The Arabidopsis LFR gene is required for the formation of anther cell layers and normal expression of key regulatory genes.

The anther is the male reproductive organ in flowering plants. Although some genes were reported to be involved in anther development, the molecular mechanisms underlying the transcriptional regulation of these genes is unclear. lfr-2 (leaf and flower related-2), the null allele of Arabidopsis thaliana LFR (LEAF AND FLOWER RELATED), was male-sterile. The anthers of lfr-2 plants were defective in sporogenous cell formation, tapetum development, and pollen development. In agreement with these phenotypes, expression studies showed that LFR was expressed in all cell layers of the anther, and that expression was particularly strong in the tapetal cells and pollen grains. Quantitative RT-PCR analysis revealed that LFR is required for the normal transcription of some anther development-related genes, such as AMS, CALS5, and DYT1, MS1 and MS2, and ROXY2. Genetic analysis showed that SPL was epistatic to LFR while LFR was epistatic to DYT1. We propose that LFR may be a crucial component in the regulation of a genetic network that modulates anther development.

Characterization of a cDNA coding for an extracellular calmodulin-binding protein from suspension-cultured cells of Angelica dahurica.

In order to characterize a specific extracellular 21-kDa calmodulin-binding protein (named: ECBP21) from Angelica dahurica L. suspension-cultured cells, the cDNA coding for the protein has been cloned. Here, Southern blot analysis shows that there are at least two copies of ECBP21 gene in Angelica genome. Using truncated versions of ECBP21 and synthetic peptide in CaM binding assays, we mapped the calmodulin-binding domain to a 16-amino acid stretch (residues 200-215) at the C-terminal region. The ECBP21 was localized in the cell wall area by the immunogold electron microscopy and by GFP labeling method. These results define ECBP21 as a kind of an extracellular calmodulin-binding protein (CaMBP). Furthermore, using Northern blot analysis, we examined the expression dynamics of ecbp21 during the incubation of Angelica suspension-cultured cells and the treatments with some growth regulators. The above studies further provide the molecular evidence for the existence of the gene coding for extracellular CaMBPs and imply a possible role for ECBP21.

Ca2+ influx into lily pollen grains through a hyperpolarization-activated Ca2+-permeable channel which can be regulated by extracellular CaM.

Confocal laser scanning microscopy (CLSM) and whole-cell patch-clamp were used to investigate the role of Ca2+ influx in maintaining the cytosolic Ca2+ concentration ([Ca2+]c) and the features of the Ca2+ influx pathway in germinating pollen grains of Lilium davidii D. [Ca2+]c decreased when Ca2+ influx was inhibited by EGTA or Ca2+ channel blockers. A hyperpolarization-activated Ca2+-permeable channel, which can be suppressed by trivalent cations, verapamil, nifedipine or diltiazem, was identified on the plasma membrane of pollen protoplasts with whole-cell patch-clamp recording. Calmodulin (CaM) antiserum and W7-agarose, both of which are cell-impermeable CaM antagonists, lead to a [Ca2+]c decrease, while exogenous purified CaM triggers a transient increase of [Ca2+]c and also remarkably activated the hyperpolarization-activated Ca2+ conductance on plasma membrane of pollen protoplasts in a dose-dependent manner. Both the increase of [Ca2+]c and the activation of Ca2+ conductance which were induced by exogenous CaM were inhibited by EGTA or Ca2+ channel blockers. This primary evidence showed the presence of a voltage-dependent Ca2+-permeable channel, whose activity may be regulated by extracellular CaM, in pollen cells.