AtPRK2 Promotes ROP1 Activation via RopGEFs in the Control of Polarized Pollen Tube Growth

The ROP1 GTPase-based signaling network controls tip growth in Arabidopsis pollen tubes. Our previous studies imply that ROP1 might be directly activated by RopGEF1, which belongs to a plant-specific family of Rho guanine nucleotide exchange factors （RopGEFs） and in turn may be activated by an unknown factor through releasing RopGEFI＇s auto-inhibition. In this study, we found that RopGEF1 forms a complex with ROP1 and AtPRK2, a receptor-like protein kinase previously shown to interact with RopGEFs. AtPRK2 phosphorylated RopGEF1 in vitro and the atprkl,2,5 tri- ple mutant showed defective pollen tube growth, similar to the phenotype of the ropgef1,9,12,14 quadruple mutant. Overexpression of a dominant negative form of AtPRK2 （DN-PRK2） inhibited pollen germination in Arabidopsis and reduced pollen elongation in tobacco. The DN-PRK2-induced pollen germination defect was rescued by overexpressing a constitutively active form of RopGEF1, RopGEF1（90-457）, implying that RopGEF1 acts downstream of AtPRK2. Moreover, AtPRK2 increased ROP1 activity at the apical plasma membrane whereas DN-PRK2 reduced ROP1 activity. Finally, two mutations at the C-terminal putative phosphorylation sites of RopGEF1 （RopGEF1S460A and RopGEF1S480A） eliminated the function of RopGEF1 in vivo. Taken together, our results support the hypothesis that AtPRK2 acts as a positive regula- tor of the ROP1 signaling pathway most likely by activating RopGEF1 through phosphorylation.

Ethylene Response Factor TERF1 Enhances Glucose Sensitivity in Tobacco through Activating the Expression of Sugar-related Genes

Ethylene response factor （ERF） proteins are important plant-specific transcription factors. Increasing evidence shows that ERF proteins regulate plant pathogen resistance, abiotic stress response and plant development through interaction with different stress responsive pathways. Previously, we revealed that overexpression of TERF1 in tobacco activates a cluster gene expression through interacting with GCC box and dehydration responsive element （DRE）, resulting in enhanced sensitivity to abscisic acid （ABA） and tolerance to drought, and dark green leaves of mature plants, indicating that TERF1 participates in the integration of ethylene and osmotic responses. Here we further report that overexpression of TERF1 confers sugar response in tobacco. Analysis of the novel isolated tomato TERF1 promoter provides information indicating that there are many cis-acting elements, including sugar responsive elements （SURE） and W box, suggesting that TERF1 might be sugar inducible. This prediction is confirmed by results of reverse transcription-polymerase chain reaction amplification, indicating that transcripts of TERF1 are accumulated in tomato seedlings after application of glucose. Further investigation indicates that the expression of TERF1 in tobacco enhances sensitivity to glucose during seed germination, root and seedling development, showing a decrease of the fresh weight and root elongation under glucose treatment. Detailed investigations provide evidence that TERF1 interacts with the sugar responsive cis-acting element SURE and activates the expression of sugar response genes, establishing the transcriptional regulation of TERF1 in sugar response. Therefore, our results deepen our understanding of the glucose response mediated by the ERF protein TERF1 in tobacco.

Actin Dynamics Regulates Voltage-Dependent Calcium-Permeable Channels of the Vicia faba Guard Cell Plasma Membrane

Free cytosolic Ca^2＋ （[Ca^2＋]cyt） is an ubiquitous second messenger in plant cell signaling, and [Ca^2＋]cyt elevation is associated with Ca^2＋-permeable channels in the plasma membrane and endomembranes regulated by a wide range of stimuli. However, knowledge regarding Ca^2＋ channels and their regulation remains limited in planta. A type of voltage- dependent Ca^2＋-permeable channel was identified and characterized for the Vicia faba L. guard cell plasma membrane by using patch-clamp techniques. These channels are permeable to both Ba^2＋ and Ca^2＋, and their activities can be inhibited by micromolar Gd^3＋. The unitary conductance and the reversal potential of the channels depend on the Ca^2＋ or Ba^2＋ gradients across the plasma membrane. The inward whole-cell Ca^2＋ （Ba^2＋） current, as well as the unitary current amplitude and NPo of the single Ca^2＋ channel, increase along with the membrane hyperpolarization. Pharmacological experiments suggest that actin dynamics may serve as an upstream regulator of this type of calcium channel of the guard cell plasma membrane. Cytochalasin D, an actin polymerization blocker, activated the NPo of these channels at the single channel level and increased the current amplitude at the whole-cell level. But these channel activations and current increments could be restrained by pretreatment with an F-actin stabilizer, phalloidin. The potential physiological significance of this regulatory mechanism is also discussed.

A Putative Calcium-Permeable Cyclic Nucleotide-Gated Channel,CNGC18,Regulates Polarized Pollen Tube Growth

A tip-focused Ca^2＋ gradient is tightly coupled to polarized pollen tube growth, and tip-localized influxes of extracellular Ca^2＋ are required for this process. However the molecular identity and regulation of the potential Ca^2＋ channels remains elusive. The present study has implicated CNGC18 （cyclic nucleotide-gated channel 18） in polarized pollen tube growth, because its overexpression induced wider and shorter pollen tubes. Moreover, CNGC18 overexpression induced depolarization of pollen tube growth was suppressed by lower extracellular calcium （[Ca^2＋]ex）. CNGC18-yellow fluorescence protein （YFP） was preferentially localized to the apparent post-Golgi vesicles and the plasma membrane （PM） in the apex of pollen tubes. The PM localization was affected by tip-localized ROP1 signaling. Expression of wild type ROP1 or an active form of ROP1 enhanced CNGC18-YFP localization to the apical region of the PM, whereas expression of RopGAP1 （a ROP1 deactivator） blocked the PM localization. These results support a role for PM-Iocalized CNGC18 in the regulation of polarized pollen tube growth through its potential function in the modulation of calcium influxes.