Ultrastructural features of Mimulus aurantiacus ( Scrophulariaceae ) pollen tubes in vivo

The aim of this study is to give information on ultrastructure of in vivo pollen tubes of Mimulus aurantiacus which were collected from the Botanical Garden of the University of California at Berkeley. Materials were prepared according to electron microscopy methods and examined under Zeiss electron microscope. Four zones were examined in the pollen tubes of Mimulus aurantiacus. Apical zone: Mitochondria, smooth endoplasmic reticulum, rough endoplasmic reticulum, dictyosomes and secretory vesicles were observed. Subapical zone: This area contained abundant rough endoplasmic reticulum and occasionally some smooth endoplasmic reticulum. The polysomes, mitochondria, proplastids that contain starch, small vacuoles and a few lipid bodies were detected. Nuclear zone: Both generative and vegetative cell nuclei lie in this zone. The vegetative cell nucleus was large and long. Rough endoplasmic reticulum, mitochondria, ribosomes, dictyosomes, and amyloplasts that are rich of starch were observed. Vacuolation and plug formation zone: Cytoplasm of the tubes was full of large vacuoles. Few organelles such as mitochondria, dictyosome and rough endoplasmic reticulum were detected along their periphery.

Those studies were generally on the ultrastructure of in vitro pollen tubes except the studies with Prunus avium (Cresti et al. 1979), (Uwate andLin 1980).It is too hard to perform and to follow the development of in vivo pollen tubes, so these studies are very important and restricted.Therefore, the number of those studies should be increased.
Some electron microscope observations on pollen grains have been done in some members of subtribe Castilleiinae (Jensen et al. 1974) and in the genus Mimulus of subtribe Gratioleae Scrophulariaceae (Argue 1980).Olgun has done embryological researches on Digitalis sp.(Scrophulariaceae) with light microscope (Olgun 1979), (Yakar andOlgun 1984).She continued her studies with TEM such as ultrastructures of embryo sac (Olgun and Jensen 1987) and endothelium (Dane et al. 2007) in Penstemon gentianoides which belongs to the same family.This study can be regarded as the follow-up of those studies.
The ultrastructure of in vivo pollen tubes of Mimulus aurantiacus (Scrophulariaceae) which is naturally grown in North America, and possible similarities related to the ultrastructure of in vitro and in vivo pollen tubes in other angiosperms (Moscatelli and Cresti 2001) were emphasized in this study.
Ultrastructural studies were generally realized with the families Liliaceae, Solonaceae, Rosaceae and in recent years with some members of Asclepiadeceae, and Magnoliaceae.However, no studies have been found on ultrastuctural level with the pollen tubes of Scrophulariaceae family members neither in vivo nor in vitro.This will be the first ultrastructural study on in vivo pollen tubes of Mimulus aurantiacus and also in Scrophulariaceae family.

MATERIALS AND METHODS
Mimulus aurantiacus (Scrophulariaceae) flowers were collected from the Botanical Garden of the University of California at Berkeley.Gynoecia were removed from flowers and they were fixed in 4% glutaraldehyde in 0.1M cacodylate buffer pH 6.8, for 2 hours.Then, the pollinated pistils were washed several times in buffer, fixed overnight with 2% buffered OsO 4 ; dehydration was applied with gradually increasing aceton-propylenoxide series with staining in 70% acetone containing 1% uranyl nitrate overnight.The material was embedded in Spurr's medium.0.5-1 m thick semi-thin sections were stained with methylene blue (1%) using 0.02 M NaOH to control pH and observed by light microscopy.Thin sections were cut on a Porter-Blum ultramicrotome with a glass or diamond knife.Sections were stained on grids with lead citrate (Reynolds 1963) for one minute and observed by a Zeiss EM 9A electron microscope.

RESULTS
In this study, four zones were examined in vivo pollen tubes of Mimulus aurantiacus as similar in the previous studies.

APICAL ZONE
The cytoplasm of that zone was rich of organelles.Many electron dense dictyosomes and spherical mitochondria were visible.Small vacuoles, abundant rough endoplasmic reticulum (RER), occasionally some smooth endoplasmic reticulum (SER) and electron dense lipid bodies were also seen (Fig. 1a).A well-developed RER (Fig. 1b) and dictyosomes were observed.Many vesicles budded out from the active dictyosomes were seen, in various sizes around cis and trans regions of dictyosomes, in the apical zone (Fig. 1c).Cell wall and cell membrane were also clearly visible in the electron micrographs of apical zone (Figs.1a, b).

SUBAPICAL ZONE
Proplastids containing starch granules, small vacuoles and spherical shaped mitochondria can be seen in this zone like apical zone (Fig. 2a).This zone also contained a few electron dense lipid bodies and abundant RER (Figs. 2a, b).The ribosomes were frequently aggregated An Acad Bras Cienc (2009) 81 (1) as polysomes.Well-developed active dictyosomes and budding vesicles were also found in the subapical zone (Fig. 2c).

NUCLEAR ZONE
Vegetative and generative nuclei were located in this zone (Figs. 3,4a,b).The vegetative cell nucleus was large and long.It contained one nucleolus and several weakly-stained chromatin aggregations.It was rich of euchromatin material.Vacuoles in different sizes, mitochondria and dictyosomes were observed around vegetative nucleus (Figs.3a, b).There were 2, 3 amyloplasts containing starch grains and spherical shaped mitochondria around generative nucleus (Figs.4a, b).Electron dense lipid bodies were seen near the amyloplast full of starch grains (Fig. 4b).

VACUOLATION AND PLUG FORMATION ZONE
Cytoplasm of the tubes was full of large and old vacuoles (Fig. 5a).Mitochondria, few ribosomes, polysomes and dictyosomes were seen along their periphery (Figs.5a, b).There were also autophagic vacuoles exactly in the same size as others.They contained fibrillar materials (Fig. 5b).

DISCUSSION
The term "zonation" was introduced to describe the functional cytoplasmic distribution in the pollen tubes of Lycopersicum perivuanum (Cresti et al. 1977); in which four different areas were identified: (i) the apical zone, rich in SVs (Secretory Vesicles); (ii) the subapical area, containing organelles; (iii) the nuclear zone, comprising the MGU (Male Germ Unite); and (iv) the zone of vacuolization and callose plug formation (Moscatelli and Cresti 2001), (Bhojwani and Soh 2001).The presence of four zones was detected in the in vivo growing pollen tubes of Mimulus aurantiacus as reported by Cresti et al. (1977), Ciampolini et al. (1982) in their in vitro and by Cresti et al. (1979Cresti et al. ( , 1980)), Uwate and Lin's (1980) in vivo studies.In general, the constitution of organelles in various zones in vitro and in vivo are quite similar as also seen in Lycopersicum (Cresti et al. 1977), (Cresti et al. 1980), in Malus (Speranza et al. 1982), in Prunus avium (Cresti et al. 1979), in cherry (Ciampolini et al. 1982), and in Conospermum (Stone et al. 2004).
It has been hypothesized that the apical zonation -applicable to most Angiosperm pollen tubes -is maintained by cytoplasmic movements within the growing tube and by the presence of a "capture mechanism" An Acad Bras Cienc ( 2009) 81 (1)   2001).Apical and subapical zones are active zones because they are rich of organelles such as: mitochondria, well-developed dictyosomes and lipid bodies.We also observed areas which would be designated as smooth and rough endoplasmic reticulum zones (Uwate and Lin 1980).Our micrographs showed small vesicles arising from the well-developed dictyosome cisternae.Besides playing a major role in the carbohydrate metabolism, dictyosomes have functioned as sorting and dispatching station for the ER products (Dupree and Sherrier 1998).According to Uwate and Lin (1980), these vesicles have been arisen from smooth endoplasmic reticulum cisternae, and rough endoplasmic reticulum circumscription of vacuoles.Lipid bodies were visible in various zones in our study.This was also mentioned by Uwate and Lin (1980).
Since pollen tubes go into the stigma and stylus, they need glycoproteins and glycolipids to build pollen tube wall.These molecules are synthesized in these welldeveloped dictyosomes.Amiloplasts detected in vivo growing pollen tubes of Mimulus auriaticus are less than in vitro ones of Prunus avium (Ciampolini et al. 1982).
In vivo growing pollen tubes can absorb nutrients from the stylar tissue surrounding them.However, this may also occur in vitro studies due to high concentration of sucrose used in culture mediums.
Vacuolization zone exhibits autolysis or any other symptoms of degradation in pollen tubes of M. auraticus in vivo because this zone contains a lot of autophagic vacuoles in various sizes.Ciampolini et al. (1982) have specially mentioned the origin, ultrastructure and the role of secretory vesicles.These vesicles are generated by the dictyosomes, and seen in all zones of the pollen tubes.The older and larger vesicles are more visible in the vacuolization zone than in other zones.This is similar to the smaller vesicles in the apical zone due to participation in the pollen wall construction.Our in vivo electron micrographs of Mimulus aurantiacus pollen tubes also exhibit autophagic vacuoles.They have also been observed in pollen tubes of Lycopersicum peruvianum in vitro (Cresti et al. 1977).
According to Cresti and Van Went (1976), there are two ways of callose deposition in Petunia pollen tubes growing in the style.The first one is callose deposition outside the plasma membrane; the second one is callose deposition within the cytoplasm as distinct callose grains, leading to the formation of callose plugs.Both pollen tube elongation and organelle distribution must be highly coordinated, so that pollen tube growth makes the oldest part of the vegetative cytoplasm move forward, and it becomes isolated by callose plug formation.The mechanism of growth in pollen tubes as well as in other cell types, requires the integrity of the secretory system, namely ER and Golgi apparatus.The secretory system involves the secretion of protein and polysaccharide components.In this study, the elements of RER, SER and Golgi apparatus dispersed through the pollen tube of Mimulus aurantiacus.
In conclusion, the ultrastructure of zonation in pollen tubes of Mimulus aurantiacus in vivo had similarities with the ultrastructure of in vivo pollen tubes in Prunus avium (Cresti et al.1979, Uwate andLin 1980).In addition, it was seen that callose deposition occurred within the cytoplasm of pollen tubes in Mimulus aurantiacus.