Calcium Signal Change In Cold-acclimating Citrus

Citrus is one of the most important economical crops in southern China as well as in the world. Environmental stresses, including cold injury, cost great economic losses in the most of the citrus product region in the world. So, it is a great challenge for us to know more about the mechanism for citrus in response to the environmental stress. Calcium play a key role in hard resistance of biology, and has been recognized widely as the second message in the plant signal system. But how and when does calcium regulate the stresss resistance in citrus plants remain to know. In this paper, marterials as leaf of ponkan(Citrus reticulate Blanco.) and trifoliate orange (Poncitrus. trifoliata Raf), protoplast of lime (C. auratifolia Swingle), the Subcellular calcium localization through EM and [Ca2+]cyt change through laser scanning confocal microscopy(LSCM) were studied respectively.1. Subcellular calcium localization of leaf cell on ponkan (C. reticulate Blanco.) and trifoliate orange (P. trifoliata Raf)were analyzed through EM-cytochemical procedures. The observation revealed that antimonite Ca2+ deposits were localized in the vacuole and intercellular space more than in cytoplasm when they were grown at 25 ℃. After 2h 4℃ chilling, deposits increased in the cytosol and chloroplast. After 48h, or 7d chilling in 4℃ the increased cytosolic and nuclear antimonite Ca2+ deposits were restored to low resting level.2. Using Fluo-4/AM as the indicator to study the spatial-temporal changes of [Ca2+]cyt in the protoplasts of lime (C. auratifolia Swingle) under resting and temperature dropping conditions respectively. The results obtained in this experiment show that Fluo-4/AM being used as a fluorescent indicator in citrus and its stain effects in protoplasm are reliable and effective. Buffer with 5μmol/L A23187, control buffer, buffer with 2mmol/L EGTA are used respectively in measuring [Ca2+]cyt with Fluo-4/AM. The results showed that when the protoplast were treated under the above three solution systems, the resulting intensity of fluorescence in cytoplasm decreased in this order, which also implyed fluorescence is elicited by specific reaction between Fluo-4/AM and Ca2+. Meantime, the resting value of [Ca2+]cyt is the key factor in deciding the cells response abilities and response levels under low temperature stimulation. When temperature drop from 15℃ to -10℃ as low chilling rate (dT/dt<0.004℃), there is no detectable [Ca2+]cyt response at all. But protoplast react to cold-shock (i.e a temperature drop of several degree within less than 1 sec) from 15℃-4℃ and show dynamic changes:the [Ca2+]cyt level of protoplast elevate transitly. Prolonged cold treatment time attenuate the [Ca2+]cyt responses to subsequent episodes of cooling .The results provide new evidence in citrus for the hypothesis "Ca2+ plays a key role as a primary physiological transducer upon chilling".