Identification And Analysis Of A Molecular Signaling Pathway That Regulates Calcium Oscillations In Root Hair Tip-Growth

Calcium, as an important second messenger, plays a crucial role in signal transduction and plant growth, especially in cell polarity growth. Now known as the polar growth cell, in plants, they include pollen tubes and root hairs. Previous studies showed that Ca2+influx through plasma-membrane Ca2+channels is necessary to form the tip-high Ca2+gradient and target the gradient oscillations of [Ca2+]cyt to control root tip-growth machinery.The Arabidopsis Cyclic Nucleotide-Gated Channel ?CNGC? family has been proposed to function in plant cells as potential calcium channels. By mutant screening, we identified an Arabidopsis CNGC14knock-out mutant cngc14-1?SALK₂06460?, which has very short, sometimes branched, stop-growing root hair. Meanwhile, miR173ts RNA interference transgenic line cngc14-2is a knock-down mutant, showing similar phenotype as cngc14-1. CNGC14is preferentially expressed in growing root hairs and root epidermis cells, as indicated from a promoter::GUS ??-glucuronidase? reporter analysis and expression profiling. YFP-tagged CNGC14was found to localize to the root hair plasma membrane. The phenotype of cngc14can be partly complemented by adding additional1.5mM Calcium into1/2MS medium. CNGC14, probably like other members of CNGC family, is a typical calcium-permeable channel in root hair.In Xenopus oocytes, CNGC14was showed to be a typical calcium-permeable channel. Using a whole-cell patch-clamp configuration, cngc14was recorded with less calcium inward current in Arabidopsis root hair protoplasts when compared with wild type. At the same time, the root hairs of cngcl4had lower levels of free [Ca2+]cyt by Ca2+measurements with Fluo-4pentaacetoxymethyl ester ?AM?. cngcl4-1barely showed any tip-gradient calcium ion in root hair. By using fluorescence resonance transfer detect calcium sensor YC3.60of cngcl4-1, we found that not only calcium FRET fluorescence of cngcl4-1was significantly lower than WT, but also Ca2+gradient oscillations frequency of cngcl'4-1was twice faster than that in WT.By yeast two-hybrid assays, CNGC14was found to interact with CaM6and7. Through Bimolec?lar fluorescence complementation ?BiFC? in tobacco and protein pulldown experiment, we confirmed the physical interactions between CaMs and CNGC14both in vivo and in vitro. Thus, CNGC14maydirectly permit Ca2+influx through plasma membrane into root hair cells and then form Ca2+gradient oscillate to regulate the root tip-growth machinery.When co-expressed CNGC14, both CaM6and CaM7was found to inhibit CNGC14calcium influx activity in in Xenopus Oocytes. Our experimental results also showed that CNGC14had stronger interactions with calcium-free CaMs ?apo-CaMs?. There is another important protein PCaP2of which gene is expressed predominantly in root hairs and pollen tubes and regulate tip-growth both polarity cell. PCaP2only interacts with Ca2+-bound CaMs ?Ca2+-CaMs?. By protein pulldown experiments, we confirmed that PCaP2can interact with CaM6and CaM7. In yeast three hybrid system, we confirmed that in the case of PCaP2exist, would weaken CNGC14and CaMs interaction. And in Xenopus oocytes system, also proved that when simultaneous expression PCaP2, CaM7and CNGC14in same time, PCaP2can be partially lifted CaM7on CNGC14inhibition. Then, it was believed that when CaMs-CNGC14complex is activated, calcium influx from outside into Cytoplasmic. Ca-bound CaMs may cause dissociation from the CNGC14, and Ca-CaM complex interact with PCaP2to promote dissociation of PtdInsPs ?specifically PIP2? from PCaP2. Through Ca2+-free and Ca2+-bound forms, CNGC14and PCaP2are engaged in a cross-talk between calcium and phosphate phosphatidylinositol two signal pathways to changes in [Ca2+]cyt and form Ca2+gradient oscillate to regulate the root tip-growth machinery.