SUMMARYManual evaluation of cellular structures is a popular approach in cell biological studies. However, such approaches are laborious and are prone to error, especially when large quantities of image data need to be analyzed. Here, we introduce an image analysis framework that overcomes these limitations by semiautomatic quantification and clustering of cytoskeletal structures. In our framework, cytoskeletal orientation, bundling and density are quantified by measurement of newly-developed, robust metric parameters from microscopic images. Thereafter, the microscopic images are classified without supervision by clustering based on the metric patterns. Clustering allows us to collectively investigate the large number of cytoskeletal structure images without laborious inspection. Application of this framework to images of GFP-actin binding domain 2 (GFP-ABD2)-labeled actin cytoskeletons in Arabidopsis guard cells determined that microfilaments (MFs) are radially oriented and transiently bundled in the process of diurnal stomatal opening. The framework also revealed that the expression of mouse talin GFP-ABD (GFP-mTn) continuously induced MF bundling and suppressed the diurnal patterns of stomatal opening, suggesting that changes in the level of MF bundling are crucial for promoting stomatal opening. These results clearly demonstrate the utility of our image analysis framework.

To examine the cross talk between the abscisic acid (ABA) and ethylene signal transduction pathways, signaling events during ABA-induced stomatal closure were examined in Arabidopsis (Arabidopsis thaliana) wild-type plants, in an ethyleneoverproducing mutant (eto1-1), and in two ethylene-insensitive mutants (etr1-1 and ein3-1). Using isolated epidermal peels, stomata of wild-type plants were found to close within a few minutes in response to ABA, whereas stomata of the eto1-1 mutant showed a similar but less sensitive ABA response. In addition, ABA-induced stomatal closure could be inhibited by application of ethylene or the ethylene precursor 1-aminocyclopropane-1-carboxylic acid (ACC). In contrast, stomata of the etr1-1 and ein3-1 mutants were able to close in response to concomitant ABA and ACC application, although to a lesser extent than in wild-type plants. Moreover, expression of the ABA-induced gene RAB18 was reduced following ACC application. These results indicate that ethylene delays stomatal closure by inhibiting the ABA signaling pathway. The same inhibitive effects of ethylene on stomatal closure were observed in ABA-irrigated plants and the plants in drought condition. Furthermore, upon drought stress, the rate of transpiration was greater in eto1-1 and wild-type plants exposed to ethylene than in untreated wild-type control plants, indicating that the inhibitive effects of ethylene on ABA-induced stomatal closure were also observed in planta.

Pteridophytes have a longer evolutionary history than any other vascular land plant and, therefore, have endured greater loss of phylogenetically informative information. This factor has resulted in substantial disagreements in evaluating characters and, thus, controversy in establishing a stable classification. To compare competing casicatis, we obtained DNA sequences of a chloroplast gene. The sequence of 1206 nt of the large subunit of the ribulose-bisphosphate carboxylase gene (rbcL) was determined from 58 species, representing almost all families of leptosporangiate ferns. Phylogenetic trees were inferred by the neighbor-joining and the parsimony methods. The two methods produced almost identical phylogenetic trees that provided insights concerning major general evolutionary trends in the leptosporangiate ferns. Interesting findings were as follows: (i) two morphologically distinct heterosporous water ferns, Marula and Salvinia, are sister genera; (iu) the tree ferns (Cyatheaceae, Dicksoniaceae, and Metaxyaceae) are monophyletic; and (ui) polypodiolds are distantly related to the gleicheniolds in spite of the imilarit of their exindusiate soral morphology and are close to the higher indusiate ferns In addition, the affiities of several "problematic genera" were assessed.The extant ferns include -10,000 species and 250 genera in the world (1). They are the most conspicuous spore-bearing land plants and the principal members of land flora after the flowering plants. Ferns Based on morphology, the leptosporangiate ferns are usually classified into three major groups, Marsileaceae, Salviniaceae including Azollaceae, and the rest, which are often treated as different orders (5-7). Although both the Marsileaceae and the Salviniaceae have distinctive morphologies, both live in aquatic habitats and are characterized by heterospory, the latter being extremely rare in the leptosporangiate ferns. The phylogenetic relationship ofthese aquatic ferns to the homosporus ferns remains unsolved (8).Classification and phylogenetic relationships of the leptosporangiate ferns above the family level are controversial (1, 5,6,[8][9][10]. The reasons for discrepancy among classification schemes include disagreements in evaluation of morphological characters used. It is often difficult to identify homologous characters because similar characters are found in apparently different phylogenetic lineages; convergent or parallel evolution probably often occurred during the long evolutionary history of ferns (11). Furthermore, frequent extinctions produced missing links, which have resulted in difficulties elucidating phylogenetic interrelationships of major groups (1, 11). Micromolecular information (12) also is not useful to infer familial relationships for the same reasons.Recently molecular systematics in plants has progressed rapidly with in vitro DNA amplification (polymerase chain reaction, PCR) mediated by thermostable DNA polymerase and the direct sequencing methods. In angiosperm systematics, this molecular approach...

SummaryThe actin cytoskeleton of higher plants plays an essential role in plant morphogenesis and in maintaining various cellular activities. In this study we established a tobacco BY-2 cell line, stably transformed with a GFPfimbrin actin-binding domain (ABD) 2 construct, that allows visualization of actin microfilaments (MFs) in living cells. Using this cell line, designated BY-GF11, we performed time-sequential observations of MF dynamics during cell-cycle progression. Detailed analyses revealed the appearance of a broad MF band in the late G 2 phase that separated to form a structure corresponding to the so-called actin-depleted zone (ADZ) in mitosis. In BY-GF11, the MF structure at the cell cortex in mitosis appeared to form two bands rather than the ADZ. Measurements of fluorescent intensities of the cell cortex indicated an MF distribution that resembled two peaks, and we therefore named the structure MF 'twin peaks' (MFTP). The cell plate formed exactly within the valley between the MFTP at cytokinesis, and this cell-plate guidance was distorted by disruption of the MFTP by an inhibitor of actin polymerization. These results suggest that the MFTP originates from the broad MF band in the G 2 phase and functions as a marker of cytokinesis.