Structure, ultrastructure and cation accumulation in quinoa epidermal bladder cell complex under high saline stress.

Quinoa is a facultative halophyte with excellent tolerance to salinity. In this study, the epidermal bladder cell complex (EBCc) of quinoa leaves was studied to determine their cellular characteristics and involvement in salt tolerance. We used light microscopy, confocal RAMAN microscopy, confocal fluorescence microscopy, transmission electron microscopy, and environmental scanning electron microscopy complemented by energy dispersive X-ray analysis. Ionic content was quantified with flame atomic absorption spectroscopy and with flame emission photometry. Results show that: (i) the number of EBCcs remains constant but their density and area vary with leaf age; (ii) stalk cells store lipids and exhibit thick walls, bladder cells present carotenes in small vesicles, oxalate crystals in vacuoles and lignin in their walls and both stalk and bladder cells have cuticles that differ in wax and cutin content; (iii) chloroplasts containing starch can be found on both stalk and bladder cells, and the latter also presents grana; (iv) plasmodesmata are observed between the stalk cell and the bladder cell, and between the epidermal cell and the stalk cell, and ectodesmata-like structures are observed on the bladder cell. Under high salinity conditions, (v) there is a clear tendency to accumulate greater amounts of K+ with respect to Na+ in the bladder cell; (vi) stalk cells accumulate similar amounts of K+ and Na+; (vii) Na+ accumulates mainly in the medullary parenchyma of the stem. These results add knowledge about the structure, content, and role of EBCc under salt stress, and surprisingly present the parenchyma of the stem as the main area of Na+ accumulation.

Increased Fat Oxidation During Arm Cycling Exercise in Adult Men With Spinal Cord Injury Compared With Noninjured Controls.

People with spinal cord injury (SCI) tend to be more sedentary and increase fat accumulation, which could have a negative influence on metabolic flexibility. The aim of this study was to investigate the capacity to oxidize fat in a homogenous sample of men with thoracic SCI compared with healthy noninjured men during an arm cycling incremental test. Forty-one men, 21 with SCI and 20 noninjured controls, performed an incremental arm cycling test to determine peak fat oxidation (PFO) and the intensity of exercise that elicits PFO (Fatmax). PFO was expressed in absolute values (g/min) and relative to whole-body and upper-body lean mass ([mg·min-1]·kg-1) through three different models (adjusting by cardiorespiratory fitness and fat mass). Gross mechanical efficiency was also calculated. PFO was higher in SCI than in noninjured men (0.27 ± 0.07 vs. 0.17 ± 0.07 g/min; 5.39 ± 1.30 vs. 3.29 ± 1.31 [mg·min-1]·kg-1 whole-body lean mass; 8.28 ± 2.11 vs. 5.08 ± 2.12 [mg·min-1]·kg-1 upper-body lean mass). Fatmax was found at a significantly higher percentage of VO2peak in men with SCI (33.6% ± 8.2% vs. 23.6% ± 6.4%). Differences persisted and even increased in the fully adjustment model and at any intensity. Men with SCI showed significantly higher gross mechanical efficiency at 35 and 65 W than the noninjured group. Men with SCI showed higher fat oxidation when compared with noninjured men at any intensity, even increased after full adjustment for lean mass, fat mass, and cardiorespiratory fitness. These findings suggest that SCI men could improve their metabolic flexibility and muscle mass for greater efficiency, not being affected by their fat accumulation.

The effects of Age, Organized Physical Activity and Sedentarism on Fitness in Older Adults: An 8-Year Longitudinal Study.

The aims of the present study were (1) to describe the changes in physical fitness during an 8 year follow-up in a large sample of Spanish adults aged 65 or over that are initially engaged in organized physical activity (OPA), (2) to compare fitness changes according to different age groups (65 to 69 vs. 70 to 74 vs. ≥75 years-old), (3) to evaluate the independent and combined effects of changes in OPA engagement and sitting time (ST) on physical fitness. A total of 642 (147 males) non-institutionalized over 65 years-old participants completed the EXERNET battery fitness tests and completed a validated questionnaire from which information regarding OPA and ST were collected. All participants completed evaluations in 2008-2009 and in 2016-2017. An impairment of fitness-related variables happens after 65 years of age in both males and females, with the older participants (≥75), showing the largest decreases. Males who continued performing OPA demonstrated lower decreases in balance, leg flexibility and agility when compared to those who stopped performing OPA during the follow-up. Females who continued performing OPA demonstrated lower decreases of all variables except for balance when compared to those who stopped performing OPA during the follow-up.

Strength and Endurance Training in Older Women in Relation to ACTN3 R577X and ACE I/D Polymorphisms.

The purpose of this study is to analyze the effect of two genetic polymorphisms, ACTN3R577X, and ACE I/D, on physical condition in a sample of active older women after a two-year training period. The sample was composed of 300 healthy women over the age of 60 who underwent a two-year training program. Adapted tests from the Senior Fitness Test were used. The genotyping of the polymorphisms was obtained from the participants' DNA via buccal swabs. The analysis of the ACE polymorphism did not reveal differences between genotypes. The analysis of the R577X polymorphism showed a favorable effect for the ACTN3 XX genotype in tests for leg strength (p: 0.001) after training, compared to the other genotypes, and also in the analysis of the combined effect of the polymorphism (ACE II + ACTN3 RX/XX). The intragroup effect revealed an improvement in arm strength for carriers of the X allele after 24 months of training (p < 0.05). The endurance values significantly worsened in all study groups. Conclusions: The R577X polymorphism of ACTN3 may have an important role in capacities related to muscle strength, providing a beneficial effect for carriers of the X allele.

Red light delays programmed cell death in non-host interaction between Pseudomonas syringae pv tomato DC3000 and tobacco plants.

Light modulates almost every aspect of plant physiology, including plant-pathogen interactions. Among these, the hypersensitive response (HR) of plants to pathogens is characterized by a rapid and localized programmed cell death (PCD), which is critical to restrict the spread of pathogens from the infection site. The aim of this work was to study the role of light in the interaction between Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and non-host tobacco plants. To this end, we examined the HR under different light treatments (white and red light) by using a range of well-established markers of PCD. The alterations found at the cellular level included: i) loss of membrane integrity and nuclei, ii) RuBisCo and DNA degradation, and iii) changes in nuclease profiles and accumulation of cysteine proteinases. Our results suggest that red light plays a role during the HR of tobacco plants to Pto DC3000 infection, delaying the PCD process.

Shotgun proteomic analysis of quinoa seeds reveals novel lysine-rich seed storage globulins.

Quinoa seeds have high protein content and an exceptional balance of amino acids, with higher contents of lysine, methionine and cysteine than common cereals. To date, only three globulins, all of which have a content of lysine mass that does not exceed 3.8%, have been identified in quinoa. To address the protein present in quinoa seeds, TCA/Acetone protein extraction was performed using four different quinoa seed genotypes with contrasting edaphoclimatic origins. Proteins were identified and analyzed using label-free shotgun proteomics followed by in silico analysis, using the three published quinoa genomes. This analysis allowed us to identify sixteen globulins, thirteen of which are novel: nine legumin-like proteins and seven vicilin-like proteins. Seven of the novel proteins contain 7.5% or more of lysine mass, justifying the high content of lysine repeatedly reported in quinoa seeds. No significant differences were found between the four genotypes here analyzed.

Effects of Zumba® and Aquagym on Bone Mass in Inactive Middle-Aged Women.

Background and objectives: Regular exercise may stimulate bone formation and reduce the loss of bone mass in premenopausal women. This study aims to evaluate the effect of high-impact physical activity (Zumba®) and low-impact physical activity (Aquagym) on bone mass in inactive middle-aged women. Materials and methods: Fifty-five healthy inactive women (30⁻50 years old) were recruited in Spain in 2016 and were randomly allocated into one of three groups: High impact group (HIG: n = 15), low impact group (LIG: n = 12) and control group (CG: n = 28). HIG and LIG were recruited from Madrid and the CG from Toledo. HIG and LIG completed a 12-week intervention program with three 40' sessions per week of Zumba® or Aquagym; respectively. Dual energy X-ray absorptiometry (DXA) measured bone mineral content (BMC) and areal bone mineral density (aBMD) at total body less head (TBLH), lumbar spine and right hip. Results: Post-intervention adjusted data showed no significant differences in BMC between any of the groups nor in aBMD between HIG and LIG. Interestingly; significant differences for the HIG vs. CG were found in the change in total hip aBMD (1.76% vs. -0.44%), femoral neck aBMD (1.80% vs. -2.71%), and intertrochanter aBMD (2.03% vs. -0.50%). Moreover, significant differences for the LIG vs. CG were also found in the change in femoral neck aBMD (-0.54% vs. -2.71%). Conclusions: The regular practice of Zumba® and Aquagym might reduce the progressive deterioration of bone mass in inactive middle-aged women.

Cardiorespiratory fitness and arm bone mineral health in young males with spinal cord injury: the mediator role of lean mass.

Spinal cord injury (SCI) derives in loss of bone mineral content (BMC) and bone mineral density (BMD). However, physical activity is an important determinant in bone mass acquisition, which is partially mediated through the lean mass (LM). The aim was to examine the effect of cardiorespiratory fitness (CRF) on BMD and BMC arms of adult males with SCI and able-bodied controls using the arm LM as a mediator variable. Thirty able-bodied men and thirty men with SCI participated. BMC and BMD were analysed by DXA, and indirect calorimetry was used to calculate VO2peak during a progressive arm-cranking test. When groups were divided by the amount of LM, the subgroup with highest LM had significantly higher arm BMC compared to the lowest LM subgroup (p ≤ 0.05) in both SCI and able-bodied groups. Moreover, same differences were found when confidence intervals were analysed. Only in the SCI group, arm LM mediated the relationship between bone mass and CRF at 30.9%, as indicated by the Sobel test (z = 2.17 and z = 2.04 for BMC and BMD, respectively). In conclusion, LM mediates the indirect association between CRF and bone health, specifically in the arms. This finding highlights the importance of having an adequate CRF for the maintenance of good bone health in SCI men.

Deterioration of willow seeds during storage.

Willow (Salix spp.) seeds are able to tolerate desiccation, but differ from typical orthodox seeds in that they lose viability in a few days at room temperature, and in that the chloroplasts in embryo tissues do not dedifferentiate during maturation drying, thus retaining chlorophyll and maintaining intact their thylakoid membranes. In the present study, we investigated the damage generated in willow seeds during storage under appropriate conditions to exclude the eventual generation of reactive oxygen species by photooxidation. To this end, we measured different indicators of molecular damage, such as changes in the fatty acid profile, protein degradation, nuclease activities, and DNA damage, and evaluated normal germination and total germination in seeds stored for one, ten and sixteen years. We found: (i) a decrease in the fraction of unsaturated fatty acids; (ii) changes in the protein profile due to a decrease in protein solubility; (iii) activation of nucleases; and (iv) DNA fragmentation. Taken together, our findings identified programmed cell death as a key mechanism in seed deterioration during storage. We also found that, although the seeds maintained high percentages of total germination, the death program had already started in the seeds stored for ten years and was more advanced in those stored for sixteen years.

Activation of Nucleases, PCD, and Mobilization of Reserves in the Araucaria angustifolia Megagametophyte During Germination.

The megagametophyte of mature seeds of Araucaria angustifolia consists of cells with thin walls, one or more nuclei, a central vacuole storing proteins, and a cytoplasm rich in amyloplasts, mitochondria and lipid bodies. In this study, we describe the process of mobilization of reserves and analyzed the dismantling of the tissue during germination, using a range of well-established markers of programmed cell death (PCD), including: morphological changes in nuclei and amyloplasts, DNA degradation, and changes in nuclease profiles. TUNEL reaction and DNA electrophoresis demonstrate that DNA fragmentation in nuclei occurs at early stages of germination, which correlates with induction of specific nucleases. The results of the present study add knowledge on the dismantling of the megagametophyte of genus Araucaria, a storage tissue that stores starch as the main reserve substance, as well as on the PCD pathway, by revealing new insights into the role of nucleases and the expression patterns of putative nuclease genes during germination.

[Organized physical activity in the elderly, a tool to improve physical fitness during ageing]. / La actividad física organizada en las personas mayores, una herramienta para mejorar la condición física en la senectud.

OBJECTIVE: An adequate level of fitness is critical to achieving healthy ageing. The aim of this study was to evaluate the impact of organized physical activity (PA) and the number of hours of practice on the fitness levels of the elderly. METHODS: A total of 3104 people over 65 years old from Spain, belonging to the EXERNET Multicenter Project (722 men and 2382 women, mean age: 72.1 ± 5.3 years) participated in the study. Fitness was evaluated by 8 specific tests for this population. Participation in organized PA was recorded through a questionnaire. Differences in fitness values depending on the participation in PA and the number of hours were measured with ANCOVA adjusting for age, hours of walking and sitting time. RESULTS: Those who did not participate in this type of activity increased their risk of having a low level of fitness in most variables evaluated, with some differences between sexes (OR: from 1.6 to 2.6 in men and from 1.4 to 2.2 in women). In men, no significant differences were observed between practicing less than 2, 2-4 or more than 4 hours (P grater than 0,05), with an exception in the strength of the left arm. However, women who performed more than 2 hours a week in organized PA had better balance, strength of lower and upper extremities, flexibility of lower extremities, walking speed and aerobic capacity than those who performed less than 2 hours of PA a week (P less than 0,05). CONCLUSIONS: The practice of organized PA produces a beneficial effect on the fitness of the elderly regardless of age, hours of walking and time sitting per day.

OBJETIVO: Un nivel adecuado de condición física (CF) es fundamental para el logro de un envejecimiento saludable. El objetivo de este estudio consistió en evaluar el impacto de la actividad física (AF) organizada y el número de horas de práctica sobre el nivel de CF de las personas mayores. METODOS: En el estudio participaron 3104 personas mayores de 65 años de España, pertenecientes al Proyecto Multicéntrico EXERNET (722 hombres y 2382 mujeres; edad media: 72,1±5,3 años). La CF se evaluó mediante 8 test específicos para esta población. La participación en AF organizada se registró mediante un cuestionario. Las diferencias en la CF en función de la práctica y número de horas de AF se midieron con ANCOVA ajustando por edad, horas de caminar y de estar sentado. RESULTADOS: Aquellos que no participaban en este tipo de actividad incrementaban su riesgo de tener un nivel bajo de CF en la mayoría de las variables evaluadas, observándose algunas diferencias entre sexos (OR: desde 1,6 hasta 2,6 en hombres y desde 1,4 hasta 2,2 en mujeres). En los hombres no se observó ninguna diferencia significativa entre practicar menos de 2, 2-4 o más de 4 horas (todos P mayor de 0,05), a excepción de la fuerza del brazo izquierdo; sin embargo, las mujeres que realizaban más de 2 horas de AF organizada a la semana tenían mayor equilibrio, fuerza de piernas y de brazos, flexibilidad de piernas, velocidad al caminar y resistencia aeróbica que aquellas que asistían a este tipo de sesiones menos de 2 horas a la semana (P menor de 0,05). CONCLUSIONES: La práctica de AF organizada produce un efecto beneficioso sobre la CF de las personas mayores independientemente de la edad, las horas que estén sentados o el tiempo que dediquen a caminar.

Death of embryos from 2300-year-old quinoa seeds found in an archaeological site.

In the 1970s, during excavations at Los Morrillos, San Juan, Argentina, quinoa seeds were found within ancient pumpkin crocks protected from the light and high temperatures, and preserved in the very dry conditions of the region. The radiocarbon dates confirmed the age of these seeds at around 2300 years. Sectioning of some of these seeds showed reddish-brown embryos, different from the white embryos of recently harvested quinoa seeds. The ancient seeds did not germinate. The structure of the embryo cells was examined using light and transmission electron microscopy; proteins were analyzed by electrophoresis followed by Coomassie blue and periodic acid Schiff staining and fatty acids by gas chromatography. The state of nuclear DNA was investigated by TUNEL assay, DAPI staining, ladder agarose electrophoresis and flow cytometry. Results suggest that, although the embryo tissues contained very low water content, death occurred by a cell death program in which heterochromatin density was dramatically reduced, total DNA was degraded into small fragments of less than 500bp, and some proteins were modified by non-enzymatic glycation, generating Maillard products. Polyunsaturated fatty acids decreased and became fragmented, which could be attributable to the extensive oxidation of the most sensitive species (linolenic and linoleic acids) and associated with a collapse of lipid bodies.

Somatic Embryogenesis in Araucaria angustifolia (Bertol.) Kuntze (Araucariaceae).

This chapter deals with the features of somatic embryogenesis (SE) in Araucaria angustifolia, an endangered and native conifer from south Brazil. In this species SE includes the induction and proliferation of embryogenic cultures composed of pro-embryogenic masses (PEMs), which precede somatic embryos development. A. angustifolia SE model encompasses induction, proliferation, pre-maturation, and maturation steps. Double-staining with acetocarmine and Evan's blue is useful to evaluate the embryonic somatic structures. In this chapter we describe A. angustifolia SE protocols and analyzes morphological features in the different SE developmental stages.

Toward establishing a morphological and ultrastructural characterization of proembryogenic masses and early somatic embryos of Araucaria angustifolia (Bert.) O. Kuntze.

Somatic embryogenesis is a morphogenetic route useful for the study of embryonic development, as well as the large-scale propagation of endangered species, such as the Brazilian pine (Araucaria angustifolia). In the present study, we investigated the morphological and ultrastructural organization of A. angustifolia somatic embryo development by means of optical and electron microscopy. The proembryogenic stage was characterized by the proliferation of proembryogenic masses (PEMs), which are cellular aggregates composed of embryogenic cells (ECs) attached to suspensor-like cells (SCs). PEMs proliferate through three developmental stages, PEM I, II, and III, by changes in the number of ECs and SCs. PEM III-to-early somatic embryo (SE) transition was characterized by compact clusters of ECs growing out of PEM III, albeit still connected to it by SCs. Early SEs showed a dense globular embryonic mass (EM) and suspensor region (SR) connected by embryonic tube cells (TCs). By comparison, early somatic and zygotic embryos showed similar morphology. ECs are round with a large nucleus, nucleoli, and many cytoplasmic organelles. In contrast, TCs and SCs are elongated and vacuolated with cellular dismantling which is associated with programmed cell death of SCs. Abundant starch grains were observed in the TCs and SCs, while proteins were more abundant in the ECs. Based on the results of this study, a fate map of SE development in A. angustifolia is, for the first time, proposed. Additionally, this study shows the cell biology of SE development of this primitive gymnosperm which may be useful in evolutionary studies in this area.

Cellular and molecular aspects of quinoa leaf senescence.

During leaf senescence, degradation of chloroplasts precede to changes in nuclei and other cytoplasmic organelles, RuBisCO stability is progressively lost, grana lose their structure, plastidial DNA becomes distorted and degraded, the number of plastoglobuli increases and abundant senescence-associated vesicles containing electronically dense particles emerge from chloroplasts pouring their content into the central vacuole. This study examines quinoa leaf tissues during development and senescence using a range of well-established markers of programmed cell death (PCD), including: morphological changes in nuclei and chloroplasts, degradation of RuBisCO, changes in chlorophyll content, DNA degradation, variations in ploidy levels, and changes in nuclease profiles. TUNEL reaction and DNA electrophoresis demonstrated that DNA fragmentation in nuclei occurs at early senescence, which correlates with induction of specific nucleases. During senescence, metabolic activity is high and nuclei endoreduplicate, peaking at 4C. At this time, TEM images showed some healthy nuclei with condensed chromatin and nucleoli. We have found that DNA fragmentation, induction of senescence-associated nucleases and endoreduplication take place during leaf senescence. This provides a starting point for further research aiming to identify key genes involved in the senescence of quinoa leaves.

Programmed cell death in seeds of angiosperms.

During the diversification of angiosperms, seeds have evolved structural, chemical, molecular and physiologically developing changes that specially affect the nucellus and endosperm. All through seed evolution, programmed cell death (PCD) has played a fundamental role. However, examples of PCD during seed development are limited. The present review examines PCD in integuments, nucellus, suspensor and endosperm in those representative examples of seeds studied to date.

Analogous reserve distribution and tissue characteristics in quinoa and grass seeds suggest convergent evolution.

Quinoa seeds are highly nutritious due to the quality of their proteins and lipids and the wide range of minerals and vitamins they store. Three compartments can be distinguished within the mature seed: embryo, endosperm, and perisperm. The distribution of main storage reserves is clearly different in those areas: the embryo and endosperm store proteins, lipids, and minerals, and the perisperm stores starch. Tissues equivalent (but not homologous) to those found in grasses can be identified in quinoa, suggesting the effectiveness of this seed reserve distribution strategy; as in cells of grass starchy endosperm, the cells of the quinoa perisperm endoreduplicate, increase in size, synthesize starch, and die during development. In addition, both systems present an extra-embryonic tissue that stores proteins, lipids and minerals: in gramineae, the aleurone layer(s) of the endosperm; in quinoa, the micropylar endosperm; in both cases, the tissues are living. Moreover, the quinoa micropylar endosperm and the coleorhiza in grasses play similar roles, protecting the root in the quiescent seed and controlling dormancy during germination. This investigation is just the beginning of a broader and comparative study of the development of quinoa and grass seeds. Several questions arise from this study, such as: how are synthesis and activation of seed proteins and enzymes regulated during development and germination, what are the genes involved in these processes, and lastly, what is the genetic foundation justifying the analogy to grasses.

[Physical fitness evolution in octogenarian population and its relationship with a sedentary lifestyle]. / Evolución de los niveles de condición física en población octogenaria y su relación con un estilo de vida sedentario.

OBJECTIVE: The aim of this study was to determine the changes in physical fitness over two years of following up in octogenarian people and to check whether a sedentary lifestyle modify these variations. METHODS: Physical fitness of 182 subject (48 men, 134 women) with a mean age of 82,3 ± 2,3 years were evaluated using 8 different tests. A repeated measures analysis was carried out to see the differences between the two evaluation periods and to see the physical fitness differences between sedentary people (sit ≥ 4 hours/day) and non sedentary people (sit < 4 hours/day). RESULTS: Between the two evaluation periods, we found a significant decrease in the agility test (p < 0.05), walking speed (p < 0.01) and endurance (p < 0.01). In relation to the subjects who spent sitting 4 hours/day there was a decrease in the walking speed test between the two evaluations (p < 0.05). Moreover, there was a decrease of walking speed and endurance between the two evaluation periods in both sedentary and nonsedentary people (p < 0.05). CONCLUSION: In two years of following up, there are adverse changes in the level of physical fitness in octogenarians. Long periods of sitting time may translate into a loss of agility. Walking speed and endurance seem to be the components of physical fitness more affected by the ageing process in this population; and this loss is not determined by the hours of sitting per day.

Objetivos: Determinar los cambios que se producen en la condición física a lo largo de dos años de seguimiento en octogenarios y comprobar si un estilo de vida sedentario modifica estas variaciones. Metodología: La condición física de 182 sujetos (48 hombres, 134 mujeres) con una media de edad de 82,3 ± 2,3 años se evaluó mediante 8 test (adaptados de las baterías "Senior Fitness Test" y "Eurofit"). Se realizó un análisis de medidas repetidas entre los dos periodos de evaluación y para ver las diferencias de condición física entre los sujetos considerados sedentarios (permanecían sentados ≥4 horas/día) y no sedentarios (permanecían sentados < 4 horas/día) Resultados: Entre las dos evaluaciones encontramos un descenso significativo en los test de agilidad (p < 0,05), velocidad (p < 0,01) y resistencia (p < 0,01) y un ligero aumento de la fuerza de brazos en los sujetos que permanecían sentados < 4 horas/día (p < 0,05). Aquellos que permanecían sentados > 4 horas/día sufrieron un descenso de la agilidad (p < 0,05). Se produjo una pérdida de velocidad y resistencia tanto en sedentarios como en no sedentarios (p < 0,05). Conclusión: En dos años de seguimiento, se producen cambios negativos en el nivel de condición física en personas octogenarias. Periodos prolongados de sedestación se traducen en una pérdida de agilidad. La velocidad de la marcha y resistencia parecen ser los componentes de la condición física más afectados por el proceso de envejecimiento en este grupo poblacional y su pérdida no viene determinada por las horas diarias de sedestación.