This review considers pollen tube growth with regard to current information on pollen tube cytoplasm, wall structure and calcium ion interactions with pollen tubes. Pollen tubes have a marked cytoplasmic Polarity with a number of distinct zones along the tube, each with a characteristic complement of cytoplasmic and nuclear structures. The cytoplasmic structures are characteristic of secretory cells with extensive endoplasmic reticulum cisternae and numerous dictyosomes. The dictyosomes produce secretory vesicles that are mainly directed to the extending tip of the tube, where they provide new plasma membrane and wall components. The rates of secretory vesicle production and delivery have been estimated, allowing quantitative assessments of the rate of delivery of materials to the tip. Pollen tubes contain cytoskeletal components, with microtubules and microfilament strands lying axially in the main tube and diffuse microfilament strands at the tip. The tube wall consists of an outer fibrous layer containing pectins and an inner, more homogeneous layer containing callose and cellulose-like microfibrils, possessing both β-1,4 and β-1,3 linkages. Protein is also present in the wall. The tube tip lacks the inner callosic wall. This type of structure is considered to be different from that of elongating sporophyte tissue cells which are enclosed by a wall containing layers of cellulose microfibrils. Calcium ions are required for pollen tube growth and, in at least some species, act as a chemotropic agent. High concentrations of calcium ions in the external medium inhibit growth. Pollen tubes contain some calcium ions bound to the cell wall and larger amounts located intracellularly, which enter the tube at the tip. This intracellular calcium is present as ions that exist freely within the cytoplasmic Matrix and as ions bound to membrane systems. The highest concentrations in both of these pools are found at the tip and in both they decline towards the base. The structure of the tip and the activity involved in providing components for plasma membrane and Wall assembly provide a basis for considering possible mechanisms of tip growth. Two hypotheses to account for the regulation of tip extension are considered, cell wall control and cytoskeletal control. In the cell wall hypothesis, control depends on an interaction between internal turgor pressure and a plastic cell wall. The mechanical properties of the wall are assumed to be partly dependent on the availability of external calcium ions to crosslink acidic pectin chains. According to this hypothesis, high external calcium ion concentrations cause cessation of tip growth due to increased mechanical resistance of the tip wall. Various observations on plant cell-wall interactions with calcium ions and on experimentally-treated pollen tubes provide evidence that does not support this hypothesis. The cytoskeletal control hypothesis of tip growth depends on the internal tip cytoskeleton to contain the tube tip cytoplasm against the internal turgor pressure during cell wall assembly. The activities and mechanical properties of the cytoskeleton are assumed to depend on the availability of external calcium ions. High external concentrations are believed to cause a state of rigor in the cytoskeleton and hence a cessation of tip growth. Some experimental evidence is presented which suggests that the effects of excess calcium ions are on intracellular processes, and not extracellular ones. The mitochondrial zone behind the tip is believed to maintain the tip calcium ion concentration at an optimal level for growth. Some comparisons are made between tip growth in pollen tubes and that in other tip growing cells. CONTENTS Summary 323 I. Introduction 324 II. Cytoplasm 326 III. Wall structure 332 IV. Calcium 335 V. Tip growth 339 VI. Conclusions 350 References 351.

Pollen tube tip growth.

Visual examination of roots from 12,000 mutagenized Arabidopsis seedlings has led to the identification of more than 40 mutants impaired in root hair morphogenesis. Mutants from four phenotypic classes have been characterized in detail, and genetic tests show that these result from single nuclear recessive mutations in four different genes designated RHD1, RHD2, RHD3, and RHD4. The phenotypic analysis of the mutants and homozygous double mutants has led to a proposed model for root hair development and the stages at which the genes are normally required. The RHD1 gene product appears to be necessary for proper initiation of root hairs, whereas the RHD2, RHD3, and RHD4 gene products are required for normal hair elongation. These results demonstrate that root hair development in Arabidopsis is amenable to genetic dissection and should prove to be a useful model system to study the molecular mechanisms governing cell differentiation in plants.

Genetic Control of Root Hair Development in Arabidopsis thaliana.

The polarized growth of cells as diverse as fungal hyphae, pollen tubes, algal rhizoids and root hairs is characterized by a highly localized regulation of cell expansion confined to the growing tip. In apically growing plant cells, a tip-focused [Ca2+]c gradient and the cytoskeleton have been associated with growth. Although actin has been established to be essential for the maintenance of elongation, the role of microtubules remains unclear. To address whether the microtubule cytoskeleton is involved in root hair growth and orientation, we applied microtubule antagonists to root hairs of Arabidopsis. In this report, we show that depolymerizing or stabilizing the microtubule cytoskeleton of these apically growing root hairs led to a loss of directionality of growth and the formation of multiple, independent growth points in a single root hair. Each growing point contained a tip-focused gradient of [Ca2+]c. Experimental generation of a new [Ca2+]c gradient in root hairs pre-treated with microtubule antagonists, using the caged-calcium ionophore Br-A23187, was capable of inducing the formation of a new growth point at the site of elevated calcium influx. These data indicate a role for microtubules in regulating the directionality and stability of apical growth in root hairs. In addition, these results suggest that the action of the microtubules may be mediated through interactions with the cellular machinery that maintains the [Ca2+]c gradient at the tip.

Microtubules regulate tip growth and orientation in root hairs of Arabidopsis thaliana

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The first descriptions of sex chromosomes in plants, of the continuity of chromosomes during the mitotic cycle and of non-Mendelian inheritance as well as the introduction of UV-mutagenesis to genetic research are landmarks in biological sciences first achieved by scientists working on bryophytes. Haploidy of the tissue facilitates mutant isolation and many developmental moss mutants have been isolated. Early moss development is triggered by auxin, by cytokinin and by light, mainly acting via phytochrome and a blue-light receptor. Due to the simplicity of the plants, development can be pinpointed to the differentiation of a single cell and be analysed in living tissue, making mosses ideal candidates for the analysis of development in an integrated approach of cell and molecular biology. Molecular genetic techniques have been applied mainly to Physcomitrella patens (Hedw.) B.S.G., where efficient protocols for transformation of nuclear DNA have been established and several nuclear, chloroplast and mitochondrial genes have been analysed. These studies reveal that Physcomitrella may be an appropriate model to study plant development in molecular terms. Recently, it has been shown that, in this species, nuclear genes can be targeted very efficiently by homologous recombination, now opening the door to reverse genetics for plant biologists.

Development, Genetics and Molecular Biology of Mosses

Publisher Summary This chapter discusses the cell wall synthesis in apical hyphal growth. It appears that the hyphal apex is best viewed as a highly polarized system of exocytosis. Wall materials, extracellular enzymes, and probably other substances are excreted at the growing end of a tubular cell. The most obvious cellular features that accompany this polarized system are (1) the unidirectional flow of vesicles in the cytoplasm fusing with the plasma membrane at the apex, (2) the gradients in wall synthesis at the apex, and (3) the cytoplasmic gradients in ion distribution that are maintained at the apex. New microscopic techniques reveal a cytoskeletal organization of the cytoplasm at the apex, which may be crucial to its polarized activity. Growth of the wall at the hyphal apex requires that the wall in this region has plastic properties, which contrast with the requirement of rigidity elsewhere in the hypha. A widely held view involves the participation of wall-lytic enzymes in plasticizing the wall at the apex and in allowing new wall material to be inserted. A critical evaluation of the evidence presented to support this view makes this hypothesis less attractive. As an alternative a steady-state model is discussed based on recent observations in the author's laboratory. This model holds that the assemblage of polymers synthesized at the apex is inherently plastic. However, this assemblage develops rigidity by interactions, in the wall, between and among the various individual polymers present while the wall segment moves in subapical directions during elongation. This model seems to fit many of the original observations made on living hyphae.

Cell-wall synthesis in apical hyphal growth

In the caulonema tip cells of Funaria hygrometrica, chloroplasts, mitochondria, and dictyosomes have differences in structure which are determined by cell polarity. In contrast to the slowly growing chloronema tip cells the apical cell of the caulonema contains a tip body. Colchicine stops tip growth; it causes the formation of subapical cell protrusions, redistribution of the plastids, and a loss of their polar differentiation. Cytochalasin B inhibits growth and affects the position of cell organelles. After treatment with ionophore A23 187, growth is slower and shorter and wider cells are formed. D2O causes a transient reversion of organelle distribution but premitotic nuclei are not dislocated. In some tip cells the reversion of polarity persists; they continue to grow with a new tip at their base. During centrifugation, colchicine has only a slight influence on the stability of organelle anchorage. The former polar organization of most cells is restored within a few hours after centrifugation, and the cells resume normal growth. In premitotic cells the nucleus and other organelles cannot be retransported, they often continue to grow with reversed polarity. Colchicine retards the redistribution of organelles generally and increases the number of cells that form a basal outgrowth. The interrelationship between the peripheral cytoplasm and the nucleus and the role of microtubules in maintaining and reestablishing cell polarity are discussed.

G. Schmiedel

Papers

Polarity and growth of caulonema tip cells of the moss Funaria hygrometrica.