Estudio de las proteínas de estrés térmico (HSPS) como indicador bioquímico de daño poscosecha en mandarinas Satsuma Okitsu

Abstract

El objetivo del presente trabajo fue evaluar la expresión de las HSPs en mandarinas Satsuma Okitsu con diferentes índices de color (IC) a cosecha sometidos a tratamiento de desverdizado y posterior conservación frigorífica, como posible indicador bioquímico de alteraciones fisiológicas. Se cosecharon dos lotes de frutos (Entre Ríos) con índices de color de -12,8 y -9,6, ambos con grado de madurez adecuados para su consumo en fresco. Estos lotes fueron sometidos a tratamiento de desverdizado (1-3 ppm etileno, 72h a 21°C), seguido de descanso (21°C 72 hs, 95% HR) y posterior conservación (30 días a 5°C) y 7 días a 20°C simulando la etapa de comercialización. El análisis se realizó a través de la expresión dos familias de HSPs: 1) la familia de HSP de bajo peso molecular (sHSPs), y 2) la familia de HSP de 70 kDa (HSP70). Se extrajeron las proteínas del flavedo de los frutos utilizando fenol buffereado. Ochenta microgramos (80 μg) de proteínas totales fueron separadas mediante SDS-PAGE y posteriormente analizadas inmunológicamente mediante la técnica de Western blot. A nivel de Western blot, el anti- HSPC1 (sHSPs) inmunoreaccionó con tres bandas proteicas con peso molecular de aprox. 22- 23, 18 y 16 kDa, en ambos lotes de frutas analizados (IC: -12,8 y -9,6). Para el lote con IC -9,6, se observaron diferencias de nivel de expresión de las proteínas de 18 y 16 kDa entre el control y los 30 días de conservación frigorífica. Respecto al lote con IC -12,8, no hubo cambios en la expresión de estas dos proteínas. Por otra parte, el anticuerpo anti-HSP70 sólo inmunoreaccionó en las muestras pertenecientes al lote con menor índice de color (-12,8), dando una única banda de 70 kDa, tanto para la muestra control como para la de 30 días de conservación frigorífica. A partir de estos resultados preliminares podríamos inferir que la expresión diferencial de las HSPs podría ser herramienta para predecir posibles alteraciones fisiológicas que minimicen las pérdidas comerciales provocadas por manchas que pudieran ocasionar el desverdizado y su posterior conservación en frío. ABSTRACT The aim of the present work was evaluate the expression of HSPs in Satsuma Okitsu mandarins in two batches of fruits with different color indexes (CI) at harvest, and subjected to degreening treatment and subsequent refrigeration conservation, as a possible biochemical indicator of physiological alterations. Two batches of fruits (Entre Ríos) were harvested with color indexes of -12.8 and -9.6, both with adequate degree of maturity for the external market. These batches were subjected to degreening treatment (1-3 ppm ethylene, 72h at 21 °C), followed by rest (72hs at 21 °C, 95% RH) and subsequent storage (30 days at 5 °C) and 7 days at 21 °C simulating the marketing stage. The analysis was carried out through the expression of two families of HSPs: 1) the family of HSPs of low molecular weight (sHSPs) and 2) the family of HSP of 70 kDa (HSP70). Proteins (flavedo) were extracted from fruits using buffered phenol. Eighty micrograms (80 μg) of total proteins were separated by SDS-PAGE and subsequently analyzed immunologically by the Western blot technique. At the Western blot level, the anti-HSPC1 (sHSPs) immunoreacted with three protein bands with molecular weight of approx. 22-23, 18 and 16 kDa, in both batches of fruits analyzed (CI: -12.8 and -9.6). For the batch with IC - 9.6, differences in the expression level of the 18 and 16 kDa proteins were observed between the control and the 30 days of refrigerated storage. Regarding the batch with IC -12.8, there were no changes in the expression of these two proteins. On the other hand, the anti-HSP70 antibody only immunoreacted in the samples belonging to the batch with the lowest color index (-12.8), giving a single band of 70 kDa, both control and for the 30-day refrigerated conservation samples. From the results obtained we can conclude that the differential expression of the HSPs could be used as a tool to define the management of the fruit, minimizing the commercial losses caused by degreening and cold storage.

The aim of the present work was evaluate the expression of HSPs in Satsuma Okitsu mandarins in two batches of fruits with different color indexes (CI) at harvest, and subjected to degreening treatment and subsequent refrigeration conservation, as a possible biochemical indicator of physiological alterations. Two batches of fruits (Entre Ríos) were harvested with color indexes of -12.8 and -9.6, both with adequate degree of maturity for the external market. These batches were subjected to degreening treatment (1-3 ppm ethylene, 72h at 21 °C), followed by rest (72hs at 21 °C, 95% RH) and subsequent storage (30 days at 5 °C) and 7 days at 21 °C simulating the marketing stage. The analysis was carried out through the expression of two families of HSPs: 1) the family of HSPs of low molecular weight (sHSPs) and 2) the family of HSP of 70 kDa (HSP70). Proteins (flavedo) were extracted from fruits using buffered phenol. Eighty micrograms (80 μg) of total proteins were separated by SDS-PAGE and subsequently analyzed immunologically by the Western blot technique. At the Western blot level, the anti-HSPC1 (sHSPs) immunoreacted with three protein bands with molecular weight of approx. 22-23, 18 and 16 kDa, in both batches of fruits analyzed (CI: -12.8 and -9.6). For the batch with IC - 9.6, differences in the expression level of the 18 and 16 kDa proteins were observed between the control and the 30 days of refrigerated storage. Regarding the batch with IC -12.8, there were no changes in the expression of these two proteins. On the other hand, the anti-HSP70 antibody only immunoreacted in the samples belonging to the batch with the lowest color index (-12.8), giving a single band of 70 kDa, both control and for the 30-day refrigerated conservation samples. From the results obtained we can conclude that the differential expression of the HSPs could be used as a tool to define the management of the fruit, minimizing the commercial losses caused by degreening and cold storage.