Transcriptome analysis reveals key defense-related genes upon SA induction in Cocos nucifera L.

BACKGROUND: Salicylic acid (SA) is an important regulator of genes involved in plant defense and pathogen-triggered systemic acquired resistance (SAR). Coconut is an important crop affected by several pathogens. Reported evidence suggests SA involvement in defense responses, including SAR in coconut. OBJECTIVE: To identified differentially expressed genes in leaf and root tissues of coconut plantlets, as a result of SA, that might be involved in coconut defense responses. METHODS: Comparative transcriptomic analysis by RNA-Seq of leaf and root tissues from in vitro coconut plantlets unexposed and exposed to SA 2.5 mM for 48 h. And in silico validation of gene expression by qRT-PCR. RESULTS: We identified 4615 and 3940 differentially expressed unigenes (DEUs) in leaf and root tissues respectively. Our GO analysis showed functional categories related to the induction of defense responses, such as "systemic acquired resistance" and highly enriched hormone categories, such as abscisic acid. The most abundant KEGG pathway in our results was "Biosynthesis of antibiotics". Our findings support that exogenous application of SA to plantlets induced the activation of PRs, RGAs, ICS2, NLTP2, PER4, TRXM and some WRKYs mediated by NPR1-dependent pathways. Also, we found DEUs, such as BZR1, HSL1, and WHY2 that support that SA could regulate defense-related genes through NPR1-independent pathways. CONCLUSION: The present study of massive data analysis carried out on coconut plantlets exposed to SA, generates valuable information that increases our understanding of defense molecular mechanisms in coconut and open new venues for research for the improvement of management of coconut diseases.

Haplaxius crudus (Hemiptera: Cixiidae) Transmits the Lethal Yellowing Phytoplasmas, 16SrIV, to Pritchardia pacifica Seem. & H.Wendl (Arecaceae) in Yucatan, Mexico.

Lethal yellowing (LY) affects several palm species in the Americas. It is caused by 16SrIV group phytoplasmas. In Florida (USA), LY was shown to be transmitted by the planthopper Haplaxius crudus ( Van Duzee ) (Hemiptera, Cixiidae) to different palm species, including Pritchardia pacifica Seem . & H. Wendl . (Arecaceae) in insect-proof cage experiments in the 1980s, a result that had never been reproduced later. LY has destroyed many coconut plantations as well as other palm species in the Caribbean and Mexico. In order to evaluate if H. crudus is a vector of LY phytoplasmas in Mexico, experiments were carried out in Yucatan (Mexico). Several H. crudus from palms infected by LY in the field were introduced into cages containing young P. pacifica palms. These insects were able to transmit 16SrIV group phytoplasmas to P. pacifica palms. According to DNA sequences comparative analysis, virtual restriction fragment length polymorphism, and phylogenetic analysis, the phytoplasmas detected in these infected P. pacifica were of subgroups A and D. All of ten P. pacifica palms infected with the subgroup D phytoplasmas developed symptoms of LY and died, whereas only one of two palms infected with subgroup A developed LY symptoms and died. This is the first time, more than 30 years later, that the role of H. crudus as a vector of LY is confirmed.

Endogenous cytokinins in Cocos nucifera L. in vitro cultures obtained from plumular explants.

Auxin induces in vitro somatic embryogenesis in coconut plumular explants through callus formation. Embryogenic calli and non-embryogenic calli can be formed from the initial calli. Analysis of endogenous cytokinins showed the occurrence of cytokinins with aromatic and aliphatic side chains. Fourteen aliphatic cytokinins and four aromatic cytokinins were analysed in the three types of calli and all the cytokinins were found in each type, although some in larger proportions than others. The most abundant cytokinins in each type of callus were isopentenyladenine-9-glucoside, zeatin-9-glucoside, zeatin riboside, isopentenyladenine riboside, dihydrozeatin and dihydrozeatin riboside in decreasing order. Total cytokinin content was compared between the three types of calli, and it was found to be lower in embryogenic calli compared to non-embryogenic calli or initial calli. The same pattern was observed for individual cytokinins. When explants were cultured in media containing exogenously added cytokinins, the formation of embryogenic calli in the explants was reduced. When 8-azaadenine (an anticytokinin) was added the formation of embryogenic calli and somatic embryos was increased. These results suggest that the difference in somatic embryo formation capacity observed between embryogenic calli and non-embryogenic calli is related to their endogenous cytokinin contents.

GA3 stimulates the formation and germination of somatic embryos and the expression of a KNOTTED-like homeobox gene of Cocos nucifera (L.).

The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 muM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem.

Detection of a SERK-like gene in coconut and analysis of its expression during the formation of embryogenic callus and somatic embryos.

Somatic embryogenesis involves different molecular events including differential gene expression and various signal transduction pathways. One of the genes identified in early somatic embryogenesis is S OMATIC E MBRYOGENESIS R ECEPTOR-like K INASE (SERK). Cocos nucifera (L.) is one of the most recalcitrant species for in vitro regeneration, achieved so far only through somatic embryogenesis, although just a few embryos could be obtained from a single explant. In order to increase efficiency of this process we need to understand it better. Therefore, the purpose of the present work was to determine if an ortholog of the SERK gene is present in the coconut genome, isolate it and analyze its expression during somatic embryogenesis. The results showed the occurrence of a SERK ortholog referred to as CnSERK. Predicted sequence analysis showed that CnSERK encodes a SERK protein with the domains reported in the SERK proteins in other species. These domains consist of a signal peptide, a leucine zipper domain, five LRR, the Serine-Proline-Proline domain, which is a distinctive domain of the SERK proteins, a single transmembrane domain, the kinase domain with 11 subdomains and the C terminal region. Analysis of its expression showed that it could be detected in embryogenic tissues before embryo development could be observed. In contrast it was not detected or at lower levels in non-embryogenic tissues, thus suggesting that CnSERK expression is associated with induction of somatic embryogenesis and that it could be a potential marker of cells competent to form somatic embryos in coconut tissues cultured in vitro.

First Report of a 16SrIV Group Phytoplasma Associated with Declining Coyol Palms in Honduras.

Lethal yellowing (LY) of coconut palm (Cocos nucifera L.) caused by a subgroup 16SrIV-A phytoplasma has been present along the northern coast and adjacent Bay Islands of Honduras since 1996. In the southern municipalities of San Esteban and Guanaco, approximately 150 km from the Atlantic coast, substantial numbers of coyol palm (Acrocomia aculeata (Jacq.) Lodd. ex Mart.) and several coconut palms growing nearby were either dead or in an advanced stage of decline during 2005. Declining palms of both species exhibited loss of fruit, withered inflorescences, and dried, discolored (grayish brown) leaves. Most or all leaves on declining palms had collapsed and hung downward around the stem. Samples (3 to 5 g) of tissue were excised from stems of 20 coyol and 2 coconut palms. DNA was extracted from each sample using a previously described protocol (2) and assayed for phytoplasma DNA using polymerase chain reaction (PCR) employing phytoplasma universal rRNA primer pair P1 (1) and P7 (4). A product of expected size (approximately 1.8 kb) was amplified from 12 of 20 diseased coyol palms, 2 of 2 diseased coconut palms and from DNA of a Florida-grown Chinese fan palm (Livistona chinensis (Jacq.) R. Br. ex Mart.) with LY symptoms included as a known positive control. Amplified P1/P7 products used in a nested PCR assay with 16SrIV-specific primer pair LY16Sf (2) and LY16Sr (5'-GCTTACGCA-GTTAGGCTGTC-3') yielded a product of approximately 1.39 kb. Neither primary nor nested PCRs generated a product from DNA of healthy coconut palm. Poor quality of tissues within stems of declining palms may have contributed to lack of amplification of any product from eight coyol samples. A reevaluation of DNA samples using PCR employing nonribosomal primer pair LYF1/LYR1, which specifically detects subgroup 16SrIV-A phytoplasmas (3), failed to amplify an expected 1-kb product from any palm sample other than the LY positive control. Digestion of nested amplification products (1.39 kb) with AluI endonuclease and electrophoresis of digests through 8% polyacrylamide gels revealed the same three-fragment restriction pattern for all phytoplasmas detected in coyol and coconut palms. The pattern differed from the five-fragment profile of LY phytoplasma rDNA included for comparative purposes. These results indicate that coyol palm is a new phytoplasma host and that decline symptoms on coyol and adjacent coconuts were not a consequence of natural dispersion of subgroup 16SrIV-A phytoplasmas from the northern coast. The 16S rDNA sequences amplified from coyol palm decline (CPD) (GenBank Accession No. DQ321818) and coconut decline (CLDO) (GenBank Accession No. DQ321819) phytoplasmas were coidentical and most similar (99.87%) to that of Yucatan coconut lethal decline (LDY), a known subgroup 16SrIV-B strain. This relationship was further supported by phylogenetic analysis of rDNA sequences. On the basis of these findings, we have tentatively concluded that strains CPD and CLDO represent new members of the coconut lethal yellows subgroup 16SrIV-B. References: (1) S. Deng and C. Hiruki, J. Microbiol. Methods 14:53, 1991 (2) N. A. Harrison et al. Plant Dis. 86:676, 2002 (3) N. A. Harrison et al. Plant Pathol. 43:998, 1994. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62:2988, 1996.

Changes in carbohydrate metabolism in coconut palms infected with the lethal yellowing phytoplasma.

ABSTRACT Lethal yellowing (LY), a disease caused by a phytoplasma, is the most devastating disease affecting coconut (Cocos nucifera) in Mexico. Thousands of coconut palm trees have died on the Yucatan peninsula while plantations in Central America and on the Pacific coast of Mexico are severely threatened. Polymerase chain reaction assays enable identification of incubating palm trees (stage 0+, phytoplasma detected but palm asymptomatic). With the development of LY, palm trees exhibit various visual symptoms such as premature nut fall (stage 1), inflorescence necrosis (stages 2 to 3), leaf chlorosis and senescence (stages 4 to 6), and finally palm death. However, physiological changes occur in the leaves and roots prior to onset of visual symptoms. Stomatal conductance, photosynthesis, and root respiration decreased in stages 0+ to 6. The number of active photosystem II (PSII) reaction centers decreased during stage 2, but maximum quantum use efficiency of PSII remained similar until stage 3 before declining. Sugar and starch concentrations in intermediate leaves (leaf 14) and upper leaves (leaf 4) increased from stage 0- (healthy) to stages 2 to 4, while root carbohydrate concentrations decreased rapidly from stage 0- to stage 0+ (incubating phytoplasma). Although photosynthetic rates and root carbohydrate concentrations decreased, leaf carbohydrate concentrations increased, suggesting inhibition of sugar transport in the phloem leading to stress in sink tissues and development of visual symptoms of LY.

First Report of a Phytoplasma-Associated Leaf Yellowing Syndrome of Palma Jipi Plants in Southern México.

The palm-like monocot Palma Jipi (Carludovica palmata, Cyclanthaceae), from which Panama hats are traditionally made, is important to the rural economy of southern Mexico and other Latin American countries. A lethal decline of C. palmata plants was first recognized by farmers at Kalkini in the state of Campeche, Mexico, during 1994. Characterized by a progressive yellowing of successively younger leaves, affected plants died within a few weeks after the onset of this primary symptom. Annual losses estimated at 10% of the naturalized C. palmata population have since occurred in the vicinity of Kalkini, an area in which coconut lethal yellowing (LY) disease is also prevalent. The close proximity and superficially similar symptomatology of these two diseases suggested that both might share a common etiology. DNA samples were obtained from five diseased and five healthy C. palmata plants by small scale extraction of immature leaf bases and assessed for phytoplasma DNA by use of the polymerase chain reaction (PCR) at laboratories in Mérida, INIFAP/Universidad Autónoma de Nuevo León (Nuevo León) and the University of Florida (Fort Lauderdale). Samples from symptomatic plants consistently tested positive by PCR employing universal rRNA primers (P1/P7), which amplify a 1.8-kb phytoplasma rDNA product (4), and negative when LY-specific primers LYF1/LYR1 (1) or MMF/MMR (3) were used. No PCR products were evident when DNAs of symptomless plants were evaluated with these primer combinations. Fragment patterns resolved by 8% polyacrylamide gel electrophoresis of rDNA digested separately with either AluI, BamHI, BstUI, DdeI, DraI, EcoRI, HaeIII, HhaI, HinfI, MspI, RsaI, Sau3AI, TaqI, or Tru9I endonucleases revealed no differences between phytoplasma isolates associated with five C. palmata plants. Collectively, restriction fragment length polymorphism (RFLP) patterns generated with key enzymes AluI, BamHI, DraI, and HaeIII clearly differentiated the C. palmata yellows (CPY) phytoplasma from LY and other known phytoplasmas previously characterized by this means (2). A sequence homology of 99.21% between 16S rDNA of CPY (1,537 bp; GenBank accession, AF237615) and LY (1,524 bp; accession, U18747) indicated that these strains were very similar. This relationship was confirmed by phylogenetic analysis of 16S rDNA sequence, which placed both strains into the same phytoplasma subclade. References: (1) N. A. Harrison et al. Plant Pathol. 43:998, 1994. (2) I.-M. Lee et al. Int. J. Syst. Bacteriol. 48:1153, 1998. (3) J. P. Martínez-Soriano et al. Rev. Mex. Fitopat. 12:75, 1994. (4) C. D. Smart et al. Appl. Environ. Microbiol. 62: 2988, 1996.

Protein phosphorylation during coconut zygotic embryo development

Evidence was obtained on the occurrence of protein threonine, serine, and tyrosine (Tyr) kinases in developing coconut (Cocos nucifera L. ) zygotic embryos, based on in vitro phosphorylation of proteins in the presence of [gamma-32P]ATP, alkaline treatment, and thin-layer chromatography analysis, which showed the presence of [32P]phosphoserine, [32P]phosphothreonine, and [32P]phosphotyrosine in [32P]-labeled protein hydrolyzates. Tyr kinase activity was further confirmed in extracts of embryos at different stages of development using antiphosphotyrosine monoclonal antibodies and the synthetic peptide derived from the amino acid sequence surrounding the phosphorylation site in pp60(src) (RR-SRC), which is specific for Tyr kinases. Anti-phosphotyrosine western blotting revealed a changing profile of Tyr-phosphorylated proteins during embryo development. Tyr kinase activity, as assayed using RR-SRC, also changed during embryo development, showing two peaks of activity, one during early and another during late embryo development. In addition, the use of genistein, a Tyr kinase inhibitor, diminished the ability of extracts to phosphorylate RR-SRC. Results presented here show the occurrence of threonine, serine, and Tyr kinases in developing coconut zygotic embryos, and suggest that protein phosphorylation, and the possible inference of Tyr phosphorylation in particular, may play a role in the coordination of the development of embryos in this species.

Regeneration of coconut (Cocos nucifera L.) from plumule explants through somatic embryogenesis.

A protocol was developed for coconut regeneration using plumules from mature zygotic embryos as explants, and media with the synthetic growth regulators 2,4-dichlorophenoxyacetic acid and 6-benzylaminopurine. Evidence for the regeneration process from these tissues occurring through somatic embryogenesis is presented. The somatic embryos were capable of germination, subsequent development into plantlets and successful transfer to the nursery. The yields were larger, nearly twofold for calli and over tenfold for calli bearing somatic embryos, than those previously reported with inflorescence explants. The present protocol thus represents an improvement in time and yield over previous protocols. Even though plumule explants are not the ideal tissue source due to possible genetic heterogeneity, the improvements made here may be applicable to tissues from mature plants. In addition, micropropagation of coconut using plumules is potentially useful when they are obtained from fruit produced from selected parents of outstanding performance, such as those resistant to diseases.

[Evaluation of Mexican biomedical journals]. / La evaluación de las revistas biomédicas mexicanas.

We present the evaluation procedures of Biomedical Journals, performed by the Journal Committee of the Centro Nacional de Información y Documentación sobre Salud (CENIDS) for the preparation of CR-ROM ARTEMISA 2 (Edited health information articles in Mexico), which contains a database of the complete text of articles appearing in the journals of the highest quality published in Mexico.

Bases para la formulación del plan de operaciones en el marco de la ley safco / sss

El esfuerzo de programación que hoy se requiere, es mas significativo que nunca. Cuanto más escasos son los recursos mayor es la necesidad de racionalizar y ésta es la tarea que debe afrontar cada unidad ejecutora, con responsabilidad, criterio y aplicando las técnicas que le permitan alcanzar los resultados acordes con las exigencias legales y con la propia responsabilidad profesional de servicios públicos

Tratamiento de las estenosis faringeas por la ingestion de causticos. / Treatment of the pharyngeal stenosis caused by caustic ingestion

Este articulo se basa en la revision de los resultados obtenidos en cinco pacientes que ingirieron causticos y que presentaron lesiones faringeas que motivaron tratamiento quirurgico mediante substitucion con tubo esofagico. Se describen diferentes procedimientos que tuvieron como finalidad tratar las lesiones laringofaringeas con la idea de aprovechar funcionalmente el esofago reconstruido