Imaging and Analysis of the Content of Callose, Pectin, and Cellulose in the Cell Wall of Arabidopsis Pollen Tubes Grown In Vitro.

To achieve fertilization, pollen tubes have to protect and properly deliver sperm cells through the pistil to the ovules. Pollen tube growth is a representative example of polarized growth where new components of the cell wall and plasma membrane are continuously deposited at the tip of the growing cell. The integrity of the cell wall is of fundamental importance to maintain apical growth. For this reason, pollen tube growth has become an excellent model to study the role of polysaccharides and structural cell wall proteins involved in polar cell expansion. However, quantification of structural polysaccharides at the pollen tube cell wall has been challenging due to technical complexity and the difficulty of finding specific dyes. Here, we propose simple methods for imaging and quantification of callose, pectin , and cellulose using specific dyes such as Aniline Blue, Propidium Iodide, and Pontamine Fast Scarlet 4B.

Structure of the style and pollen tube pathway in the Ziziphoid and Rhamnoid clades of Rhamnaceae.

The ultrastructure of the style and pollen tube pathway before, during and after anthesis were studied in 13 species belonging to the tribes Pomaderreae, Paliureae, Colletieae and Gouanieae (Ziziphoid clade) and Rhamneae (Rhamnoid clade) using light microscopy and transmission electron microscopy. The aim of this study is to provide new morphological characters useful for phylogenetic analysis at suprageneric level in Rhamnaceae. The patterns of pollen tube growth and the ultrastructural changes undergone by cells of the style were also described. Species of Rhamneae (Scutia buxifolia and Condalia buxifolia) have a solid style, with the transmitting tissue forming three independent strands (S. buxifolia) or a central, single horseshoe-shaped strand as seen in transversal section (C. buxifolia) which could derive from the fusion of formerly independent strands. In contrast, Pomaderreae, Gouanieae and Paliureae showed semi-solid styles, while in Colletieae, as previously reported, the style is hollow with two or three stylar canals. The style anatomy and the ultrastructure of the pollen tube pathway show that there is a tendency towards a solid style with a single strand of transmitting tissue within the family. The three-canalled hollow style could be the plesiomorphic state of the character "type of style" in the family, the semi-solid style the synapomorphic state and the solid style with three strands of transmitting tissue the apomorphic state, with the solid style with a single strand of transmitting tissue as the most derived state. Therefore, Colletieae would be the most basal tribe of the Ziziphoid clade.

ROS Regulation of Polar Growth in Plant Cells.

Root hair cells and pollen tubes, like fungal hyphae, possess a typical tip or polar cell expansion with growth limited to the apical dome. Cell expansion needs to be carefully regulated to produce a correct shape and size. Polar cell growth is sustained by oscillatory feedback loops comprising three main components that together play an important role regulating this process. One of the main components are reactive oxygen species (ROS) that, together with calcium ions (Ca(2+)) and pH, sustain polar growth over time. Apoplastic ROS homeostasis controlled by NADPH oxidases as well as by secreted type III peroxidases has a great impact on cell wall properties during cell expansion. Polar growth needs to balance a focused secretion of new materials in an extending but still rigid cell wall in order to contain turgor pressure. In this review, we discuss the gaps in our understanding of how ROS impact on the oscillatory Ca(2+) and pH signatures that, coordinately, allow root hair cells and pollen tubes to expand in a controlled manner to several hundred times their original size toward specific signals.

Ultrastructure of the stigma and style of Cabomba caroliniana Gray (Cabombaceae).

Cabomba Aubl. is a genus that presents a range of features that have made it to be considered a potential genetic model for studies of early angiosperm evolution. Therefore, any study that expands our knowledge of this genus is potentially useful for the understanding of the evolution of early angiosperms. This paper reports the study of the anatomy and the ultrastructure of the stigma and the style of Cabomba caroliniana Gray during the 2 days of anthesis using bright-field microscope, fluorescence microscope and transmission electron microscope. The stigma is dry and has pluricellular papillae. The style is hollow with a central canal coated by an epithelium. The papillae have fewer organelles than those typical of glandular cells, and they are covered by a cuticle that is broken when pollen germinates. The ultrastructure of epithelial cells indicates that the cells lining the canal are secretory. The canal is filled with a fibrillar and granular substance. The pollen tubes grow inside the canal through this substance. The results are discussed in the context of what is known for other species of angiosperms.

Using hyper as a molecular probe to visualize hydrogen peroxide in living plant cells: a method with virtually unlimited potential in plant biology.

Reactive oxygen species (ROS) are highly reactive reduced oxygen molecules that play a myriad of roles in animal and plant cells. In plant cells, the production of ROS occurs as a result of aerobic metabolism during respiration and photosynthesis. Therefore mitochondria, chloroplasts, and peroxisomes constitute an important source of ROS. However, they can be produced in response to many physiological stimuli such as pathogen attack, hormone signaling, abiotic stresses, or during cell wall organization and plant morphogenesis. Monitoring ROS in plant cells has been limited to biochemical assays and use of fluorescent probes, however, the irreversible oxidation of the fluorescent dyes make it impossible to visualize dynamic changes of ROS. Hyper is a recently developed live cell probe for H2O2 and consists of a circularly permutated YFP (cpYFP) inserted into the regulatory domain of the Escherichia coli hydrogen peroxide (H2O2) sensor OxyR rendering it a H2O2 specific ratiometric, and therefore quantitative probe. Herein, we describe a protocol for using Hyper as a dynamic probe for H2O2 in Arabidopsis with virtually unlimited potential to detect H2O2 throughout the plant and under a broad range of developmental and environmental conditions.

Histochemical map of the ectopic expression of the Arabidopsis atExt1 extensin gene in transgenic tobacco.

Histochemical analysis of transgenic tobacco plants harboring the promoter of the Arabidopsis extensin gene atExt1 fused to the ß-glucuronidase gene coding sequence demonstrated expression of the transgene in stem tissues. The transgene was expressed in cells of the internal and external phloem at the nodal and internodal regions of the older parts of mature stems. In younger parts of the same stem, expression of the transgene was slightly modified: expression was detected in the internal phloem in the internodal areas. In the nodal areas, the phloem tissue and the surrounding parenchyma were stained, demonstrating transgene expression. Expression was also seen in the phloem of the inflorescence; it was non-specific at the junctions of the flowers to the inflorescence. ß-glucuronidase (reporter gene) staining was also observed in the pollen grains and at the base of the corolla.

Effects of low-energy argon ion implantation on the dynamic organization of the actin cytoskeleton during maize pollen germination.

The relationship between pollen germination and the dynamic organization of the actin cytoskeleton during pollen germination is a central theme in plant reproductive biology research. Maize (Zea mays) pollen grains were implanted with 30 keV argon ion (Ar(+)) beams at doses ranging from 0.78 x 10(15) to 13 x 10(15) ions/cm(2). The effects of low-energy ion implantation on pollen germination viability and the dynamic organization of the actin cytoskeleton during pollen germination were studied using confocal laser scanning microscopy. Maize pollen germination rate increased remarkably with Ar(+) dose, in the range from 3.9 x 10(15) to 6.5 x 10(15) ions/cm(2); the germination rate peaked at an Ar(+) dose of 5.2 x 10(15) ions/cm(2). When the implantation dose exceeded 7.8 x 10(15) ions/cm(2), the rate of pollen germination decreased sharply. The actin filaments assembled in pollen grains implanted with 5.2 x 10(15) ions/cm(2) Ar(+) much earlier than in controls. The actin filaments organized as longer parallel bundles and extended into the emerging pollen tube in treated pollen grains, while they formed random and loose fine bundles and were gathered at the pollen aperture in the control. The reorganization of actin cytoskeleton in the pollen implanted with 9.1 x 10(15) ions/cm(2) Ar(+) was slower than in controls. There was a positive correlation between pollen germination and the dynamic organization of the actin cytoskeleton during pollen germination. Ion implantation into pollen did not cause changes in the polarization of actin filaments and organelle dynamics in the pollen tubes. The effects of Ar(+) implantation on pollen germination could be mediated by changes in the polymerization and rearrangement of actin polymers.