Starch degradation in the bean fruit pericarp is characterized by an increase in maltose metabolism.

The bean fruit pericarp accumulates a significant amount of starch, which starts to be degraded 20 days after anthesis (DAA) when seed growth becomes exponential. This period is also characterized by the progressive senescence of the fruit pericarp. However, the chloroplasts maintained their integrity, indicating that starch degradation is a compartmentalized process. The process coincided with a transient increase in maltose and sucrose levels, suggesting that ß-amylase is responsible for starch degradation. Starch degradation in the bean fruit pericarp is also characterized by a large increase in starch phosphorylation, as well as in the activities of cytosolic disproportionating enzyme 2 (DPE2, EC 2.4.1.25) and glucan phosphorylase (PHO2, EC 2.4.1.1). This suggests that the rate of starch degradation in the bean fruit pericarp 20 DAA is dependent on the transformation of starch to a better substrate for ß-amylase and the increase in the rate of cytosolic metabolism of maltose.

Phosphorylation of S11 in PHR1 negatively controls its transcriptional activity.

Plant responses to phosphate starvation (-Pi) are very well characterized at the biochemical and molecular levels. The expression of thousands of genes is modified under this stress condition, depending on the action of Phosphate starvation response 1 (PHR1). Existing data indicate that neither the PHR1 transcript nor the quantity or localization of its protein increase during nutrient stress, raising the question of how its activity is regulated. Here, we present data showing that SnRK1 kinase is able to phosphorylate some phosphate starvation response proteins (PSRs), including PHR1. Based on a model of the three-dimensional structure of the catalytic subunit SnRK1α1, docking simulations predicted the binding modes of peptides from PHT1;8, PHO1 and PHR1 with SnRK1. PHR1 recombinant protein interacted in vitro with the catalytic subunits SnRK1α1 and SnRK1α2. A BiFC assay corroborated the in vivo interaction between PHR1 and SnRK1α1 in the cytoplasm and nucleus. Analysis of phosphorylated residues suggested the presence of one phosphorylated site containing the SnRK1 motif at S11, and mutation in this residue disrupted the incorporation of 32 P, suggesting that it is a major phosphorylation site. Electrophoretic mobility shift assay results indicated that the binding of PHR1 to P1BS motifs was not influenced by phosphorylation. Importantly, transient expression assays in Arabidopsis protoplasts showed a decrease in PHR1 activity in contrast with the S11A mutant, suggesting a role for Ser11 as a negative regulatory phosphorylation site. Taken together, these findings suggest that phosphorylation of PHR1 at Ser11 is a mechanism to control the PHR1-mediated adaptive response to -Pi.

Starch accumulation in bean fruit pericarp is mediated by the differentiation of chloroplasts into amyloplasts.

The sucrose supply to bean fruits remains almost constant during seed development, and the early stages of this process are characterized by a significant amount of starch and soluble sugars (glucose, fructose and sucrose) accumulated in the pericarp. Bean fruits are photosynthetically active; however, our results indicated that starch synthesis in the pericarp was largely dependent on the photosynthetic activity of the leaves. The photosynthetic activity and the amount of the Rubisco large subunit were gradually reduced in the fruit pericarp, and a large increase in the amount of the ADP-glucose pyrophosphorylase small subunit (AGPase SS) was observed. These changes suggested differentiation of chloroplasts into amyloplasts. Pericarp chloroplasts imported glucose 1-P to support starch synthesis, and their differentiation into amyloplasts allowed the surplus sucrose to be used in the synthesis of starch, which was later degraded to meet the needs of fast-growing seeds. Starch stored in the bean fruit pericarp was not degraded in response to drought stress, but it was rapidly used under severe nutrient restriction. Together, this work indicated that starch accumulation in the pericarp of bean fruits is important to adjust the needs of developing seeds to the amount of sucrose that is provided to fruits.

Review: How do SnRK1 protein kinases truly work?

The AMPK/SNF1/SnRK1 family of protein kinases is involved in cellular responses to energy stress. They also interact with molecules of other signaling pathways to regulate many aspects of growth and development. The biochemical, genetic and molecular knowledge of SnRK1 in plants lags behind that of AMPK and SNF1 and is freely extrapolated such that, in many cases, it is assumed that plant enzymes behave in the same way as homologs in other organisms. In this review, we present data that support the evidence that the structural characteristics of the SnRK1 subunits determine the functional properties of the complex. We also discuss results suggesting that the SnRK1 subunits participate in the assembly of different complexes and that not all combinations are equally important. The activity of SnRK1 is dependent on the phosphorylation of SnRK1αThr175 found in the activation loop of the catalytic domain. However, we propose that the phosphorylation of sites close to SnRK1αThr175 might contribute to the fine-tuned regulation of SnRK1 activity and thus requires further evaluation. Finally, we also call attention to the interaction of the SnRK1α with regulatory proteins that are not typically identified as putative substrates. The additional functions of the SnRK1 subunits, in addition to those of the active complex, may be necessary for the cell to respond to the complicated conditions presented by energy stress.

Effect of catalytic subunit phosphorylation on the properties of SnRK1 from Phaseolus vulgaris embryos.

Legume seed development represents a high demand for energy and metabolic resources to support the massive synthesis of starch and proteins. However, embryo growth occurs in an environment with reduced O2 that forces the plant to adapt its metabolic activities to maximize efficient energy use. SNF1-related protein kinase1 (SnRK1) is a master metabolic regulator needed for cells adaptation to conditions that reduce energy availability, and its activity is needed for the successful development of seeds. In bean embryo extracts, SnRK1 can be separated by anion exchange chromatography into two pools: one where the catalytic subunit is phosphorylated (SnRK1-p) and another with reduced phosphorylation (SnRK1-np). The phosphorylation of the catalytic subunit produces a large increase in SnRK1 activity but has a minor effect in determining its sensitivity to metabolic inhibitors such as trehalose 6-P (T6P), ADP-glucose (ADPG), glucose 1-P (G1P) and glucose 6-P (G6P). In Arabidopsis thaliana, upstream activating kinases (SnAK) phosphorylate the SnRK1 catalytic subunit at T175/176, promoting and enhancing its activity. Recombinant Phaseolus vulgaris homologous to SnAK proteins (PvSnAK), can phosphorylate and activate the catalytic domains of the α-subunits of Arabidopsis, as well as the SnRK1-np pool purified from bean embryos. While the phosphorylation process is extremely efficient for catalytic domains, the phosphorylation of the SnRK1-np complex was less effective but produced a significant increase in activity. The presence of SnRK1-np could contribute to a quick response to unexpected adverse conditions. However, in addition to PvSnAK kinases, other factors might contribute to regulating the activation of SnRK1.

Detección del virus del papiloma humano en muestras obtenidas mediante técnica de autotoma en un grupo de universitarias peruanas / Detection of the human papillomavirus in samples obtained by self-collection technique in a group of peruvian college students

RESUMEN Estudio transversal que determinó la frecuencia y genotipos del virus del papiloma humano de alto riesgo (VPH-AR) a través de la técnica de autotoma en un grupo de universitarias de Lima. Participaron 221 estudiantes y se detectó el ADN del VPH-AR con el método de reacción en cadena de la polimerasa (PCR). La frecuencia del VPH-AR en las participantes fue de 43,4%; de este grupo se encontraron los genotipos VPH 16 en el 15,6% y VPH 18 en el 4,2% y otros VPH-AR en el 80,2%. Se concluye que la frecuencia del VPH-AR es mayor en el grupo de universitarias de este estudio en comparación a investigaciones nacionales previas.

ABSTRACT Cross-sectional study that determined the frequency and the genotypes of the (HR-HPV) high-risk human papillomavirus through the self-collection technique in a group of college students of Lima. Two hundred twenty-one (221) students participated and the DNA of the HR-HPV was detected with polymerase chain reaction (PCR). The frequency of HR-HPV in participants was 43.4%; of this group, genotype HPV 16 was present in 15.6%, HPV 18 in 4.2%, and another HR-HPV in 80.2%. We can conclude that the frequency of HR-HPV is greater in the group of college students of this study in comparison with previous national investigations.

[Detection of the human papillomavirus in samples obtained by self-collection technique in a group of peruvian college students]. / Detección del virus del papiloma humano en muestras obtenidas mediante técnica de autotoma en un grupo de universitarias peruanas.

Cross-sectional study that determined the frequency and the genotypes of the (HR-HPV) high-risk human papillomavirus through the self-collection technique in a group of college students of Lima. Two hundred twenty-one (221) students participated and the DNA of the HR-HPV was detected with polymerase chain reaction (PCR). The frequency of HR-HPV in participants was 43.4%; of this group, genotype HPV 16 was present in 15.6%, HPV 18 in 4.2%, and another HR-HPV in 80.2%. We can conclude that the frequency of HR-HPV is greater in the group of college students of this study in comparison with previous national investigations.

Estudio transversal que determinó la frecuencia y genotipos del virus del papiloma humano de alto riesgo (VPH-AR) a través de la técnica de autotoma en un grupo de universitarias de Lima. Participaron 221 estudiantes y se detectó el ADN del VPH-AR con el método de reacción en cadena de la polimerasa (PCR). La frecuencia del VPH-AR en las participantes fue de 43,4%; de este grupo se encontraron los genotipos VPH 16 en el 15,6% y VPH 18 en el 4,2% y otros VPH-AR en el 80,2%. Se concluye que la frecuencia del VPH-AR es mayor en el grupo de universitarias de este estudio en comparación a investigaciones nacionales previas.

Amenazas a la seguridad de la paciente por la conducción del parto con oxitocina. Experiencia en un hospital público de Lima, Perú / Threats to the safety of the patient due to the delivery management with oxytocin. Experience in a public hospital in Lima, Peru

Resumen OBJETIVO Identificar las amenazas a la seguridad de la paciente en la práctica de la conducción del trabajo de parto con oxitocina y las repercusiones maternas y perinatales en un hospital público de tercer nivel de Lima, Perú. MATERIALES Y MÉTODOS Estudio cuantitativo, descriptivo, retrospectivo y transversal efectuado en pacientes embarazadas atendidas en un hospital público, de tercer nivel, de Lima, Perú, a quienes se indicó conducción del parto con oxitocina. Los datos se tabularon en Excel y SPSS v21. El análisis univariado incluyó frecuencia y porcentaje para las variables cualitativas; se emplearon medidas de tendencia central, dispersión y desviación estándar para variables cuantitativas. RESULTADOS Se revisaron 112 historias clínicas de pacientes sin alteraciones en el trabajo de parto a quienes se indicó conducción con oxitocina (29.5%; n = 33). La principal vía de término fue el parto vaginal (73.2%; n = 82) y del grupo con cesárea el motivo más común fue la desproporción céfalo pélvica (56.7%; n = 17). Se observaron complicaciones obstétricas en 25% (n = 28), de este grupo la principal fue la hemorragia posparto (46.4%; n = 13). El peso de los recién nacidos fue 3401 ± 394 gramos, 93.7 y 96.4% obtuvieron un Apgar entre 10 a 7 al primero y quinto minutos, respectivamente. CONCLUSIONES Las amenazas identificadas a la seguridad de la paciente fueron: conducción del parto con oxitocina en quienes no tenían alteraciones en el trabajo de parto y en quienes tuvieron desproporción céfalo-pélvica, a pesar de que es una contraindicación para el procedimiento.

Abstract OBJECTIVE Identify the threats to the safety of the patient in the practice of conducting labor with oxytocin and maternal and perinatal outcomes in a public tertiary hospital in Lima, during 2016. MATERIALS AND METHODS Quantitative, descriptive, retrospective and cross-sectional. A total 112 pregnants' medical histories were review who underwent augmentation of labour with oxytocin. Inclusion criteria's: term gestation, hospitalization with a 4 cm dilatation, fetuses in cephalic presentation. Exclusion criteria's: patients with previous or intercurrent pathologies to pregnancy. RESULT The augmentation with oxytocin made in pregnant women without alterations in labor (29.5%; n = 33). The main type childbirth was vaginal delivery (73.2%; n = 82) and of the group that underwent cesarean section, the most usual reason was cephalopelvic disproportion (56.7%; n = 17). Obstetric complications were observed (25%; n = 28), of this group the main was postpartum hemorrhage (46.4%; n = 13). The weight of the newborns oscillated between 3401 ± 394 g, 93.7% and 96.4% obtained an Apgar 10 to 7 at the 1st and 5th minute respectively. CONCLUSIONS The threats to the safety of the patient identified were the practice of augmentation of labor with oxytocin in pregnant women without alterations in labor and in pregnant women with cephalopelvic disproportion, although it is a contraindication to the procedure.

Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINßγ-ß3 complex.

The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associated with the control of energy balance, in addition to being involved in the regulation of other aspects of plant growth and development. Analysis of complex formation indicates that increased activity is achieved when the catalytic subunit is phosphorylated and bound to regulatory subunits. SnRK1.1 subunit activity is higher than that of SnRK1.2, which also exhibits reduced activation due to the regulatory subunits. The catalytic phosphomimetic subunits (T175/176D) do not exhibit high activity levels, which indicate that the amino acid change does not produce the same effect as phosphorylation. Based on the mammalian AMPK X-ray structure, the plant SnRK1.1/AKINßγ-ß3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINßγ and the ß3 subunit. While the AKINßγ prediction retains the 4 CBS domain organization of the mammalian enzyme, significant differences are found in the putative nucleotide binding pockets. Docking and MD studies identified two sites between CBS 3 and 4 which may bind adenine nucleotides, but only one appears to be functional, as judging from the predicted binding energies. The recombinant AKINßγ-ßs complexes were found to bind adenine nucleotides with dissociation constant (Kd) in the range of the AMP low affinity site in AMPK. The saturation binding data was consistent with a one-site model, in agreement with the in silico calculations. As has been suggested previously, the effect of AMP was found to slow down dephosphorylation but did not influence activity.

Changes in nutrient distribution are part of the mechanism that promotes seed development under severe nutrient restriction.

When bean fruits are detached from a plant at 20 days after anthesis (DAA), the material accumulating in the pod is relocalized to the seeds. This mobilization is more active during the first five days after the fruits are removed, which allows some seeds to continue their development. In freshly removed fruits, (14)C-sucrose was evenly distributed among seeds; however, in fruits that were removed three days before, the labeled sugar was concentrated in 1-2 seeds. In addition, in removed pods, embryos dissected from seeds that no longer continue development can assimilate and efficiently use sucrose for protein and starch synthesis. Our results support the hypothesis that most embryos in removed fruits are forced to stop developing by removal of the nutrient supply. We also observed that SnRK1 activity increased in embryos that were subjected to nutrient deprivation, supporting the role of SnRK1 in the metabolic adaptation to stress conditions.

SnRK1 is differentially regulated in the cotyledon and embryo axe of bean (Phaseolus vulgaris L) seeds.

SnRK1 activity is developmentally regulated in bean seeds and exhibits a transient increase with the highest value at 20 days after anthesis (DAA), which coincides with the beginning of protein and starch accumulation. The catalytic subunit of SnRK1 shows a consistent decrease throughout the seed development period. However, by 15 DAA a significant proportion of the catalytic subunit appears phosphorylated. The increase in activity and phosphorylation of the catalytic subunit coincides with a decrease in hexoses. However, SnRK1 activity is differentially regulated in the cotyledon and embryo axe, where a larger proportion of the catalytic subunit is phosphorylated. SnRK1 obtained from endosperm extract is inhibited by T6P and to a lesser extent by ADPG and UDPG, whereas the enzyme isolated from embryo is virtually insensitive to T6P but exhibits some inhibition by ADPG and UDPG. In cotyledon extracts, the effects of T6P and ADPG on SnRK1 activity are additive, whereas in embryo extract, T6P inhibits the enzyme only when ADPG is present. After fractionation on Sephacryl-S300, SnRK1 activity obtained from cotyledon extracts is detected as a single peak associated with a molecular weight of 250 kDa whereas that obtained form embryo axe extracts detected as 2 peaks associated with molecular weight of 250 and 180 kDa. In both cases, the catalytic subunit exhibits a wide distribution but is concentrated in the fractions with the highest activity. To analyse the composition of the complex, cotyledon and embryo extracts were treated with a reversible crosslinker (DSP). DSP induced the formation of complexes with molecular weights of 97 and 180 kDa in the cotyledon and embryo extracts, respectively. Since all the phosphorylated catalytic subunit is present in the complexes induced by DSP, it appears that the phosphorylation favors its interaction with other proteins.

Evidence that abscisic acid promotes degradation of SNF1-related protein kinase (SnRK) 1 in wheat and activation of a putative calcium-dependent SnRK2.

Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) form a major family of signalling proteins in plants and have been associated with metabolic regulation and stress responses. They comprise three subfamilies: SnRK1, SnRK2, and SnRK3. SnRK1 plays a major role in the regulation of carbon metabolism and energy status, while SnRKs 2 and 3 have been implicated in stress and abscisic acid (ABA)-mediated signalling pathways. The burgeoning and divergence of this family of protein kinases in plants may have occurred to enable cross-talk between metabolic and stress signalling, and ABA-response-element-binding proteins (AREBPs), a family of transcription factors, have been shown to be substrates for members of all three subfamilies. In this study, levels of SnRK1 protein were shown to decline dramatically in wheat roots in response to ABA treatment, although the amount of phosphorylated (active) SnRK1 remained constant. Multiple SnRK2-type protein kinases were detectable in the root extracts and showed differential responses to ABA treatment. They included a 42 kDa protein that appeared to reduce in response to 3 h of ABA treatment but to recover after longer treatment. There was a clear increase in phosphorylation of this SnRK2 in response to the ABA treatment. Fractions containing this 42 kDa SnRK2 were shown to phosphorylate synthetic peptides with amino acid sequences based on those of conserved phosphorylation sites in AREBPs. The activity increased 8-fold with the addition of calcium chloride, indicating that it is calcium-dependent. The activity assigned to the 42 kDa SnRK2 also phosphorylated a heterologously expressed wheat AREBP.

The sucrose non-fermenting-1-related (SnRK) family of protein kinases: potential for manipulation to improve stress tolerance and increase yield.

Sucrose non-fermenting-1 (SNF1)-related protein kinases (SnRKs) take their name from their fungal homologue, SNF1, a global regulator of carbon metabolism. The plant family has burgeoned to comprise 38 members which can be subdivided into three sub-families: SnRK1, SnRK2, and SnRK3. There is now good evidence that this has occurred to allow plants to link metabolic and stress signalling in a way that does not occur in other organisms. The role of SnRKs, focusing in particular on abscisic acid-induced signalling pathways, salinity tolerance, responses to nutritional stress and disease, and the regulation of carbon metabolism and, therefore, yield, is reviewed here. The key role that SnRKs play at the interface between metabolic and stress signalling make them potential candidates for manipulation to improve crop performance in extreme environments.

Characterization of a type A response regulator in the common bean (Phaseolus vulgaris) in response to phosphate starvation.

Type A response regulators are a family of genes in Arabidopsis thaliana involved primarily in cytokinin signal transduction. A member of this family was isolated from a cDNA library constructed from bean plants (Phaseolus vulgaris) grown under conditions of phosphate starvation. The complete cDNA sequence showed the presence of the DDK domain, which is the hallmark of the response regulator family. Expression of the P. vulgaris response regulator 1 (PvRR1) showed clear regulation based on phosphate availability because transcript levels increased during phosphate starvation and returned to basal levels after resupplementation with phosphorus. Nitrogen and potassium starvation also upregulated PvRR1, indicating that cross talk with other nutrient signaling pathways might occur. Addition of cytokinins to plants growing under phosphate-sufficient conditions stimulated PvRR1 transcript levels both in detached leaves and in roots. However, cytokinins strongly inhibited PvRR1 expression in phosphate-starved plants after 24 h of incubation. At the protein level, subcellular localization of PvRR1 indicated that it is a nuclear protein and that phosphate starvation modified protein levels but not the localization.

Possible role played by R1 protein in starch accumulation in bean (Phaseolus vulgaris) seedlings under phosphate deficiency.

The effect of phosphate (Pi) deficiency on starch accumulation was studied in bean (Phaseolus vulgaris). After 3 weeks of Pi deprivation total Pi concentration in root and shoot was reduced by 68% and 42%, respectively; however, only shoot growth was affected. In leaves, Pi deprivation induced glucose, fructose and starch accumulation. Pi deficiency did not affect starch synthesis, but it reduced its mobilization during the dark period. At the same time, starch produced by Pi deficient plants have fewer Pi bound and was also less susceptible to beta-amylase hydrolysis. R1 protein is the protein responsible of phosphorylating C3 and C6 glucosyl residues of the polyglucan, increasing the hydration capacity and the interaction with amylolytic enzymes. Pi deprivation did not change the amount of R1 protein detected in total extracts but decreased its association with starch granules.