific populations segregating for a large number of morphological characters suitable for quantitative trait loci analysis. METHODS: A qualitative comparison of eight described diploid Fragaria species was performed and measurements were taken of 23 morphological traits from 19 accessions including eight described species and one previously undescribed species. A principal components analysis was performed on 14 mathematically unrelated traits from these accessions, which partitioned the species accessions into distinct morphological groups. Interspecific crosses were performed with accessions of species that displayed significant quantitative divergence and, from these, populations that should segregate for a range of quantitative traits were raised. KEY RESULTS: Significant differences between species were observed for all 23 morphological traits quantified and three distinct groups of species accessions were observed after the principal components analysis. Interspecific crosses were performed between these groups, and F2 and backcross populations were raised that should segregate for a range of morphological characters. In addition, the study highlighted a number of distinctive morphological characters in many of the species studied. CONCLUSIONS: Diploid Fragaria species are morphologically diverse, yet remain highly interfertile, making the group an ideal model for the study of the genetic basis of phenotypic differences between species through map-based investigation using quantitative trait loci. The segregating interspecific populations raised will be ideal for such investigations and could also provide insights into the nature and extent of genome evolution within this group.
Asunto(s)
Diploidia , Fragaria/anatomía & histología , Fragaria/genética , Cruzamientos Genéticos , Flores/anatomía & histología , Flores/genética , Frutas/anatomía & histología , Frutas/genética , Variación Genética , Fenotipo , Hojas de la Planta/anatomía & histología , Hojas de la Planta/genética , Especificidad de la EspecieRESUMEN
Diploid Fragaria provide a potential model for genomic studies in the Rosaceae. To develop a genetic linkage map of diploid Fragaria, we scored 78 markers (68 microsatellites, one sequence-characterised amplified region, six gene-specific markers and three morphological traits) in an interspecific F2 population of 94 plants generated from a cross of F.vesca f. semperflorens x F. nubicola. Co-segregation analysis arranged 76 markers into seven discrete linkage groups covering 448 cM, with linkage group sizes ranging from 100.3 cM to 22.9 cM. Marker coverage was generally good; however some clustering of markers was observed on six of the seven linkage groups. Segregation distortion was observed at a high proportion of loci (54%), which could reflect the interspecific nature of the progeny and, in some cases, the self-incompatibility of F. nubicola. Such distortion may also account for some of the marker clustering observed in the map. One of the morphological markers, pale-green leaf (pg) has not previously been mapped in Fragaria and was located to the mid-point of linkage group VI. The transferable nature of the markers used in this study means that the map will be ideal for use as a framework for additional marker incorporation aimed at enhancing and resolving map coverage of the diploid Fragaria genome. The map also provides a sound basis for linkage map transfer to the cultivated octoploid strawberry.
Asunto(s)
Fragaria/genética , Secuencia de Bases , Mapeo Cromosómico/métodos , Cromosomas de las Plantas/genética , Cartilla de ADN , Diploidia , Marcadores Genéticos , Repeticiones de MicrosatéliteRESUMEN
Fragaria vesca is a short-lived perennial with a seasonal-flowering habit. Seasonality of flowering is widespread in the Rosaceae and is also found in the majority of temperate polycarpic perennials. Genetic analysis has shown that seasonal flowering is controlled by a single gene in F. vesca, the SEASONAL FLOWERING LOCUS ( SFL). Here, we report progress towards the marker-assisted selection and positional cloning of SFL, in which three ISSR markers linked to SFL were converted to locus-specific sequence-characterized amplified region (SCAR1-SCAR3) markers to allow large-scale screening of mapping progenies. We believe this is the first study describing the development of SCAR markers from ISSR profiles. The work also provides useful insight into the nature of polymorphisms generated by the ISSR marker system. Our results indicate that the ISSR polymorphisms originally detected were probably caused by point mutations in the positions targeted by primer anchors (causing differential PCR failure), by indels within the amplicon (leading to variation in amplicon size) and by internal sequence differences (leading to variation in DNA folding and so in band mobility). The cause of the original ISSR polymorphism was important in the selection of appropriate strategies for SCAR-marker development. The SCAR markers produced were mapped using a F. vesca f. vesca x F. vesca f. semperflorens testcross population. Marker SCAR2 was inseparable from the SFL, whereas SCAR1 mapped 3.0 cM to the north of the gene and SCAR3 1.7 cM to its south.
Asunto(s)
Flores/genética , Fragaria/genética , Genes de Plantas/genética , Polimorfismo Genético , Estaciones del Año , Secuencia de Bases , Mapeo Cromosómico , Cruzamientos Genéticos , Cartilla de ADN , Electroforesis en Gel de Poliacrilamida , Marcadores Genéticos/genética , Repeticiones de Minisatélite/genética , Datos de Secuencia Molecular , Polimorfismo de Nucleótido SimpleRESUMEN
The seasons are astronomical, astrological, meteorological, biological, and agricultural. From a perspective outside the biological sciences, the questions of interest about plant seasonality are linked to this wider context. In this review I try to see flowering time, as one important aspect of seasonality, from an outsider's point of view, and describe what is known about it in different types of plants. What is known about it is conditioned by what particular scientists have asked about it, so the variety of approaches to seasonality is another point of emphasis. Detailed consideration is given to flowering seasonality in perennials compared with annuals, and both molecular and whole plant perspectives are presented.
Asunto(s)
Magnoliopsida/fisiología , Estaciones del AñoRESUMEN
The completion of flower development in Impatiens balsamina requires continuous inductive (short-day) conditions. We have previously shown that a leaf-derived signal has a role in floral maintenance. The research described here analyzes the role of the leaf in flower development. Leaf removal treatments, in which plants were restricted to a specified number of leaves, resulted in flowers with increased petal number, up to double that of the undefoliated control. Similar petal number increases (as well as changes in bract number or morphology) were recorded when plants began their inductive treatment at a late developmental age or when plants of a nonreverting line (capable of floral maintenance in the absence of continuous short days) were transferred from short days to long days. Our data imply that the increased petal number was neither a response to stress effects associated with leaf removal nor a result of alterations in primordium initiation rates or substitutions of petals for stamens. Rather, the petal initiation phase was prolonged when the amounts of a leaf-derived signal were limiting. We conclude that a leaf-derived signal has a continuous and quantitative role in flower development and propose a temporal model for the action of organ identity genes in Impatiens. This work adds a new dimension to the prevailing ABC model of flower development and may provide an explanation for the wide variety and instabilities of floral form seen among certain species in nature.
Asunto(s)
Magnoliopsida/fisiología , Modelos Biológicos , Hojas de la Planta/fisiología , Tallos de la Planta/fisiología , Transducción de SeñalRESUMEN
We analyzed the process of inflorescence formation in Impatiens balsamina by studying the architecture of the plant under different photoperiod treatments. Floral reversion under noninductive conditions in this species is caused by the lack of persistence of the induced state in the leaf. This can be used to control the amount of inductive signal and to examine its quantitative influence on morphological changes in the plant. The floral transition was characterized by a continuum of variation at the level of meristem identity, primordium initiation, and floral organ identity. This continuum was enhanced during reversion, suggesting that the establishment of a continuum partly reflects limiting amounts of inductive signal exported from the leaf to the meristem. The transcription patterns of two homologs of genes involved in the control of floral meristem identity, Imp-FLO and Imp-FIM, were similar in terminal and axillary flowers and may be associated with the continuum exhibited by I. balsamina. By analyzing the fate of axillary meristem primordia initiated before and after the beginning of the inductive period, we showed that de novo initiation of axillary meristem primordia by the evoked meristem is not required and that primordia initiated before evocation can adopt different fates, depending on the amount of inductive signal. The influence of age and/or position on primordium responsiveness to the inductive signal is discussed.
RESUMEN
Exocytosis is the final event in the secretory pathway and requires the fusion of the secretory vesicle membrane with the plasma membrane. It results in the release to the outside of vesicle cargo from the cell interior and also the delivery of vesicle membrane and proteins to the plasma membrane. An electrophysiological assay that measures changes in membrane capacitance has recently been used to monitor exocytosis in plants. This complements information derived from earlier light and electron microscope studies, and allows both transient and irreversible fusion of single exocytotic vesicles to be followed with high resolution in protoplasts. It also provides a tool to investigate bulk exocytotic activity in single protoplasts under the influence of cytoplasmic modulators. This research highlights the role of intracellular Ca2+, GTP and pressure in the control of exocytosis in plants. In parallel to these functional studies, plant proteins with the potential to regulate exocytosis are being identified by molecular analysis. In this review we describe these electrophysiological and molecular advances, and emphasise the need for parallel biochemical work to provide a complete picture of the mechanisms controlling vesicle fusion at the plasma membrane of plant cells.
Asunto(s)
Exocitosis/fisiología , Células Vegetales , Animales , Membrana Celular/fisiología , Conductividad Eléctrica , Electrofisiología , Endosomas/metabolismo , Endosomas/fisiología , Fusión de Membrana , Técnicas de Placa-ClampRESUMEN
Flowering and reversion in Impatiens are characterised by gradual transitions of organ identity and constitute a unique system for the molecular and physiological study of floral organogenesis. The authors have isolated an Impatiens homologue of the FIM gene of Antirrhinum (UFO in Arabidopsis), Imp-FIM, and analysed its expression in three states of the terminal meristem: vegetative, floral, and reverted. In floral meristems, Imp-FIM transcription is associated with petal identity, as in Antirrhinum and Arabidopsis, but this is achieved through a novel transcription pattern, characterised by a high level of transcript within petal primordia. This novel transcription pattern could contribute to the more diffuse boundaries between organ types in Impatiens. In vegetative meristems, Imp-FIM is expressed in the axils of leaf primordia which are arranged in a spiral. A similar pattern is observed in reverted meristems in which leaf primordia are initiated in a whorled arrangement. This result indicates that the maintenance of floral phyllotaxis is not associated with a specific pattern of Imp-FIM transcription. Transcription of Imp-FIM in a non-reverting line is no different from that in the reverting line. Therefore, the lack of floral commitment in the reverting line does not seem to be responsible for Imp-FIM transcription within petals. The novel transcription pattern in petals, together with features of Impatiens that are reminiscent of fim and ufo mutant phenotypes suggest an evolutionary divergence for Imp-FIM regulation in this species.
Asunto(s)
Proteínas de Plantas/genética , Plantas/genética , Transcripción Genética , Secuencia de Aminoácidos , Arabidopsis , Clonación Molecular , Meristema/genética , Meristema/crecimiento & desarrollo , Datos de Secuencia Molecular , Fragmentos de Péptidos/genética , Desarrollo de la Planta , Reacción en Cadena de la Polimerasa , Reproducción/genética , Alineación de Secuencia , Homología de Secuencia de AminoácidoRESUMEN
The mechanisms that establish the floral meristem are now becoming clearer, but the way in which flowering is maintained is less well understood. Impatiens balsamina provides a unique opportunity to address this question because reversion to vegetative growth can be obtained in a predictable way by transferring plants from inductive to non-inductive conditions. Following increasing amounts of induction, reversion takes place at progressively later stages of flower development. Partial flower induction and defoliation experiments show that a floral signal is produced in the cotyledon in response to inductive conditions and that this signal progressively diminishes after transfer to non-inductive conditions, during reversion. Therefore reversion in Impatiens is most likely due to the failure of leaves to become permanent sources of inductive signal in addition to the lack of meristem commitment to flowering. Analysis of the expression of the Impatiens homologues of the meristem identity genes floricaula and squamosa indicates that a change in floricaula transcription is not associated with the establishment or maintenance of the floral meristem in this species. Squamosa transcription is associated with floral development and petal initiation, and is maintained in existing petal or petaloid primordia even after the meristem has reverted. However, it is not expressed in the reverted meristem, in which leaves are initiated in whorled phyllotaxis and without axillary meristems, both characteristics usually associated with the floral meristem. These observations show that squamosa expression is not needed for the maintenance of these floral characters. The requirement for the production of the floral signal in the leaf during the process of flower development may reflect an additional function separate to that of squamosa activation; alternatively the signal may be required to ensure continued transcriptional activation in the meristem.
Asunto(s)
Desarrollo de la Planta , Plantas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular , Cartilla de ADN/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Genes de Plantas , Hibridación in Situ , Datos de Secuencia Molecular , Fenotipo , Fotoperiodo , Hojas de la Planta/crecimiento & desarrollo , Proteínas de Plantas/genética , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Transducción de Señal , Transcripción GenéticaRESUMEN
The isolation, cloning, and sequencing of two full-length cDNAs corresponding to the root tip forms of the maize (Zea mays L. cv Clipper) annexins p33 and p35 are described. These are the first complete sequences for the widely reported doublet of plant annexins. The predicted sequences can be divided into four repeat domains characteristic of the annexin family, but Ca2+ binding by the type-II site typical of annexins would be predicted to occur only in repeats 1 and 4. This reduced number of sites is consistent with previously reported biochemical data indicating a high Ca2+ requirement for membrane association. Although the two annexins are very similar (80% amino acid identity), their genes are quite distinct, as demonstrated by their different 3' noncoding regions and Southern blotting. The predicted sequences of the root tip proteins are very similar to regions known from peptide sequencing of the coleoptile proteins. Because a rather small gene family is indicated, the implication is that there may be less functional diversity than in animal cells. Furthermore, the sequence data clearly show that plant annexins form a very distinct group compared with those from other kingdoms.
Asunto(s)
Anexinas/biosíntesis , ADN Complementario/aislamiento & purificación , Zea mays/metabolismo , Secuencia de Aminoácidos , Animales , Anexinas/química , Genes de Plantas , Humanos , Datos de Secuencia Molecular , Familia de Multigenes , Proteínas de Plantas/biosíntesis , Proteínas de Plantas/química , Raíces de Plantas , Homología de Secuencia de Aminoácido , Zea mays/genéticaRESUMEN
An ATPase activity is associated with maize (Zea mays) annexins. It has a pH optimum of 6.0, shows Michaelis-Menten kinetics and is not stimulated by Ca2+, Mg2+, EDTA or KCl; it is not inhibited by vanadate, molybdate, nitrate or azide, but N-ethylmaleimide inhibits by approximately 30% at 1-2 mM. These properties indicate that the activity is unlike other ATPases, although it has many features in common with the myosin ATPase. Gel filtration shows that the ATPase activity is mainly associated with a 68 kDa protein that is extracted with the p33/p35 annexins and cross-reacts with antibodies to these proteins.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Anexinas/metabolismo , Zea mays/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Especificidad por Sustrato , Zea mays/enzimologíaRESUMEN
We have examined the characteristics of Ca(2+)-dependent phospholipid-binding proteins (annexins) in maize (Zea mays L.) coleoptiles and tip-growing pollen tubes of Lilium longiflorum. In maize, there are three such proteins, p35, p33, and p23. Partial sequence analysis reveals that peptides from p35 and p33 have identity to members of the annexin family of animal proteins and to annexins from tomato. Interestingly, multiple sequence alignments reveal that the domain responsible for Ca(2+) binding in animal annexins is not conserved in these plant peptide sequences. Although p33 and p35 share the annexin characteristic of binding to membrane lipid, unlike annexins II and VI they do not associate with detergent-insoluble cytoskeletal proteins or with F-actin from either plants or animals. Immunoblotting with antiserum raised to p33/p35 from maize reveals that cross-reactive polypeptides of 33 to 35 kilodaltons are also present in protein extracts from pollen tubes of L. longiflorum. Immunolocalization at the light microscope level suggests that these proteins are predominantly confined to the nongranular zone at the tube tip, a region rich in secretory vesicles. Our hypothesis that plant annexins mediate exocytotic events is supported by the finding that p23, p33, and p35 bind to these secretory vesicles in a Ca(2+)-dependent manner.
RESUMEN
Calcium-dependent hydrophobic interaction chromatography has been widely used for the purification of calcium-binding proteins, following the report that calmodulin could be purified using this proce dure (1). The method makes use of the fact that proteins such as calmodulin, undergo a conformational change and expose a hydrophobic region on binding calcium (2). This means that they bind to a hydrophobic resin, such as phenyl Sepharose, in the presence of calcium, and can be eluted with the calcium chelator EGTA. The procedure has been developed to allow separation of calmodulin from other calcium-binding proteins, exploiting differences in affinity for calcium and in hydrophobicity, and hence elution time in EGTA (3,4). Changes in pH in conjunction with EGTA elution have also been used for fractionation of calcium-regulated proteins on phenyl Sepharose (5).
RESUMEN
There is evidence that Ca(2+) can regulate vesicle-mediated secretion in plant cells, but the mechanism for this is not known. One possibility is that Ca(2+) -dependent phospholipid-binding proteins (annexins) couple the Ca(2+) stimulus to the exocytotic response. Using a protocol developed for the isolation of animal annexins we have identified proteins in maize (Zea mays L.) coleoptiles that have similar characteristics to annexins. The predominant polypeptide species run as a doublet of relative molecular mass (Mr) 33000-35000 on sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE); another less-abundant protein of Mr 23000 is also present. In the presence of Ca(2+) these proteins bind to liposomes composed of acidic phospholipids. Calcium-sensitivity of binding differs for each protein and is also influenced by the pH of the buffer used for the liposome-binding assay. Antiserum raised to the 33 to 35-kDa doublet purified on SDS-PAGE recognises the doublet in crude extracts from maize and proteins of similar Mr in Tradescantia virginiana and tobacco Nicotiana tabacum L. The antiserum also recognises p68 (Annexin VI) from chicken gizzard extracts, indicating homology between animal annexins and the maize proteins. For the maize proteins to be involved in the regulation of exocytosis, binding to phospholipids would be expected to occur at physiological levels of Ca(2+). The characteristics of the maize annexin-like proteins are described and attention drawn to the marked effect of pH in lowering the requirement for Ca(2+) for phospholipid binding.
RESUMEN
This paper describes the results of experiments in which phenyl Sepharose was used to partially purify Ca2(+)-activated protein kinase (CPK) from maize soluble and membrane-solubilized proteins. It is shown that CPK has very similar properties to Ca2(+)-activated, calmodulin independent protein kinase from other plant tissues, and that chromatography on phenyl Sepharose resolves two closely related forms of CPK from both soluble and membrane-solubilized proteins. The amount of each of these forms differs in the two fractions, and it is suggested that the kinase requiring EGTA for elution from phenyl Sepharose at high pH may be either a non-proteolitically digested form or an acylated form of CPK.
Asunto(s)
Calcio/metabolismo , Isoenzimas/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Proteínas de Plantas/metabolismo , Proteína Quinasa C/aislamiento & purificación , Cromatografía Liquida , Isoenzimas/metabolismo , Proteína Quinasa C/metabolismo , Sefarosa/análogos & derivados , Zea mays/enzimologíaRESUMEN
To determine whether a Ca2+-activated protein kinase is regulated by calmodulin, it is necessary to separate it from endogenous calmodulin and from protein kinase activity that is not calcium dependent. We describe here a procedure for achieving these goals using Ca2+-dependent hydrophobic interaction chromatography on phenyl Sepharose in combination with a pH change. The procedure is based on the observation that while calmodulin solubilized from apple fruit membranes binds to phenyl Sepharose in a Ca2+-dependent fashion at both pH 7.0 and 8.5, Ca2+-activated protein kinase from the same source only shows a Ca2+-dependent interaction above pH 7.5. The implications of this finding for the regulation of this Ca2+-activated protein kinase are briefly discussed.