Analysis of the tip-to-base gradient of CaM in pollen tube pulsant growth using in vivo CaM-GFP system.

Ca(2+)-CaM signaling is involved in pollen tube development. However, the distribution and function of CaM and the downstream components of Ca(2+)-CaM signal in pollen tube development still need more exploration. Here we obtained the CaM-GFP fusion protein transgenic line of Nicotiana tobacum SRI, which allowed us to monitor CaM distribution pattern in vivo and provided a useful tool to observe CaM response to various exogenous stimulations and afforded solid evidences of the essential functions of CaM in pollen tube growth. CaM-GFP fusion gene was constructed under the control of Lat52-7 pollen-specific promoter and transformed into Nicotiana tobacum SRI. High level of CaM-GFP fluorescence was detected at the germinal pores and the tip-to-base gradient of fluorescence was observed in developing pollen tubes. The distribution of CaM at apical dome had close relationship with the pulsant growth mode of pollen tubes: when CaM aggregated at the apical dome, pollen tubes stepped into growth state; When CaM showed non-polarized distribution, pollen tubes stopped growing. In addition, after affording exogenous Ca(2+), calmidazolium (antagonism of CaM) or Brefeldin A (an inhibitor of membrane trafficking), CaM turned to a uniform distribution at the apical dome and pollen tube growth was held back. Taken together, our results showed that CaM played a vital role in pollen tube elongation and growth rate, and the oscillation of tip-to-base gradient of CaM was required for the normal pulsant growth of pollen tube.

[Gene expression and activity regulation of two calmodulin binding protein kinases in tobacco seedling].

Two different calmodulin-binding protein kinase cDNAs (NtCBK1/2) have been isolated from tobacco. To understand the CBK protein activity regulation, we compared the activity regulation of NtCBK1 and NtCBK2 by pH, Mg(2+) concentration and Na(+) concentration. We found the autophosphorylation of NtCBK1/2 reached the maximum in pH 7.5 and 8 respectively; Mg(2+) and Na(+) shown different effects on the activity of NtCBKs, high and low Mg(2+) concentrations both inhibited the activity of NtCBKs, but Na+ had little effect on the kinase activity. In addition, to obtain further insight about the physiological roles of individual NtCBKs, we detected the expression profiles of CBKs. The results revealed different patterns of expression of NtCBK1 and NtCBK2. Both are largely expressed in leaf and flower; but in stem and root, NtCBK1 gene had stronger expression than NtCBK2. NtCBK2 expression was induced by GA treatment, while NtCBK1 expression remained unchanged under GA treatment. Expression of both NtCBK1 and NtCBK2 increased in response to salt stress, the former to a greater extent, and both expressions did not change under high/low temperature, drought, NAA and ABA treatments.