Study On The Working Mechanism Of Ca²⁺ And CaM During The Ethylene-induced Flower Bud Physiological Differentiation And Initiation In Guzmania 'Amaranth'

Ca is the intracellular information transfer substances, regulating a number of important physiological processes in plants, and known as the "second messenger". Most of the reports on flower bud differentiation were limited to changes in the level of substances. Studies on the internal machanism were seldom.This paper was to clarify the dynamics and the mechanism of Ca and CaM in the process of flower bud differentiation by using Guzmania'Amaranth'as materials. Treated with the Ca2+ accelerator A23187, calcium ion-specific chelating agent EGTA, calmodulin (CaM) antagonists W-7 and TFP respectively, the morphological characteristics of Guzmania 'Amaranth'meristem were observed by paraffin section. And the contents of IAA, GAS, ZR, ABA and CaM were detected by ELISA. Ca contents were analized by Atomic Absorption Spectrophotometry (AAS). The CaM expression characteristics were studied by using Northern blot and RT-PCR methods.The major results were as follows:(1) About 9 days after ethphon treatment, the shoot tips of G.'Amaranth'began to expanding. After further developments of 14 days, there appeared the formation of floral premium. The flower buds were obvious after 30 days. The functions of exogenous ethylene increased with the treatment of calcium ionophore A23187. This result indicated that Ca2+ and CaM may participate in the process of exogenous ethylene on flower induction of G.'Amaranth'. Calmodulin antagonist trifluoperizine (TFP), W-7 and Ca2+ chelator EGTA all inhibited or delayed the flower bud differentiation induced by ethylene to some extents. It shows that Ca2+ and CaM may be involved in the process of ethylene-induced flower bud differentiation, regulates bud differentiation process.(2) Endogenous hormones levels and flower bud differentiation had closely relations. Low level of ABA promoted the differentiation of G.'Amaranth'flower buds, while high level of ABA can increase the development of flower buds. There was a certain negative corelation between GA3 contents and flower bud differention at the early stage of the flower bud differentiation, which was low levels of GA3 was good at the the flower bud physiological differentiation. High levels of ZR, IAA had the functions of promoting the flower buds differentiation. This result indicated that the balance and dynamics of ZR+ ABA/GA3, ZR+IAA/GA3 played an important role in regulating the ethylene-induced flower bud differentiation. We speculated that Ca2+-CaM signal system ultimately achieve the regulation of ethylene-induced flower bud differentiation by regulating the level of endogenous hormones or their synergies.(3) A23187 treatment enhanced the contents of Ca and CaM of during the flower bud differentiation, and promoted the flower bud differentiation. EGTA, W-7 and TFP treatment reduced the contents of Ca and CaM during the flower bud differentiation, and delayed the accumulation of Ca and CaM, which inhibit the flower bud differentiation. This fully shows the boot process needs to the involvement of Ca2+ and CaM.(4) A cDNA fragment with 454 bp of calmodulin in G.'Amaranth'was firstly isolated.(5) The expression level of CaM gene was analized with semi-quantitaty RT-PCR techniques and Northern blot. The results showed that ethylene treatment accelerated the expression of CaM gene. Treated with ethylene, the CaM gene expression began to increase after at 1 d with dealed. After, reached the peak at 3 d, the CaM gene expressed the largest amount, and then the first expression of a declined from 4 d. The treatment of blank expression of CaM genes has been maintained at a relatively low level of expression and has not the trend of increased expression. The results of Semi-quantitative RT-PCR were consistent with those of Northern blot. The research proved at the molecular level that the CaM genes involved in the flower bud differentiation process of G.'Amaranth'.At physiological and molecular levels, the above two experiments confirmed that the Ca2+ -CaM channels involved in the ethylene-induced flower bud differentiation process of G.'Amaranth'and supplied referrences for the research of the interactions between plant hormone, Ca2+-CaM system and flower bud differentiation.