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= Pollen Tube =

Overview
In order for successful fertilization to occur, there is rapid tip growth in pollen tubes which delivers the male gametes into the ovules. A pollen tube consists of three different regions: the apex which is the growth region, the subapex which is the transition region, and the shank which acts like normal plant cells with the specific organelles. The apex region is where tip growth occurs and requires the fusion of secretory vesicles. There is mostly pectin and homogalacturonans (part of the cell wall at the pollen tube tip) inside these vesicles. The homogalacturonans accumulate in the apex region via exocytosis in order to loosen the cell wall. A thicker and softer tip wall with a lower stress yield will form and this allows cell expansion to occur, which leads to an increase in tip growth. Reverse-fountain cytoplasmic streaming occurs during the tip growth which is essential for the cellular expansion, because it is transporting organelles and vesicles between the shank region and subapex region.

The actin cytoskeleton is an important factor in pollen tube growth, because there are different patterns of actin cytoskeleton within the different regions of the pollen tube for the maintenance of polarized cell growth. For instance, there are longitudinal actin cables in the shank region in order to regulate reverse-fountain cytoplasmic streaming. The F-actin controls the accumulation of the homogalacturonans full vesicles- essentially mediating tip growth- in the subapex region. The actin filaments controls the apical membrane and cytoplasm interactions while the pollen tube is growing in the apex region. The F-actin from the apical membrane makes an actin binding protein called formin which is essential for pollen tube tip growth. Formins are expressed in the tip growth cells and are divided into two subgroups: type I and type II. The type I formins make the actin structures and partake in cytokinesis. The type II formins on the other hand contribute to the growth of polarized cells which is necessary for tip growth. Tip growth is a form of extreme polarized growth and this polarized process requires actin-binding protein-mediated organization of actin cytoskeleton. An essential protein required for this tip growth is the actin-organizing protein and type II formin protein called Rice Morphology Determinant (RMD). RMD is localized in the tip of the pollen tube and controls pollen tube growth by regulating the polarity and organization of F-actin array.

RMD Promotes Pollen Tube Growth
RMD promotes pollen germination and pollen tube growth, and this is proven through numerous experiments. The first experiment compares the features of the pistil and the stigma of rmd-1 mutant (rice plant without a functional RMD) and the wild-type rice plant (with a functional RMD). The anther and pistil were shorter in the rmd-1 mutants than the wild-type. This experiment showed that RMD is critical for pollen development. Wild-type rice plants have increased germination rates while rmd-1 mutants have decreased germination rates. This was seen when both were germinated in a liquid germination medium. After the germination rates were tested, there was a comparison of the lengths and widths of the pollen tubes between the two plants. The pollen tubes of the wild-type plants had a greater pollen tube length than the mutants, but the mutants had a greater tube width. This greater pollen tube width within the mutants indicates the decrease in the growth of polarized cells and thus decrease in tip growth. Next, pollen grains from the wild type and mutants were collected to compare the pollination activities between the wild types and mutants. There was decreased activity and minimal penetration within the mutants whereas an increased activity and penetration through the style and to the bottom of the pistils within the wild types. These observations indicated the delayed pollen tube growth in the rmd-1 mutants. Additionally, there was no effect on fertilization rates between the wild type and the mutant and this was tested by measuring the seed-setting rates between the wild type and mutant. It was found that both had similar seed-setting rates. Therefore, RMD does not affect fertilization and has an effect only on tip growth.

RMD Expression in the Pollen Tube
Total RNA extractions from the whole flower, lemma, palea, lodicule, pistil, anther, and mature pollen grains of the wild type plants took place in order to discover where RMD is specifically expressed in the plant as a whole. Using RT-qPCR (reverse transcription quantitative PCR), it was evident that there were different amounts of RMD transcripts within each part of the plant. And then it was evident where RMD was present in each part of the plant using RT-PCR (reverse transcription PCR) and using UBIQUITIN as a control. These two methods demonstrated that there was an abundant presence of the RMD transcripts in the lemma, pistil, anther, and mature pollen grains. In order to confirm these results, another method was performed. This method used transgenic plants that had an RMD promoter region fused with a reporter gene encoding GUS. Histochemical staining of the tissues of these transgenic plants then showed high GUS activity within the pistil, anther wall, and mature pollen grains. Therefore, these combined results demonstrated that RMD is expressed in these specific organs of the plant.

Detection of GUS signals were employed once again in order to study where RMD is specifically expressed within the pollen tube. First, pollen grains were collected from proRMD::GUS trangenic plants, and it was noted that there was a strong GUS signal within these mature pollen grains. These pollen grains were then germinated in vitro and GUS signals were observed within the tip growth of the pollen tubes. However, the strength of these GUS signals varied at different germination stages. The GUS signals were weak within the pollen tube tip at the early germination stage, but stronger at the later germination stages. Therefore, these results support that RMD is involved in pollen germination and pollen tube growth.

RMD Localization in the Pollen Tube
RMD, which are type II formins, consist of a phosphatase, (PTEN)-like domain (responsible for protein localization), and FH1 and FH2 domains (promotes actin polymerization). In order to discover the localization of RMD in the pollen tube, transient assays of growing pollen tubes of tobacco was performed and the fluorescent protein-GFP was used. Many confocal images of various pollen tubes under specific conditions were observed: pLat52::eGFP (single eGFP driven by the pollen specific Lat52 promoter and this acts as a control); pLat52::RMD-eGFP (RMD protein fused with eGFP); pLat52::PTEN-eGFP (the PTEN domain fused with eGFP); and pLat52::FH1FH2-eGFP (the FH1 and FH2 domains fused with eGFP). By comparing the images of the control with pLat52::RMD-eGFP, it is observed that the single GFP was spread throughout the entire tube whereas RMD-eGFP accumulated in the tip region of the tube. Therefore, this shows that RMD is localized within the tip of the pollen tube.

In order to discover whether the PTEN-like domain is responsible for the localization of RMD, there was a comparison between the confocal images of GFP fused with PTEN domain and shortened RMD without the PTEN domain (pLat52::FH1FH2-eGFP). The PTEN-eGFP signals were localized in the tip of the pollen tubes like the RMD-eGFP signals, whereas the FH1FH2-eGFP signals were present throughout the pollen tube and not localized in a polar manner. Therefore, these combined results demonstrate that the PTEN-like domain is responsible for the tip localization of RMD in the pollen tubes.

RMD Controls F-Actin Distribution and Polarity in the Pollen Tube
In order to determine if RMD controls F-actin organization within the pollen tube, F-actin arrays in wild type and rmd-1 mature pollen grains were observed using Alexa Fluor 488-phalloidin staining. Strongly bundled actin filaments were present around the apertures of the wild type pollen grains although there was no accumulation of actin filaments around the apertures in the rmd-1 pollen grains. Additionally, there were weak signals and random organization of the actin filaments within the rmd-1 pollen grain. Therefore, these results support that RMD is essential for controlling pollen germination.

Fluorescent intensity was measured using statistical analysis in order to observe the actin filament densities within the pollen tubes. There was greater fluorescence intensity in the shank region of the rmd-mutant tubes which means there was a higher density of F-actin within this region. But, there was a lower density of F-actin observed in the tip region of the rmd-mutant tubes compared to the wild type tubes. This demonstrates that the F-actin distribution pattern of pollen tubes is altered without a functional RMD.

In order to determine the polarity of the actin cables, the angles between the actin cables and elongation axis of the pollen tube were measured. The angles in the shank region of the wild type pollen tubes were predominantly less than 20° whereas the angles for the rmd-mutant pollen tubes were greater than 60°. These results support the fact that RMD is essential for polarized tip growth, because the rmd-mutant pollen tubes (without a functional RMD) exhibited an increased width, and thus a decrease in tip growth. The maximum length of the single cables of F-actin filaments from the apical to the shank region of elongating pollen tubes were also measured to test the polarity within the pollen tube. The maximum length of the F-actin cables were shorter in the rmd-mutant pollen tubes compared to those in the wild type tubes. Therefore, these combined results support that the proper organization of actin cables as well as normal F-actin densities within the tip of the tube can only be achieved if RMD is present.