Interpolation of microtubules into cortical arrays during cell elongation and differentiation in roots of Azolla pinnata

Longitudinal sections of roots of Azolla pinnata R. Br. were prepared for electron microscopy so that cortical microtubules could be counted along the longitudinal walls in cell files in the endodermis, pericycle, and inner and outer cortex, and in sieve and xylem elements. With the exception of the xylem, where there are no transverse cell divisions, each file of cells commences with its initial cell and then possesses a zone of concomitant cell expansion and transverse cell division, followed, after completion of the divisions, by a zone of terminal cell differentiation. The cells augment their population of cortical microtubules as they elongate and divide, showing a net increase of up to 0.6 micron of polymerized microtubule length per min. Two main sub-processes were found: (i) When a longitudinal wall is first formed it is supplied with a higher number of microtubules per unit length of wall than it will have later, when it is being expanded. This initial quota becomes diluted as the second sub-process commences. (ii) The cells interpolate new microtubules at a rate which is characteristic of the cell, and, in the endodermis, of the face of the cell, while the cell elongates. Most cell types thus maintain a set density of cortical microtubules while they elongate and divide. Comparisons of endodermal cells in untreated controls, and roots that had been treated with colchicine, low temperature, or high pressure indicate that the initial quota of microtubules, and the later interpolations, and differentially sensitive to microtuble perturbations. Three types of behaviour, all related to changes in the cell walls, were noted as cortex, xylem and sieve element cells entered their respective phases of cell differentiation. The cortical cells expanded in all dimensions, and the interpolation of microtubules diminished or ceased. The sieve elements continued to elongate, and interpolated at a high rate, reaching unusually high densities of microtubules when the cell walls were being thickened. During this period a net increase of 2.0 micron of polymerized microtubule length per min was calculated. Thereafter interpolation ceased and the density of microtubules declined. The sample applied to developing xylem except that, because wall-thickening is localized rather than widespread, the rise and subsequent fall in the density of microtubules was less marked. The data are discussed in relation to the participation of microtubules in wall deposition and to the hypothesis that cortical microtubules arise in discrete zones along the edges of cells.</jats:p

Structure and development of cortical microtubule arrays in plant cells

Microtubules in the plant cell cortex are usually aligned parallel to microfibrils of cellulose that are being deposited in the cell wall, and are considered to function in guiding or orienting cellulose synthetase complexes that lie in or on the plasma membrane. The cellulose component is largely responsible for the mechanical reaction of the wall to turgor forces, thereby determining cell size and shape, and therefore the role of the cortical microtubules is a fundamental part of the overall morphogenetic process in plants. It is important to determine the structure of cortical arrays of microtubules and to learn how the cell regulates their development, neither of these aspects having been investigated adequately since the original description likened the microtubules to “hundreds of hoops around the cell”.</jats:p

Isolation and characterization of four genes encoding pyruvate, phosphate dikinase in the oomycete plant pathogen Phytophthora cinnamomi

The oomycete genus Phytophthora contains some of the world's most devastating plant pathogens. We report here the existence in P. cinnamomi of four genes encoding the pyrophosphate-utilizing glycolytic/gluconeogenic enzyme pyruvate, phosphate dikinase (PPDK). The coding regions of the four genes are >99% identical. At least three of the genes comprise a small gene cluster, which may have arisen through recent gene duplication and inversion events. Levels of Pdk mRNA are low in vegetative hyphae, but increase rapidly and transiently upon transfer of cultures to nutrient-free media, conditions that trigger asexual sporulation. PPDK protein and enzyme activity levels do not show a similar increase during sporulation. Assays of PPDK activity in P. cinnamomi hyphal extracts suggest that the majority of glycolytic flux in sporulating hyphae probably occurs via PPDK, rather than pyruvate kinase. This finding, combined with the existence of Phytophthora-expressed sequence tags encoding two other pyrophosphate-utilizing enzymes, indicates that pyrophosphate-based metabolism may be important in Phytophthora. The possibility that PPDK and other enzymes of pyrophosphate-based metabolism may provide targets for the development of novel control measures for Phytophthora and other oomycete pathogens is discussed

Ancient origin of elicitin gene clusters in Phytophthora genomes

The genus Phytophthora belongs to the oomycetes in the eukaryotic stramenopile lineage and is comprised of over 65 species that are all destructive plant pathogens on a wide range of dicotyledons. Phytophthora produces elicitins (ELIs), a group of extracellular elicitor proteins that cause a hypersensitive response in tobacco. Database mining revealed several new classes of elicitin-like (ELL) sequences with diverse elicitin domains in Phytophthora infestans, Phytophthora sojae, Phytophthora brassicae, and Phytophthora ramorum. ELIs and ELLs were shown to be unique to Phytophthora and Pythium species. They are ubiquitous among Phytophthora species and belong to one of the most highly conserved and complex protein families in the Phytophthora genus. Phylogeny construction with elicitin domains derived from 156 ELIs and ELLs showed that most of the diversified family members existed prior to divergence of Phytophthora species from a common ancestor. Analysis to discriminate diversifying and purifying selection showed that all 17 ELI and ELL clades are under purifying selection. Within highly similar ELI groups there was no evidence for positively selected amino acids suggesting that purifying selection contributes to the continued existence of this diverse protein family. Characteristic cysteine spacing patterns were found for each phylogenetic clade. Except for the canonical clade ELI-1, ELIs and ELLs possess C-terminal domains of variable length, many of which have a high threonine, serine, or proline content suggesting an association with the cell wall. In addition, some ELIs and ELLs have a predicted glycosylphosphatidylinositol site suggesting anchoring of the C-terminal domain to the cell membrane. The eli and ell genes belonging to different clades are clustered in the genomes. Overall, eli and ell genes are expressed at different levels and in different life cycle stages but those sharing the same phylogenetic clade appear to have similar expression pattern

Immunocytochemical comparison of peripheral vesicles in zoospores of Phytophthora and Pythium species

An ultrastructural and immunocytochemical comparison has been made of vesicles in the peripheral cytoplasm of zoospores of Phytophthora cinnamomi and species of the related Oomycete genus, Pythium. Our results give evidence of three morphologically and immunologically distinct vesicle populations in Pythium aphanidermatum and Py. butleri. Large peripheral vesicles can be recognized by their size and morphology, and by labeling with monoclonal antibody, Cpa-2 raised against a P cinnamomi antigen. They occur predominantly on the dorsal surface of the zoospores and are retained within the cell during encystment. After encystment, the large peripheral vesicles move away from the plasma membrane and become distributed throughout the cyst cytoplasm, in a manner similar to that observed in P. cinnamomi. There are also small vesicles in the Pythium zoospore cortex. Some are identified as ventral vesicles through their reaction with monoclonal antibody Vsv-1, raised against a P. cinnamomi antigen. The ventral vesicles are concentrated along the ridges of the groove on the ventral surface of the zoospores, and their contents are secreted during encystment. The secreted material remains localized on the cyst surface and marks the site of germ tube emergence, as it does in P. cinnamomi. Other small peripheral vesicles occur on the dorsal surface of the Pythium zoospores. These vesicles are not labeled by any of the three antibodies tested but disappear during encystment, suggesting that their contents are secreted. Our results give strong support to the idea that three types of peripheral vesicles are a common feature of zoospores throughout the Peronosporales, and that they have similar fates during encystment