Comparative analysis of ultrastructural and metabolic changes in the transmitting tissue of  Ananas comosus (L.) Merr. after various pollinations.

doi:10.21203/rs.3.rs-3334095/v1

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Comparative analysis of ultrastructural and metabolic changes in the transmitting tissue of Ananas comosus (L.) Merr. after various pollinations.

https://doi.org/10.21203/rs.3.rs-3334095/v1

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The study of enzyme activity during pollen development can provide an idea of the role of different enzymes in pollen tube growth and elucidate certain basic patterns and mechanisms of plant growth.The tissue-specific peroxidases in the style transmitting tissue may play a direct role in regulating pollen tube growth. Coordination between pollen and pistil is essential for successful pollination in flowering plants. A.comosus (pineapple) in Bromeliaceae prevents self-fertilization rates due to gametophytic self-incompatibility. The stigma of two commercial cultivars of Ananas was pollinated with its pollen and also pollen from other cultivar. Cross-pollinated stigma showed higher peroxidase activity compared to self-pollinated and open pollinated pistil of the flower. In peroxidase localization reddish color appeared on the stigma of cross-pollinated pistil.TEM analysis showed ultrastructural and metabolic changes in the transmitting cells of styles in Ananas after various pollinations. The cells in the transmitting tissue of Ananas is rich in cytoplasm and numerous large rough endoplasmic reticulum, large vacuole, golgi vesicles and prominent nucleus in cross-pollinated pistil.

Ananas comosus

Peroxidase

TEM

Transmitting tisue

Pollination

Peroxidases are members of a broad family of isoenzymes in almost all living organisms. They are generally heme-containing enzymes (O’Brien 2000). Peroxidases catalyze the breakdown of hydrogen peroxide to yield highly oxidizing intermediates, which oxidize a variety of organic and inorganic reducing substrates. Peroxidase enzyme uses hydrogen peroxide as an electron acceptor to catalyze substrates and is widely present in plants and animal cells. Most plant peroxidases are members of a superfamily of class III peroxidases. They are enzymes that play various physiological and biochemical roles in higher plant cells, making them exciting objects for current biochemical related research (Has-Schon et al. 2005; Pandey et al.2017). Plant peroxidases are involved in a wide variety of physiological processes, including lignin production (Gross 2008), wound healing (Kumar et al. 2007), fruit ripening (Huang et al. 2007), auxin metabolism and disease resistance (Veitch 2004). Plant peroxidases are often present in flowers ( Lopez-Molina et al. 2003) and leaves (Maciel et al. 2007).

Pandey (1967) identified peroxidase as a significant enzyme component of angiosperm stigmas, and there have been many speculations about the function of the peroxidase enzyme in the stigma of a flower. The biological role of stigmatic peroxidases has yet to be. The Peroxidase occurrence in the stylar part of the pistil has been reported in only a few plant species (Brewbaker 1971; Bredemeijer and Blaas 1975). An extensive study of pistil peroxidase has been carried out in Nicotiana alata about incompatibility genotype, localization, development, and senescence. According to Pandey 1967, the peroxidase isoenzyme composition of style of Nicotiana alata was assumed to be related to the self-incompatible (S) genotype. The activity of peroxidase has been detected in stigma extracts or stigma diffusates of several species, but differences exist (Martin 1968; Nasrallah et al. 1970; Bredemeijer and Blaas 1975). Peroxidase is a defense-induced enzyme ( Cheong 2002; Delannoy 2003; Do et al. 2003; McInnis et al. 2006). Pollen grains may land on the stigma,which produces signals that activate peroxidase as a defense strategy. The activity of this enzyme is modified by germinating pollen grains (Bredemeijer 1984). The activity of several enzymes examined in different species, such as peroxidase, esterase, alcohol dehydrogenase and acid phosphatase, were used to characterize stigma receptivity (Heslop-Harrison and Shivanna 1977;Shivanna and Sastri 1981;McInnis et al 2005; Serrano and Olmedilla 2012). Tissue-specific peroxidases in the stylar transmitting tissue may be essential in regulating growth of pollen tube. At maturity, when the stigmas are prepared for pollination, they show high levels of peroxidase activity (Dupuis and Dumas 1990; Dafni and Motte Maues 1998). During the Pollination,the pistil might be able to actively recognize and inhibit self-pollen, either on the stigma surface in sporophytic self-incompatibility (SSI) or during the growth of the pollen tube through the pistil in gametophytic self-incompatibility (GSI) before reaching the ovule.

The interactions between the pollen grain and the various tissues of the pistil, including pollen adhesion,pollen germination through style and fertilization are crucial for the success of sexual reproduction in angiosperms. The pollen-pistil interaction is thus a fundamental process in the reproductive biology of the flowering plants and has been the subject of intense research for many decades (Hiscock and Allen 2008).In recent years there has been much progress in identifying molecules that mediate specific events during the pollen - pistil interaction, such as pollen adhesion and hydration, pollen tube growth and navigation through the pistil, and self-incompatibility in particular (Edlund et al. 2004; Swanson et al. 2004; Takayama and Isogai 2005; Hiscock and Allen 2008)

The pathway for compatible pollen tube growth is defined by the specialized transmitting tissue in the pistil,which produces a dense extracellular matrix (ECM) through which pollen tubes extend. While most commonly located in style, the transmitting tissue can be prominent in other parts of the pistil. In angiosperms, pollen-pistil interaction leads to fertilization and seed set. The proteins and secretary fluids present on the stigma play a crucial role in pollen germination, pollen tube growth,and successful fertilization( Heslop-Harrison 1975). The present study was conducted on Ananas comosus (Pineapple), an economically important fruit plant belonging to Bromeliaceae. Ananas comosus produces functional germ cells but is self-sterile through gametophytic incompatibility (Brewbaker and Gorrez 1967), and fruit development is parthenocarpic (py et al. 1987). Ananas comosus var comosus shows strong self-incompatibility than in other botanical varieties. Self-incompatibility occurs due to the inhibition of the growth of the pollen tube in the upper third of the style (Kerns 1932; Majumdar et al. 1964). The present study aimed to understand the presence and distribution of peroxidase in the pollinated and unpollinated pistils of Ananas comosus var comosus and the ultrastructural changes in the transmitting tissues after self,open and cross-pollinations. There is no report, however, on the cell organelles in the transmitting tissue cells of the pistil in Ananas comosus var comosus. In this study a comparative analysis was made on the changes in stigma and styles of Ananas after open ,self and cross-pollination.

Plant material

The plant material selected for the present study includes two commercial cultivars of A. comosus var. comosus (Mauritius and Kew). Ananas comosus cv.Mauritius and A.comosus cv. Kew are maintained in the Botanical garden, Department of Botany, University of Kerala, Thiruvananthapuram,Kerala.

Pollination experiments

For the pollination experiments, self-pollination of A.comosus cv Mauritius and cross-pollination with A. comosus cv Kew were done, where cultivar Mauritius is the female parent. For the experiments, 25 flowers were selected for each pollination. In both the cultivars,anther dehiscence occurred at 5:30 − 6:30 a.m and stigma become receptive from 6:00 to 7:00 a.m. Self and cross-pollination carred out separately in different inflorescence at 6:30 am, and the whole inflorescence was covered with butter paper bag. Since A.comosus cv Mauritius flowers persist for one day,stigma for in vivo germination was collected at 2:00p.m,for each pollination experiments 5 flowers were collected for in vivo studies, and remaining flowers after cross-pollination were retained for setting seeds.

In vivo germination studies using Cotton Blue

Pistils collected after 8 hrs of self, open, and cross-pollinations were fixed in Carnoy’s fluid. Fixed pistils were washed and then transferred to lactophenol solution supplemented with few drops of cotton blue stain, and incubated at 60°C for 15–20 mins. in a hot air oven. Stained pistils were mounted in glycerin on a microslide and a cover glass was placed over the specimen and pressed gently. The microslides were observed under a light microscope.The number of pollen grains deposited on the stigma surface was measured using image analyzer software(LEICA DM 2000) and estimated the percentage of pollen germination.

Localization of Peroxidase

Peroxidase was localized on the stigma surface according to Graham and Karnovesky (1966). It was carried out by localized by immersing excised pistils after pollination in a solution containing 0.1 M guaiacol, 0.1 M H₂O₂ in 20 mM phosphate buffer pH 4.5 until an orange/red color was observed (approximately 1–3 min).

Peroxidase Assay:- The peroxidase activity was analyzed according to Malik and Singh (1980). The assay mixture contained 2.5 ml of phosphate buffer (pH 6.5, 0.1 M), 0.2 ml diluted enzyme extract and 0.1 ml o-dianisidine (1 mg/ml methanol). It was extracted the stigma and upper portion of the style. It was incubated at 28°C for 2 min.in a water bath. To begin the reaction, 0.2 ml of H₂O₂ was added. The change in absorbance was recorded at 430 nm using a stopwatch at 30 s interval for 5 min. The enzyme activity was expressed in terms of the rate of increase of absorbance per hour per mg protein.

Transmission Electron Microscopic analysis

The various steps in TEM are

Sample Processing

Tissue samples harvested were grossed into 1 mm³ size and fixed in 3% glutaraldehyde in phosphate buffer for over 24 hours at 4^oC. Diluted 25% glutaraldehyde (Polysciences Inc., USA) stock to 3% using Sorenson’s phosphate buffer (0.1 M, pH 7.4).

Fixation

Glutaraldehyde was discarded, samples were washed with cold phosphate buffer (pH 7.4), and remain sample vials standing in an ice bath. Samples were placed in 1% OsO₄ for 2 hours; OsO₄ acts as a fixative cum primary stain and washed with phosphate buffer.

Dehydration

The samples were dehydrated in ascending grades of acetone.

Infiltration

Samples were placed in propylene oxide for five minutes each, then transferred into propylene oxide-epoxy resin mixtures of ratio 3:1, 1:1, and finally kept overnight in propylene: epoxy resin mixture of the ratio of 1:3. Final infiltration was carried out in pure resin prior to embedding.

Embedding

The samples were then embedded in molds containing the epoxy resin - Polybed 812 mixed with dodecenyl succinic anhydride (DDSA – Hardener), Nadic methyl anhydride (NMA -Hardener), Dimethylaminomethyl phenol (DMP - Accelerator) in appropriate ratios as per the kit instructions (Polysciences Inc., USA) and polymerized at 60°C in an oven for three days.

Sectioning

Light microscopy semithin sections (~ 1µm) were cut using a glass knife in an ultramicrotome (Leica ultracut UCT). Once the area of interest was identified, the remaining areas of the block were trimmed off. Ultrathin sections (50–70 nm) were cut using a diamond knife (Diatome®) and collected onto the shiny side of the copper grids of 300 mesh size.

Samples were then viewed in TEM (Hitachi H 7650) at 75-80KV.

Statistical analysis

Statistical analysis were performed using one way Anova software.

In the present study, in vivo pollen germination was conducted to understand the pollen germination and the number of pollen grains deposited on the surface of stigma. After cross-pollination, it was observed that a higher percentage of pollen germination was observed on the stigmaand pollen tubes started penetrated through style, whereas pollen grains deposited on the stigmatic surface after various pollinations (Table 1).This indicates the self incompatibility nature of the pistil in Ananas. Pollen tube growth through the tissues of the pistil was quantitatively and qualitatively related to the developmental state of the pistil and pollen. In peroxidase localization, a reddish-brown color appears on the surface of the cross-pollinated stigmaNo color change was observed in the open-pollinated pistil (Fig. 1).Localization of peroxidase showed the presence of peroxidase enzyme in cross-pollinated styles compared to self-pollinated ones, indicating its involvement in regulating pollen tube growth through the styles (Table 2). Peroxidase has been implicated as an indicator of stigma receptivity (Galen and Plowright 1987; Dupuis and Dumas 1990; Dafni and MotteMaues 1998 ; Stpiczynska 2003), and adherence of pollen grains to stigma has been shown to increase peroxidase activity independent of the penetration of pollen tube (Linskens 1969; Bredemeyer and Blaas 1975, Bredemeyer 1975; Bredemeyer 1979 ; Bredemeijer 1982; Bredemeijer 1984;Galen and Plowright 1987). Peroxidase, esterase, and acid-phosphatase activities have already been found on the surface of receptive stigmas of many species( Shivanna and Sastri 1981; Dupuis and Dumas 1990;Kandasamy et al. 1993;Dafni and Maués 1998; Seymour and Blaylock 2000 ;M. Stpiczynska 2003). Moreover, their presence on papillar cells may be due to their active secretory function. In the present study, significantly higher peroxidase activity was noticed in cross-pollinated pistils of Ananas compared to self-pollinated and unpollinated pistils in both the varieties studied. According to McInnis et al (2006),Stigma-specific peroxidase( SSP) in Senecio squalidus is expressed in the specialized secretory cells (papillae) of the stigma epidermis and is developmentally regulated. The expression of stigma-specific peroxidase is undetectable in small flower buds but increases during flower development and reaches a maximum when stigmas are most receptive to pollen. Bredemeijer (1974) reported that peroxidase activity in the compatible style was less compared to the incompatible one. Peroxidase activity increases with the age of pistils and reaches a maximum at the stage of stigma receptivity.

Table 1

*in vivo* pollen germination using cotton blue
Sl.No	Type of Pistil	Number of pollen grains deposited on stigma	Percentage of pollen germination
1	Open -pollinated pistil	64.4 ± 0.12	42.1 ± 0.16
2	Self -pollinated pistil	75.16 ± 0.16	56.5 ± 0.12
3	Cross-pollinated pistil	74.23 ± 0.12	60.2 ± 0.12

Table 2

Peroxidase Assay
SL.No	Type of Pistil	Peroxidase Activity (Units/min/gFW)
1	Open -pollinated pistil	0.73 ± 0.003
2	Self -pollinated pistil	0.160.003
3	Cross-pollinated pistil	1.31 ± 0.157
	Df = 2 F-value	64.63***

To understand the nature of pistils in Ananas after artificial self-pollination,open pollination and cross-pollination,Transmission electron microscopy of pistils were done. Styles range in degrees of closure from open or hollow styles, which contain few to no cells in the center portion, to closed or solid styles filled with densely packed cells (Gotelli et al. 2017).

TEM analysis of style cross section shows that transmitting tissue of cross-pollinated pistil of Ananas was characterized by spherical cells with large vacuoles and intercellular spaces. Intercellular spaces of various shapes appear among the cells. The style epidermis of open- pollinated pistil is characterized by a thick cuticle. The transmitting tissue of open-pollinated pistils showed intercellular spaces(Fig. 2A-B). Towards the epidermis,the cells contain fewer cell organelles and a large number of vacuole (Fig. 2C-D). Nucleus,mitochondria,golgi bodies and rough endoplasmic reticulum are seen after various pollinations. In the transmitting tissue of self and open-pollinated pistils,sections of pollen tubes are characterized by a dense cytoplasm with a large number of vacuoles. In the C.S of the style of cross pollinated pistil transmitting tissue is composed of round shaped cells with a central vacuole. The Nucleus, rough endoplasmic reticulum is seen prominently(Fig. 2E-F). The stylar transmitting tissue is composed of highly secretory cells characterized by the production of an extensive extracellular matrix (ECM) (Sassen 1974; Lennon et al. 1998).

The cells of the transmitting tissue that line the path of pollen tubes are metabolically active secretory cells to provide nutrients for promoting pollen tube growth. (Herrero and Hormaza 1996). In the stigma of Grevillea banksii, the peripheral cytoplasm contains mitochondria, dictyosomes, and associated vesicles starch-containing plastids and Rough endoplasmic reticulum in both vesiculate form and as narrow cisternal strands. (Herscovitch and Martin 1990). Lobed nuclei, plastid clusters, rough and smooth endoplasmic reticulum, microbodies, dictyosomes, mitochondria, multivesicular bodies and lipids in the stigma secretion and intercellular substance were all present. The stylar canal of Polygala vayredae is surrounded by metabolically active cells and lipid rich mucilage (Castro et al. 2008). In Cabomba caroliniana,the ultrastructure of the glandular epidermal cells of open type style shows abundant mitochondria, plastids, Rough endoplasmic reticulum, and dictyosomes (Galati et al. 2016). Ismailoglu and Unal (2012) described the ultrastructure of the open style of Ornithogalum sigmoideum. They considered that the presence of abundant endoplasmic reticulum, dictyosomes, mitochondria, plastids, and ribosomes would indicate the secretory function of these cells. Transmitting tissue in Nicotiana tabacum is chlorophyllous, and its cells contain numerous mitochondria, dictyosomes, RER,amyloplasts, ribosomes, as well as crystal-containing microbodies and myelin-like formations (Bell and Hicks 1976). In cross-pollinated pistils of Ananas, the Endoplasmic reticuam is prominently seen compared to open and self-pollinated pistils, inticating that the proteins produced in style and stigma help pollen tube growth and fertilization. Further detailed studies are necessary to understand the inhibiting factors present in self and open-pollinated pistils in Ananas.

The present study shows that the changes occur in the stigma-style tissues affected by a self-incompatibility mechanism that affects the metabolic status of these tissues. The open pollinated and self pollinated pistils showed pollen tube inhibition on the surface of stigma where as pollen tube growth was observed after cross pollination. A factor related to self incompatibility,presence of peroxidase also analysed in self,open pollinated and cross pollinated pistils and found more in cross pollinated pistils.In order to study the ultrastructural changes in the stylar tissues. TEM analysis was conducted in pollinated pistils and compared.The study concluded that in cross pollinated pistils cells are metabolically active to support the growing pollen tubes,it is confirmed in localization of peroxidase and TEM analysis. There were more ultrastructural studies of the pistil at different developmental stages are necessary to know the way of pollen tube germination and the factors in the pistil responsible for growth.

Author’s Contribution

VV data analyzed and wrote the manuscript. VV and PM designed the study. PM supported and guided the entire work. VV done the pollination experiments and acquired the Light microscopic images in image analyzer software(LEICA DM 2000).

Acknowledgements

We thank Science and Engineering Research Board ,New Delhi for financial Support.(Empowerment and Equity opportunities for Excellence in Science, SERB Scheme project).We thank Prof. Dr. E.A. Siril,.Head, Department of Botany, Prof.Dr. T.S Swapna, Former Head, Department of Botany for providing facilities to do the work.

We thank Susan Mani, TEM, Sree Chitra Tirunal Institute for Medical Sciences and Technology, Trivandrum for technical support and acquired images of TEM.

Conflict of interest

The authors declare no conflict of interest within this work

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Comparative analysis of ultrastructural and metabolic changes in the transmitting tissue of Ananas comosus (L.) Merr. after various pollinations.

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Abstract

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Introduction

Materials and Methods

Plant material

Pollination experiments

Localization of Peroxidase

Statistical analysis

Results and Discussions

Conclusions

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References

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