Search

Methylobacterium sp. 2A, a plant growth-promoting rhizobacteria (PGPR) able to produce indole-3-acetic acid (IAA), significantly promoted the growth of Arabidopsis thaliana plants in vitro. We aimed to understand the determinants of Methylobacterium sp. 2A-A. thaliana interaction, the factors underlying plant growth-promotion and the host range. Methylobacterium sp. 2A displayed chemotaxis to methanol and formaldehyde and was able to utilise 1-aminocyclopropane carboxylate as a nitrogen source. Confocal microscopy confirmed that fluorescent protein-labelled Methylobacterium sp. 2A colonises the apoplast of A. thaliana primary root cells and its inoculation increased jasmonic and salicylic acid in A. thaliana, while IAA levels remained constant. However, inoculation increased DR5 promoter activity in root tips of A. thaliana and tomato plants. Inoculation of this PGPR partially restored the agravitropic response in yucQ mutants and lateral root density was enhanced in iaa19, arf7, and arf19 mutant seedlings. Furthermore, Methylobacterium sp. 2A volatile organic compounds (VOCs) had a dose-dependent effect on the growth of A. thaliana. This PGPR is also able to interact with monocots eliciting positive responses upon inoculation. Methylobacterium sp. 2A plant growth-promoting effects can be achieved through the regulation of plant hormone levels and the emission of VOCs that act either locally or at a distance., (© 2024 John Wiley & Sons Ltd.)

The role of Calcium ions (Ca 2+ ) is extensively documented and comprehensively understood in eukaryotic organisms. Nevertheless, emerging insights, primarily derived from studies on human pathogenic bacteria, suggest that this ion also plays a pivotal role in prokaryotes. In this review, our primary focus will be on unraveling the intricate Ca 2+ toolkit within prokaryotic organisms, with particular emphasis on its implications for plant growth-promoting rhizobacteria (PGPR). We undertook an in silico exploration to pinpoint and identify some of the proteins described in the existing literature, including prokaryotic Ca 2+ channels, pumps, and exchangers that are responsible for regulating intracellular Calcium concentration ([Ca 2+ ] i ), along with the Calcium-binding proteins (CaBPs) that play a pivotal role in sensing and transducing this essential cation. These investigations were conducted in four distinct PGPR strains: Pseudomonas chlororaphis subsp. aurantiaca SMMP3, P. donghuensis SVBP6, Pseudomonas sp. BP01, and Methylobacterium sp. 2A, which have been isolated and characterized within our research laboratories. We also present preliminary experimental data to evaluate the influence of exogenous Ca 2+ concentrations ([Ca 2+ ] ex ) on the growth dynamics of these strains.

The serine/threonine protein phosphatases type 2A (PP2A) are involved in several physiological responses in plants, playing important roles in developmental programs, stress responses and hormone signaling. Six PP2A catalytic subunits (StPP2Ac) were identified in cultivated potato. Transgenic potato plants constitutively overexpressing the catalytic subunit StPP2Ac2b (StPP2Ac2b-OE) were developed to determine its physiological roles. The response of StPP2Ac2b-OE plants to the oomycete Phytophthora infestans, the causal agent of late blight, was evaluated. We found that overexpression of StPP2Ac2b enhances susceptibility to the pathogen. Further bioinformatics, biochemical, and molecular analyses revealed that StPP2Ac2b positively regulates developmental and pathogen-induced senescence, and that P. infestans infection promotes senescence, most likely through induction of StPP2Ac2b expression., Competing Interests: The authors have declared that no competing interests exist.

Key Message: StCDPK2 is an early player in the salt stress response in potato plants; its overexpression promoted ROS scavenging, chlorophyll stability, and the induction of stress-responsive genes conferring tolerance to salinity. The salinity of soils affects plant development and is responsible for great losses in crop yields. Calcium-dependent protein kinases (CDPKs) are sensor-transducers that decode Ca 2+ signatures triggered by abiotic stimuli and translate them into physiological responses. Histochemical analyses of potato plants harboring StCDPK2 promoter fused to the reporter gene β-glucuronidase (Pro StCDPK2 :GUS) revealed that GUS activity was high in the leaf blade and veins, it was restricted to root tips and lateral root primordia, and was observed upon stolon swelling. Comparison with Pro StCDPK1 :GUS and Pro StCDPK3 :GUS plants revealed their differential activities in the plant tissues. Pro StCDPK2 :GUS plants exposed to high salt presented enhanced GUS activity in roots which correlated with the numerous stress-responsive sites predicted in its promoter sequence. Moreover, StCDPK2 expression increased in in vitro potato plants after 2 h of high salt exposure and in greenhouse plants exposed to a dynamic stress condition. As inferred from biometric data and chlorophyll content, plants that overexpress StCDPK2 were more tolerant than wild-type plants when exposed to high salt. Overexpressing plants have a more efficient antioxidant system; they showed reduced accumulation of peroxide and higher catalase activity under salt conditions, and enhanced expression of WRKY6 and ERF5 transcription factors under control conditions. Our results indicate that StCDPK2 is an early player in the salt stress response and support a positive correlation between StCDPK2 overexpression and tolerance towards salt stress., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Four members of the potato (Solanum tuberosum L.) calcium-dependent protein kinase (CDPK) family StCDPK22/23/24 and StCDPK27, present three functional EF-hands motifs in their calmodulin-like domain (CLD). StCDPK22/23/24 are clustered in clade III-b1 with tomato and Arabidopsis CDPKs that lack the first EF-hand motif, while StCDPK27 is clustered in clade III-b3 with CDPKs that lack EF-hand 2. Members of each clade share similar intron-exon structures and acylation profiles. 3D model predictions suggested that StCDPK22 and StCDPK24 are active kinases that undergo a conformational switch in the presence of Ca 2+ even when lacking one functional EF-hand motif; however, assays performed with recombinant proteins indicated that StCDPK24:6xHis was active in all the conditions tested, and its activity was enhanced in the presence of Ca 2+ , but StCDPK22:6xHis had scarce or null activity. Both kinases share with AtCPK8 the same autophosphorylation pattern in the autoinhibitory (AD) and C-terminal variable (CTV) domains, suggesting that it could be a characteristic of clade III-b1. RT-qPCR analysis revealed that StCDPK22 is mainly expressed in early stages of tuberization, but not limited to, while StCDPK24 expression is more ubiquitous. In silico analysis predicted several abiotic stress-responsive elements in its promoters. Accordingly, StCDPK24 expression peaked at 10 h in in vitro plants exposed to salt shock and then declined. Moreover, a significant increase was observed at 2 h in stems of salt-treated greenhouse plants, suggesting that this CDPK could participate in the early events of the signaling cascade triggered in response to salt., (Copyright © 2021 Elsevier Masson SAS. All rights reserved.)

This data article describes the dataset of the International COVID-19 Impact on Parental Engagement Study (ICIPES). ICIPES is a collaborative effort of more than 20 institutions to investigate the ways in which, parents and caregivers built capacity engaged with children's learning during the period of social distancing arising from global COVID-19 pandemic. A series of data were collected using an online survey conducted in 23 countries and had a total sample of 4,658 parents/caregivers. The description of the data contained in this article is divided into two main parts. The first part is a descriptive analysis of all the items included in the survey and was performed using tables and figures. The second part refers to the construction of scales. Three scales were constructed and included in the dataset: 'parental acceptance and confidence in the use of technology', 'parental engagement in children's learning' and 'socioeconomic status'. The scales were created using Confirmatory Factor Analysis (CFA) and Multi-Group Confirmatory Analysis (MG-CFA) and were adopted to evaluate their cross-cultural comparability (i.e., measurement invariance) across countries and within sub-groups. This dataset will be relevant for researchers in different fields, particularly for those interested in international comparative education., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2021 The Author(s).)

A Gram-negative pink-pigmented bacillus (named 2A) was isolated from Solanum tuberosum L. cv. Desirée plants that were strikingly more developed, presented increased root hair density, and higher biomass than other potato lines of the same age. The 16S ribosomal DNA sequence, used for comparative gene sequence analysis, indicated that strain 2A belongs to the genus Methylobacterium . Nucleotide identity between Methylobacterium sp. 2A sequenced genome and the rest of the species that belong to the genus suggested that this species has not been described so far. In vitro, potato plants inoculated with Methylobacterium sp. 2A had a better performance when grown under 50 mM NaCl or when infected with Phytophthora infestans . We inoculated Methylobacterium sp. 2A in Arabidopsis thaliana roots and exposed these plants to salt stress (75 mM NaCl). Methylobacterium sp. 2A-inoculated plants, grown in control or salt stress conditions, displayed a higher density of lateral roots (p < 0.05) compared to noninoculated plants. Moreover, under salt stress, they presented a higher number of leaves and larger rosette diameter. In dual confrontation assays, Methylobacterium sp. 2A displayed biocontrol activity against P. infestans , Botrytis cinerea , and Fusarium graminearum , but not against Rhizoctonia solani , and Pythium dissotocum . In addition, we observed that Methylobacterium sp. 2A diminished the size of necrotic lesions and reduced chlorosis when greenhouse potato plants were infected with P. infestans . Methylobacterium sp. 2A produces indole acetic acid, solubilizes mineral phosphate and is able to grow in a N 2 free medium. Whole-genome sequencing revealed metabolic pathways associated with its plant growth promoter capacity. Our results suggest that Methylobacterium sp. 2A is a plant growth-promoting rhizobacteria (PGPR) that can alleviate salt stress, and restricts P. infestans infection in potato plants, emerging as a potential strategy to improve crop management., (Copyright © 2020 Grossi, Fantino, Serral, Zawoznik, Fernandez Do Porto and Ulloa.)

The complex formed by the cyclin-dependent kinase A (CDKA) and cyclin D is responsible for the G1-S transition in the plant cell cycle. Maize (Zea mays L) CDKA; 1 and CycD6; 1 were cloned and expressed in E. coli. The present study describes the optimization of both proteins production using a statistical approach known as response surface methodology (RSM). The experimental design took into account the effects of four variables: optical density of the culture (OD 600 ) before induction, isopropyl β-d-1-thiogalactopyranoside (IPTG) concentration, post-induction temperature, and post-induction time. For each protein, a 2 4 full factorial central composite rotary design for these four independent variables (at five levels each) was employed to fit a polynomial model; which indicated that 30 experiments were required for this procedure. An optimization of CDKA; 1 and CycD6; 1 production levels in the soluble fraction was achieved. Protein conformation and stability were studied by circular dichroism and fluorescence spectroscopy. Finally, in vitro Cyc-CDK complex formation and its kinase activity were confirmed., (Published by Elsevier Inc.)

Protein kinases constitute one of the largest protein families in nature. Current methods to assay their activity involve the use of radioactive ATP or very expensive reagents. In this work, we developed a highly sensitive, cost-effective and straightforward protocol to measure protein kinase activity using a microplate layout. Released ADP is converted into NAD + , which is quantified by its fluorescent properties after alkaline treatment (linear range 0-10 nmol ADP). To validate our protocol, we characterized a recombinant calcium-dependent protein kinase from potato. Overall, this tool represents a critical step forward in the functional characterization of protein kinases., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Key Message: We describe the potato CDPK family and place StCDPK7 as a player in potato response to Phytophthora infestans infection, identifying phenylalanine ammonia lyase as its specific phosphorylation target in vitro. Calcium-dependent protein kinases (CDPKs) decode calcium (Ca 2+ ) signals and activate different signaling pathways involved in hormone signaling, plant growth, development, and both abiotic and biotic stress responses. In this study, we describe the potato CDPK/CRK multigene family; bioinformatic analysis allowed us to identify 20 new CDPK isoforms, three CDPK-related kinases (CRKs), and a CDPK-like kinase. Phylogenetic analysis indicated that 26 StCDPKs can be classified into four groups, whose members are predicted to undergo different acylation patterns and exhibited diverse expression levels in different tissues and in response to various stimuli. With the aim of characterizing those members that are particularly involved in plant-pathogen interaction, we focused on StCDPK7. Tissue expression profile revealed that StCDPK7 transcript levels are high in swollen stolons, roots, and mini tubers. Moreover, its expression is induced upon Phytophthora infestans infection in systemic leaves. Transient expression assays showed that StCDPK7 displays a cytosolic/nuclear localization in spite of having a predicted chloroplast transit peptide. The recombinant protein, StCDPK7:6xHis, is an active Ca 2+ -dependent protein kinase that can phosphorylate phenylalanine ammonia lyase, an enzyme involved in plant defense response. The analysis of the potato CDPK family provides the first step towards the identification of CDPK isoforms involved in biotic stress. StCDPK7 emerges as a relevant player that could be manipulated to deploy disease resistance in potato crops.

Among many factors that regulate potato tuberization, calcium and calcium-dependent protein kinases (CDPKs) play an important role. CDPK activity increases at the onset of tuber formation with StCDPK1 expression being strongly induced in swollen stolons. However, not much is known about the transcriptional and posttranscriptional regulation of StCDPK1 or its downstream targets in potato development. To elucidate further, we analyzed its expression in different tissues and stages of the life cycle. Histochemical analysis of StCDPK1::GUS (β-glucuronidase) plants demonstrated that StCDPK1 is strongly associated with the vascular system in stems, roots, during stolon to tuber transition, and in tuber sprouts. In agreement with the observed GUS profile, we found specific cis-acting elements in StCDPK1 promoter. In silico analysis predicted miR390 to be a putative posttranscriptional regulator of StCDPK1. Quantitative real time-polymerase chain reaction (qRT-PCR) analysis showed ubiquitous expression of StCDPK1 in different tissues which correlated well with Western blot data except in leaves. On the contrary, miR390 expression exhibited an inverse pattern in leaves and tuber eyes suggesting a possible regulation of StCDPK1 by miR390. This was further confirmed by Agrobacterium co-infiltration assays. In addition, in vitro assays showed that recombinant StCDPK1-6xHis was able to phosphorylate the hydrophilic loop of the auxin efflux carrier StPIN4. Altogether, these results indicate that StCDPK1 expression is varied in a tissue-specific manner having significant expression in vasculature and in tuber eyes; is regulated by miR390 at posttranscriptional level and suggest that StPIN4 could be one of its downstream targets revealing the overall role of this kinase in potato development., (© 2016 Scandinavian Plant Physiology Society.)

Calcium-dependent protein kinases, CDPKs, decode calcium (Ca2+) transients and initiate downstream responses in plants. In order to understand how CDPKs affect plant physiology, their specific target proteins must be identified. In tobacco, the bZIP transcription factor Repression of Shoot Growth (NtRSG) that modulates gibberellin (GA) content is a specific target of NtCDPK1. StCDPK3 from potato is homologous (88% identical) to NtCDPK1 even in its N-terminal variable domain. In this work, we observe that NtRSG is also phosphorylated by StCDPK3. The potato RSG family of transcription factors is composed of three members that share similar features. The closest homologue to NtRSG, which was named StRSG1, was amplified and sequenced. qRT-PCR data indicate that StRSG1 is mainly expressed in petioles, stems, lateral buds, and roots. In addition, GA treatment affected StRSG1 expression. StCDPK3 transcripts were detected in leaves, petioles, stolons, roots, and dormant tubers, and transcript levels were modified in response to GA. The recombinant StRSG1-GST protein was produced and tested as a substrate for StCDPK3 and StCDPK1. 6xHisStCDPK3 was able to phosphorylate the potato StRSG1 in a Ca2+-dependent way, while 6xHisStCDPK1 could not. StCDPK3 also interacts and phosphorylates the transcription factor StABF1 (ABRE binding factor 1) involved in ABA signaling, as shown by EMSA and phosphorylation assays. StABF1 transcripts were mainly detected in roots, stems, and stolons. Our data suggest that StCDPK3 could be involved in the cross-talk between ABA and GA signaling at the onset of tuber development., Competing Interests: The authors have declared that no competing interests exist.

Potato (Solanum tuberosum L.) tubers are an excellent staple food due to its high nutritional value. When the tuber reaches physiological competence, sprouting proceeds accompanied by changes at mRNA and protein levels. Potato tubers become a source of carbon and energy until sprouts are capable of independent growth. Transcript profiling of sprouts grown under continuous light or dark conditions was performed using the TIGR 10K EST Solanaceae microarray. The profiles analyzed show a core of highly expressed transcripts that are associated to the reactivation of growth. Under light conditions, the photosynthetic machinery was fully activated; the highest up-regulation was observed for the Rubisco activase (RCA), the glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the Photosystem II 22 kDa protein (CP22) genes, among others. On the other hand, sprouts exposed to continuous darkness elongate longer, and after extended darkness, synthesis of chloroplast components was repressed, the expression of proteases was reduced while genes encoding cysteine protease inhibitors (CPIs) and metallocarboxypeptidase inhibitors (MPIs) were strongly induced. Northern blot and RT-PCR analysis confirmed that MPI levels correlated with the length of the dark period; however, CPI expression was strong only after longer periods of darkness, suggesting a feedback loop (regulation mechanism) in response to dark-induced senescence. Prevention of cysteine protease activity in etiolated sprouts exposed to extended darkness could delay senescence until they emerge to light.

Calcium-dependent protein kinases (CDPKs) are key components of calcium regulated signaling cascades in plants. In this work, isoform StCDPK3 from Solanum tuberosum was studied and fully described. StCDPK3 encodes a 63 kDa protein with an N-terminal variable domain (NTV), rich in prolines and glutamines, which presents myristoylation and palmitoylation consensus sites and a PEST sequence indicative of rapid protein degradation. StCDPK3 gene (circa 11 kb) is localized in chromosome 3, shares the eight exons and seven introns structure with other isoforms from subgroup IIa and contains an additional intron in the 5'UTR region. StCDPK3 expression is ubiquitous being transcripts more abundant in early elongating stolons (ES), leaves and roots, however isoform specific antibodies only detected the protein in leaf particulate extracts. The recombinant 6xHis-StCDPK3 is an active kinase that differs in its kinetic parameters and calcium requirements from StCDPK1 and 2 isoforms. In vitro, StCDPK3 undergoes autophosphorylation regardless of the addition of calcium. The StCDPK3 promoter region (circa 1,800 bp) was subcloned by genome walking and fused to GUS. Light and ABRE responsive elements were identified in the promoter region as well as elements associated to expression in roots. StCDPK3 expression was enhanced by ABA while GA decreased it. Potato transgenic lines harboring StCDPK3 promoter∷GUS construct were generated by Agrobacterium tumefaciens mediated plant transformation. Promoter activity was detected in leaves, root tips and branching points, early ES, tuber eyes and developing sprouts indicating that StCDPK3 is expressed in actively growing organs.

ABF/AREB bZIP transcription factors mediate plant abiotic stress responses by regulating the expression of stress-related genes. These proteins bind to the abscisic acid (ABA)-responsive element (ABRE), which is the major cis-acting regulatory sequence in ABA-dependent gene expression. In an effort to understand the molecular mechanisms of abiotic stress resistance in cultivated potato (Solanum tuberosum L.), we have cloned and characterized an ABF/AREB-like transcription factor from potato, named StABF1. The predicted protein shares 45-57% identity with A. thaliana ABFs proteins and 96% identity with the S. lycopersicum SlAREB1 and presents all of the distinctive features of ABF/AREB transcription factors. Furthermore, StABF1 is able to bind to the ABRE in vitro. StABF1 gene is induced in response to ABA, drought, salt stress and cold, suggesting that it might be a key regulator of ABA-dependent stress signaling pathways in cultivated potato. StABF1 is phosphorylated in response to ABA and salt stress in a calcium-dependent manner, and we have identified a potato CDPK isoform (StCDPK2) that phosphorylates StABF1 in vitro. Interestingly, StABF1 expression is increased during tuber development and by tuber-inducing conditions (high sucrose/nitrogen ratio) in leaves. We also found that StABF1 calcium-dependent phosphorylation is stimulated by tuber-inducing conditions and inhibited by gibberellic acid, which inhibits tuberization.

Calcium-dependent protein kinases (CDPKs) are essential calcium sensors. In this work, we have studied StCDPK2 isoform from potato both at gene and protein level. StCdpk2 genomic sequence contains eight exons and seven introns, as was observed for StCdpk1. There is one copy of the gene per genome located in chromosome 7. StCDPK2 encodes an active CDPK of 515 aminoacids, with an apparent MW of 57 kDa, which presents myristoylation and palmitoylation consensus in its N-terminus. StCDPK2 is highly expressed in leaves and green sprouts; enhanced expression was detected under light treatment, which corresponds well with light responsive cis-acting elements found in its promoter sequence. Antibodies against the recombinant StCDPK2::6xHis protein detected this isoform in soluble and particulate fractions from leaves. StCDPK2 autophosphorylation and kinase activity are both calcium dependent reaching half maximal activation at 0.6 μM calcium. The active kinase is autophosphorylated on serine and tyrosine residues and its activity is negatively modulated by phosphatidic acid (PA). Our results reveal StCDPK2 as a signalling element involved in plant growth and development and show that its activity is tightly regulated.

StCDPK1 is a calcium dependent protein kinase expressed in tuberizing potato stolons and in sprouting tubers. StCDPK1 genomic sequence contains eight exons and seven introns, the gene structure is similar to Arabidopsis, rice and wheat CDPKs belonging to subgroup IIa. There is one copy of the gene per genome and it is located in the distal portion of chromosome 12. Western blot and immunolocalization assays (using confocal and transmission electron microscopy) performed with a specific antibody against StCDPK1 indicate that this kinase is mainly located in the plasma membrane of swelling stolons and sprouting tubers. Sucrose (4-8%) increased StCDPK1 protein content in non-induced stolons, however the amount detected in swelling stolons was higher. Transgenic lines with reduced expression of StCDPK1 (beta 7) did not differ from controls when cultured under multiplication conditions, but when grown under tuber inducing conditions some significant differences were observed: the beta 7 line tuberized earlier than controls without the addition of CCC (GA inhibitor), developed more tubers than wild type plants in the presence of hormones that promote tuberization in potato (ABA and BAP) and was more insensitive to GA action (stolons were significantly shorter than those of control plants). StCDPK1 expression was induced by GA, ABA and BAP. Our results suggest that StCDPK1 plays a role in GA-signalling and that this kinase could be a converging point for the inhibitory and promoting signals that influence the onset of potato tuberization.

Medicago spp. are able to develop root nodules via symbiotic interaction with Sinorhizobium meliloti. Calcium-dependent protein kinases (CDPKs) are involved in various signalling pathways in plants, and we found that expression of MtCPK3, a CDPK isoform present in roots of the model legume Medicago truncatula, is regulated during the nodulation process. Early inductions were detected 15 min and 3-4 days post-inoculation (dpi). The very early induction of CPK3 messengers was also present in inoculated M. truncatula dmi mutants and in wild-type roots subjected to salt stress, indicating that this rapid response is probably stress-related. In contrast, the later response was concomitant with cortical cell division and the formation of nodule primordia, and was not observed in wild-type roots inoculated with nod (-) strains. This late induction correlated with a change in the subcellular distribution of CDPK activities. Accordingly, an anti-MtCPK3 antibody detected two bands in soluble root extracts and one in the particulate fraction. CPK3::GFP fusions are targeted to the plasma membrane in epidermal onion cells, a localization that depends on myristoylation and palmitoylation sites of the protein, suggesting a dual subcellular localization. MtCPK3 mRNA and protein were also up-regulated by cytokinin treatment, a hormone linked to the regulation of cortical cell division and other nodulation-related responses. An RNAi-CDPK construction was used to silence CPK3 in Agrobacterium rhizogenes-transformed roots. Although no major phenotype was detected in these roots, when infected with rhizobia, the total number of nodules was, on average, twofold higher than in controls. This correlates with the lack of MtCPK3 induction in the inoculated super-nodulator sunn mutant. Our results suggest that CPK3 participates in the regulation of the symbiotic interaction.

StCDPK1 encodes a calcium-dependent protein kinase (CDPK) from Solanum tuberosum, which is transiently induced upon tuberization in swelling stolons. In situ hybridization determined that StCDPK1 mRNA is localized in the apical dome of tuberizing stolon tips, close to the region where sucrose was reported to accumulate. The expression of StCDPK1, and other tuber-specific genes was enhanced when in vitro-cultured potato plants were transferred to high sucrose or high sorbitol containing media. Glucose, fructose or a mixture of both showed no effect on CDPK expression. Okadaic acid blocked sucrose-inducible gene expression, suggesting that phosphatases from the PP1/PP2A family could also participate in the regulation of StCDPK1 and other tuberization-related genes.

The effect of sucrose on tuber formation, calcium-dependent protein kinase (CDPK) and phosphatase activities was analysed using in vitro cultured potato plants. In short treatments, sucrose induced CDPK and phosphatase activities. In long treatments, sucrose induced tuber formation in the absence of other tuber inducing stimuli. Sorbitol caused a minor increase in CDPK activity and affected plant morphology but did not induce tuber development. The addition of the protein kinase inhibitor Staurosporine precluded sucrose-induced tuberization. Altogether, our results suggest that phosphorylation/dephosphorylation events are involved in sucrose-induced tuber development.

CDPK activities present during tuber development were analysed. A high CDPK activity was detected in the soluble fraction of early stolons and a lower one was detected in soluble and particulate fractions of induced stolons. The early and late CDPK activities displayed diverse specificity for in vitro substrates and different subcellular distribution. Western blot analysis revealed two CDPKs of 55 and 60 kDa that follow a precise spatial and temporal profile of expression. The 55 kDa protein was only detected in early-elongating stolons and the 60 kDa one was induced upon stolon swelling, correlating with early and late CDPK activities. A new member of the potato CDPK family, StCDPK3, was identified from a stolon cDNA library. Gene specific RT-PCR demonstrated that this gene is only expressed in early stolons, while the previously identified StCDPK1 is expressed upon stolon swelling. This expression profile suggests that StCDPK3 could correspond to the 55 kDa isoform while StCDPK1 could encode the 60 kDa isoform present in swelling stolons. StCDPK1 has myristoylation and palmitoylation consensus possibly involved in its dual intracellular localization. Transient expression studies with wild-type and mutated forms of StCDPK1 fused to GFP were used to show that subcellular localization of this isoform is controlled by myristoylation and palmitoylation. Altogether, our data suggest that sequential activation of StCDPK3 and StCDPK1 and the subcellular localisation of StCDPK1 might be critical regulatory steps of calcium signalling during potato tuber development.

The effect of jasmonic acid (JA) on plant growth and on calcium-dependent protein kinase (CDPK) activity and expression was studied in non-photoperiodic potato plants, Solanum tuberosum L. var. Spunta, grown in vitro. Stem cuttings were grown for 45 days (long treatment, LT) in MS medium with increasing concentrations of JA. For short treatments (ST) adult plants grown in MS were transferred for 1, 4 and 20 h to JA containing media. During the LT, low concentrations of JA promoted cell expansion and shoot elongation while higher concentrations caused growth inhibition. Under these conditions, treated plants showed root shortening and tuber formation was not induced. Morphological and histochemical studies using light microscopy and TEM analysis of leaves from treated plants revealed that JA also affected subcellular organelles of mesophyll cells. Peroxisomes increased in size and number, and an autophagic process was triggered in response to high concentrations of the hormone. CDPK activity, determined in crude extracts of treated plants (LT), was inhibited (up to 80%). Plant growth and CDPK inhibition were reverted upon transfer of the plants to hormone-free medium. Soluble CDPK activity decreased in response to JA short treatment. Concomitantly, a decline in the steady state levels of StCDPK2 mRNA, a potato CDPK isoform that is expressed in leaves, was observed. These data suggest that the phytohormone down-regulated the expression and activity of the kinase.

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks.

We isolated a full-length cDNA clone (StCDPK1) encoding a calcium-dependent protein kinase (CDPK) by screening a stolon tip cDNA library from potato plants (Solanum tuberosum L.). The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family except in the N-terminal region. As described for other CDPKs, StCDPK1 has a putative N-terminal myristoylation sequence. A coupled transcription/translation system was used to demonstrate that this post-translational modification occurs in vitro. The behaviour of the myristoylated form of StCDPK1 during its purification on a phenyl-Sepharose column mimics that of the endogenous potato enzyme suggesting that this modification occurs in vivo. In addition, a possible palmitoylation site is present in StCDPK1. Southern blot analysis suggests that more than one CDPK isoform is present in potato plants. Northern blot analysis of steady-state mRNA levels for StCDPK1 in different tissues of potato plants shows that the transcript is differentially expressed in tuberizing stolons. The transcript appears in the early steps of tuber formation before the induction of other genes, such as Pin2 and patatin. This result parallels previous data on CDPK activity in potato plants which was highest at the beginning of tuberization. Our results suggest that StCDPK1 is developmentally regulated. The early and transient expression of this CDPK isoform in the tuberization process suggests that this kinase could trigger a cascade of phosphorylation events involved in tuber induction.

In vitro culture was used to study morphogenetic aspects of the tuberization process under controlled conditions in potato (Solanum tuberosum L.) plants. This paper accurately defines four stages of tuber development and their correlation to external morphological characteristics and histological structures. Protein kinase activity, assayed in each stage using Historic HAS as substrate, was differentially expressed during the tuberization process. Phosphorylation was maximum in the first stages of tuber formation. The incorporation of [ 32 PO 4 -1 ] to endogenous peptides containing serine/threonine amino acidic residues followed the same pattern that the protein kinase activity did.

A soluble Ca2+-dependent protein kinase (CDPK) was purified to homogeneity in potato (Solanum tuberosum L.) plants. Potato CDPK was strictly dependent on Ca2+ (one-half maximal activation 0.6 [mu]M) and phosphorylated a wide diversity of substrates, in which Syntide 2 was the best phosphate acceptor (Michaelis constant = 30 [mu]M). The kinase was inhibited by Ca2+-chelating agents, phenotiazine derivatives, and N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (one-half maximal inhibition = 0.25 mM). Polyclonal antibodies directed against the regulatory region of the soybean CDPK recognized a 53-kD polypeptide. In an autophosphorylation assay, this same band was strongly labeled with [[gamma]-32P]ATP in the presence of Ca2+. CDPK activity was high in nontuberized plants, but increased 2.5-fold at the onset of tuber development and was reduced to one-half of its original activity when the tuber had completed formation. In the early stages of tuberization, Ca2+-dependent phosphorylation of endogenous targets (specific bands of 68, 51, and 46 kD) was observed. These polypeptides were not labeled in nontuberizing plants or in completely formed tubers, indicating that this phosphorylation is a stage-specific event. In addition, dephosphorylation of specific polypeptides was detected in tuberizing plants, suggesting the involvement of a phosphatase. Preincubation of crude extracts with phosphatase inhibitors rendered a 100% increase in CDPK activity.

A soluble nucleoside diphosphate kinase (NDP kinase) was purified and characterized in epimastigote forms of Trypanosoma cruzi. The enzyme was purified by affinity chromatography on Blue-agarose and Q-Sepharose columns and by FPLC on a Superose 12 column. A membrane-associated NDP kinase was identified which accounts for 30% of total enzymatic activity. Western blot analysis of the soluble NDP kinase revealed a 16.5-kDa monomer recognized by polyclonal antibodies to NDP kinase from Dictyostelium discoideum, Candida albicans or human. Most of the T. cruzi NDP kinase is found in the cell as a hexamer composed of 16.5-kDa monomers. The Km values of the enzyme for ATP, GDP and dTDP were 0.2 +/- 0.008 mM, 0.125 +/- 0.012 mM and 0.4 +/- 0.009 mM, respectively. The parasite enzyme was stable, remained active at 65 degrees C and was found to tolerate up to 2.5 M urea. The 16.5-kDa subunit was phosphorylated with [gamma-32P]ATP or thiophosphorylated with [35S]GTP gamma S. The incubation of the 32P-labelled phosphoenzyme with unlabelled nucleoside 5'-diphosphates resulted in the formation of 32P-labelled nucleoside 5'-triphosphates without strict base specificity, indicating that the reaction mechanism of the T. cruzi enzyme is the same as reported for other NDP kinases. When the phosphoenzyme was incubated with a mixture of nucleoside 5'-diphosphates, GTP was preferentially formed.

The catalytic subunit of cyclic AMP-dependent protein kinase from Trypanosoma cruzi epimastigote forms was purified by ionic-exchange chromatography, affinity chromatography and sucrose gradient centrifugation. Upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the purified preparations showed a main polypeptide band with a mobility of about 40 kDa. In Western blots this band immunoreacted with a polyclonal antibody specific for the catalytic subunit of bovine heart protein kinase A. Hydrodynamic and molecular parameters of this subunit are as follows: molecular weight, 40,000 +/- 3000; sedimentation constant, 2.8 +/- 0.3 S; Stokes' radius, 2.8 +/- 0.2 nm; frictional ratio, 1.28 +/- 0.05. Purified preparations of T. cruzi catalytic and regulatory subunits reconstitute a holoenzyme with a sedimentation constant. 8.6 +/- 1.17 S. This data together with those previously reported by Ulloa et al. [8] indicate that the T. cruzi cyclic AMP-dependent protein kinase holoenzyme is a tetramer with the structure R2C2 of about 200 kDa. The apparent Km of the catalytic subunit for ATP and histone IIA or kemptide as phosphate donor and acceptor, respectively, were 40 microM, 48.6 microM and 26 microM, respectively.

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII. PKII activity is stimulated by Ca2+ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 microM-free Ca2+ and 1 microgram/ml of the modulator. The stimulatory effect of the Ca(2+)-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80. PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca(2+-)-calmodulin dependent protein kinase, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.

A cyclic AMP-dependent protein kinase activity from epimastigote forms of Trypanosoma cruzi was characterized. Cytosolic extracts were chromatographed on DEAE-cellulose columns, giving two peaks of kinase activity, which were eluted at 0.15 M- and 0.32 M-NaCl respectively. The second activity peak was stimulated by nanomolar concentrations of cyclic AMP. In addition, a cyclic AMP-binding protein co-eluted with the second kinase activity peak. Cyclic AMP-dependent protein kinase activity was further purified by gel filtration, affinity chromatography on histone-agarose and cyclic AMP-agarose, as well as by chromatography on CM-Sephadex. The enzyme ('holoenzyme') could be partially dissociated into two different components: 'catalytic' and 'regulatory'. The 'regulatory' component had specific binding for cyclic AMP, and it inhibited phosphotransferase activity of the homologous 'catalytic component' or of the 'catalytic subunit' from bovine heart. Cyclic AMP reversed these inhibitions. A 'holoenzyme preparation' was phosphorylated in the absence of exogenous phosphate acceptor and analysed by polyacrylamide-gel electrophoresis. A 56 kDa band was phosphorylated. The same preparation was analysed by Western blotting, by using polyclonal antibodies to the regulatory subunits of protein kinases type I or II. Both antibodies reacted with the 56 kDa band.

Calmodulin has been purified from Trypanosoma cruzi epimastigote forms by ion-exchange chromatography, gel filtration and affinity chromatography on 2-chloro-10-(3-aminopropyl)phenotiazine-Sepharose. Upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the factor showed a polypeptide band with an apparent molecular weight of 16 000. In addition, cyclic AMP phosphodiesterase activity from T. cruzi epimastigote forms was purified by ion-exchange chromatography and affinity chromatography on a brain calmodulin-Sepharose column. The enzyme was activated by homologous calmodulin as well as by bovine brain and Neurospora crassa calmodulins. The activation required micromolar concentrations of Ca2+ and it was blocked by EGTA and by some neuroleptic drugs such as chlorpromazine, fluphenazine and compound 48/80. Activations were observed at micromolar concentrations of cyclic AMP as substrate. In addition, T. cruzi calmodulin was also active in bringing about the stimulation of brain phosphodiesterase.

An endogenous thermostable activator of Protein kinase III (PKIII) was purified from 100,000 X g supernatants of Neurospora crassa mycelial extracts. This 38,000 dalton polypeptide, clearly separable from calmodulin on P-60 gel filtration, specifically stimulated N. crassa PKIII activity on casein or phosvitin "in vitro" phosphorylation. The factor was only present in the initial growth phase of the fungus. The mechanism of PKIII activation and its possible regulatory role are discussed.

Activation of cyclic AMP phosphodiesterase I by brain or Neurospora calmodulin was studied. The stimulation required micromolar concentrations of Ca2+, and it was observed at cyclic AMP concentrations between 0.1 and 500 microM. Activation was blocked by EDTA and some neuroleptic drugs such as chlorpromazine and fluphenazine. These drugs inhibit the elongation of N. crassa wild-type aerial hyphae. These results reinforce the evidence towards the recognition of Ca2+-calmodulin as one of the systems controlling cyclic nucleotide concentrations in Neurospora.

Monoclonal antibodies to Neurospora crassa cyclic nucleotide phosphodiesterase (PDE I) were selected by their capacity to inhibit the enzyme activity. The monoclonal immunoglobulin, coupled to Sepharose 4B, was used for the affinity purification of PDE I activity. After SDS-polyacrylamide gel electrophoresis the affinity purified PDE I fractions showed a single polypeptide band of about 41 kDa. This band reacted in Western blots with the above mentioned monoclonal immunoglobulin.