Sebastián Rubio

An Improved Chemiluminescence Method for Hydrogen Peroxide Determination in Plant Tissues

As a consequence of the increasing importance of hydrogen peroxide in plant metabolism, more efficient methods are required for accurate determinations of its concentration in plant tissue and organs. Here we present a highly sensitive chemiluminescence (CL) method based on the Co (II) catalysed oxidation of luminol by H2O2. The replacement of ferricyanide, the traditional catalyst of luminol luminescence by Co (II), enhanced the sensitivity of the reaction towards H2O2 in three orders of magnitude. Thus, plant extracts can be diluted to such a level that quenching effects of phenols and ascorbic acid (ASA), which are normally present at high concentrations in plant tissues is avoided, and therefore, pre-treatments with PVP and ascorbate oxidase to remove these quenchers from plant-extracts become unnecessary. To exemplified the high performance of the method, measurements of H2O2 were carried out in PVP treated and non-treated extracts of grapevine leaf, a plant tissue that contain high levels of phenols and ASA. Moreover, increases in H2O2 levels were detected in disc-leaf treated with aminotriazole, a specific Cat inhibitor, showing the importance of Cat as a H2O2 scavenging enzyme in leaves of grapevine.

H2O2 is involved in the dormancy-breaking effect of hydrogen cyanamide in grapevine buds

Hydrogen cyanamide (HC) is widely used to induce the breakage of endodormancy (ED) in grape and other deciduous fruit crop, though its mechanism of action is poorly understood. Applications of HC to grapevine buds produce oxidative stress and transient respiratory disturbances which are related to the breakage of ED. Moreover, since the expression and activity of catalase (Cat) is inhibited by HC, enhancements in the levels of H2O2 have also been associated to the breakage of ED in grapevine buds. Here, we reported that increases in H2O2 level in HC-treated grapevine buds are due to the inhibition of Cat activity and enhancement of the respiratory activity of buds. In addition, exogenous applications of H2O2 partially reproduced the inducing effect of HC in the breakage of ED, thus providing further support for the hypothesis that H2O2 mediates the effects of HC. On the other hand, Mit isolated from both control and HC-treated buds respired equally well when NADH was used as a respiratory substrate, but when succinate was used as an electron donor Mit respiration was non-detected, suggesting that the stimulatory effect of HC on bud respiration is related to metabolic alterations leading to increase of the concentration of NADH rather than to changes in Mit functionality.

Use of the dynamic model for the assessment of winter chilling in a temperate and a subtropical climatic zone of Chile

A B S T R A C T Accumulated chilling was estimated by applying three different models to the hourly autumn-winter temperature records from Santiago (33°34 S lat; 625 m.a.s.l.) and Vicuna (30°02´ S lat; 643 m.a.s.l.) for the years 2005 and 2006. The model of chilling hours, currently used in Chile as an agroclimatic indicator, was of limited use for effectively contrasting a subtropical condition (Vicuna) with a temperate area such as Santiago. The application of the Utah model gave negative values from March to May, and even up to June in Vicuna, since in this model the chilling effect is “negated” by warmer temperatures. However, a modified version of the Utah model named Positive Chilling Units (PCU), in which negative values are omitted, showed differences in the accumulated chilling between both regions, although these differences were of small magnitude and were noted only from July onwards. The Dynamic Model, which considers that chilling is irreversibly accumulated as quantum or Chill Portions (CP), showed that chilling in Santiago doubled that of Vicuna, and that these differences in location were already expressed at the beginning of autumn, confirming, thus, the suitability of the model for subtropical conditions. In this work the advantages of the dynamic model over other models are discussed.

Is erratic bud-break in grapevines grown in warm winter areas related to disturbances in mitochondrial respiratory capacity and oxidative metabolism?

Bud-break and the length and depth of endodormancy (ED) were studied in grapevine (Vitis Vinifera L.) cv. Thompson Seedless (Sultana) grown in the Elqui (warm winter) and in the Maipo (temperate winter) valleys of north and central Chile, respectively. High maximum daily winter temperatures, ordinarily occurring in the Elqui valley, reduced the depth without affecting the length of ED in comparison to buds grown in the Maipo valley. Furthermore, high winter temperatures during the ED period altered the oxidative metabolism of buds by increasing its mitochondrial respiratory capacity and increasing its levels of H2O2. Moreover, a reduced expression in alternative oxidase transcript was also observed at the end of the ED period in buds collected from the warmer Elqui valley in relation to those collected from the temperate Maipo valley. In controlled environments, the bud-break response of ecodormant (ECD) buds depended on the climatic zones from which buds were sampled (temperate or warm winter), and on whether growth chamber temperatures were held constant or fluctuated. Mitochondrial respiratory capacity of dormant grapevine buds was raised by warmer winter temperatures, and higher subsequent H2O2 levels at the ECD phase appeared to be related to the erratic breaking of latent buds in subtropical areas such as the Elqui valley.

The dormancy-breaking stimuli “chilling, hypoxia and cyanamide exposure” up-regulate the expression of α-amylase genes in grapevine buds

It has been suggested that respiratory stress is involved in the mechanism underlying the dormancy-breaking effect of hydrogen cyanamide (H2CN2) and sodium azide in grapevine buds; indeed, reductions in oxygen levels (hypoxia) and inhibitors of respiration promote bud-break in grapevines. In this study, we showed that, hypoxia increased starch hydrolysis soluble sugar consumption and up-regulated the expression of α-amylase genes (Vvα-AMYs) in grapevine buds, suggesting that these biochemical changes induced by hypoxia, may play a relevant role in the release of buds from endodormancy (ED). Three of the four Vvα-AMY genes that are expressed in grapevine buds were up-regulated by hypoxia and a correlation between changes in sugar content and level of Vvα-AMY gene expression during the hypoxia treatment was found, suggesting that soluble sugars mediate the effect of hypoxia on Vvα-AMY gene expression. Exogenous applications of soluble sugars and sugar analogs confirmed this finding and revealed that osmotic stress induces the expression of Vvα-AMY1 and Vvα-AMY3 and that soluble sugars induces Vvα-AMY2 and Vvα-AMY4 gene expression. Interestingly, the plant hormone gibberellic acid (GA3) induced the expression of Vvα-AMY3 and Vvα-AMY4 genes, while dormancy breaking stimuli, chilling and cyanamide exposure, mainly induced the expression of Vvα-AMY1 and Vvα-AMY2 genes, suggesting that these two α-amylase genes might be involved in the release of grapevine buds from the ED.

The expression of VvPHYA and VvPHYB transcripts is differently regulated by photoperiod in leaves and buds of grapevines.

Light signals perceived by phytochromes (Phys) and cryptochromes (Crys) play key roles in plant growth and development and in photoperiod dependant process such as flowering, tuberization, seasonal growth cessation and dormancy. The integration of the light signals with the endogenous circadian oscillator provides plants with a mechanism to monitor changes in photoperiod or day-length. In a recent report, we established that in Vitis vinifera L. cv Thompson Seedless, photoperiod drives the entrance of buds into endodormancy (ED) and modifies the expression of VvPHYA and VvPHYB transcripts in grapevine leaves, suggesting that both VvPHYs could play crucial roles in SD-induced transition of bud into ED. Here, we aimed to establish whether the transition of grapevine buds into ED is a mere consequence of a decision taken in the leaf or whether the bud responds by itself to photoperiod. Results show that in defoliated grapevine canes, bud-ED development is delayed compared with non-defoliated control canes, and that under LD-photoperiod both VvPHYA and VvPHYB transcripts are highly expressed in grapevine buds, whilst under SD-photoperiod both VvPHYs are downregulated and expression can not be detected. Overall, the results suggest that grapevine bud behaves as semi-autonomous organ in sensing the photoperiod signal, and that VvPHYA and VvPHYB gene expression is differently regulated by photoperiod in leaf and bud of grapevines.

Prolonged Exposure of Grapevine Buds to Low Temperatures Increases Dormancy, Cold Hardiness, and the Expression of Vvα-AMYs Genes

Grapevine buds, like the buds of most temperate fruit trees, require a certain amount of chilling during the winter in order to emerge homogeneously in spring. This phenomenon is referred to as the chilling requirement (CR). The underlying mechanism of CR is unknown. As previously reported, low temperatures have a dual effect on grapevine buds, favoring release of buds from endodormancy (ED) while at the same time increasing cold hardiness (CH). To analyze whether CR is related to one or both of these processes, the BR50 (time required to reach 50 % bud break under forced conditions) and the low-temperature exotherm (LTE), which measures depth of ED and level of CH, respectively, were determined over the annual cycle of grapevine, and the results demonstrated that ED and CH occur sequentially. Using single-bud cuttings, the effect of low temperatures on both starch content and the expression of Vvα-AMYs were studied, and low temperatures resulted in increased ED and CH and stimulated starch breakdown through the activation of Vvα-AMYs, similar to that described in storage organs of higher plants such as tubers and bulbs. It has also been reported that abscisic acid (ABA) increases ED and CH in grapevine buds. Here, we confirmed that ABA synergizes the effect of low temperatures on CH and that low temperatures resulted in the up-regulation of VvNCED1, a gene encoding for a key enzyme of ABA biosynthesis. These results suggest that low temperature-induced increases in ED and CH could be mediated by ABA.

Derechos y Humanos sobre la exclusión a través del derecho

En el presente texto el autor desarrolla la idea que el Derecho en la modernidad se caracteriza por intentar abarcar toda la existencia del ser humano; normando desde su nacimiento hasta su muerte (I). En este contexto el concepto de persona es clave y ha sido determinante para poder acceder a los beneficios de las comunidades; organizadas en Estados; a traves de derechos (II). Ante ello; en las ultimas decadas se ha suscitado una serie de discusiones sobre la inclusion o exclusion de individuos y grupos; ya sea ampliando o reduciendo el acceso a derechos (III). En esto se han desarrollado ciertas herramientas juridicas destinadas a la exclusion; como lo son la condicionalidad de los derechos; la denegacion organizada de justicia y la creacion de subestatutos e identidades (IV).