**Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism**

Jose C. Jimenez-Lopez, Sonia Morales, Dieter Volkmann, Juan D. Alché and María I. Rodriguez-Garcia

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46170

#### **1. Introduction**

70 Current Insights in Pollen Allergens

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> Profilin was first identified in plants as a birch allergen (Valenta et al. 1991). Plants have several genes encoding highly divergent profilin isoforms (Kovar et al. 2000; Kandasamy et al. 2002), differentially expressed, and with biochemical and functional diversity (Huang et al. 1996), particularly physiological roles in actin-based processes. Profilins are divided in two classes: one is ubiquitously present, and constitutively expressed in all plant tissues (vegetative), whereas the second class is restricted to the reproductive tissues (Kandasamy et al. 2002). At biochemical level, plant profilins are placed into two distinct classes: Class I profilins bind to phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] much stronger than class II profilins, whereas class II have stronger affinity for actin and PLP (Gibbon et al 1998; Kovar et al 2000).

> The complexity of profilin expression and the number of isoforms in higher plants is correlated with the observation that the actin family is also more complex in plants than in other kingdoms (McDowell et al. 1996). Structurally, Overall look to plant profilins indicates that they are similar to these from yeast and vertebrate, though the identity of primary amino acid sequence is only about 30% (Fedorov et al. 1997; Thorn et al. 1997), which implicate profilins in key conserved functions throughout different kingdoms. However, the *in vitro* biochemical data suggested that different profilin isoform functions distinctly (Kovar et al. 2000), which supports an isovariant dynamics model where particular isoforms have differential functions/activities. Supporting this idea, it has been proposed that plant profilin family multi-functionality might be inferred by natural variation through profilin isovariants

© 2012 Jimenez-Lopez et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Jimenez-Lopez et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

generated among germplasm, as a result of polymorphism. The high variability might result in both differential profilin properties and differences in the regulation of the interaction with natural partners, suggesting that isovariant dynamics may expand the responses of the actin cytoskeleton or buffer it to against stress (Jimenez-Lopez 2008, Jimenez-Lopez et al. 2012).

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 73

Other different profilin localizations have been described, like the chloroplast outer membrane, which interacts with CHUP1 protein during chloroplast movement in response to light (Schmidt von Braun and Schleiff 2008), and in amyloplasts (Fischer et al. 1996), in addition to a preferential localization in association with the plasma membrane. Moreover, a differential expression of different profilin isoforms has been reported in microspores and maize pollen (von Witsch et al. 1998). Profilin isoforms PRF1 and PRF2 were localized differentially in *Arabidopsis* epidermal cell (Wang et al. 2009), which emphasizes the existence of differentially regulated and localized profilin isoforms, as well as the necessity

In the present study, we have used two experimental approaches: immune-reactivity in blotting experiments, and immunogold experiments for profilin cellular localization at TEM, with the aim of analyzing the differential immune-reactivity of profilins as result of their

Olive (*Olea europaea* L.) pollen was individually collected during May and June from olive trees of 24 cultivars, grown in the olive germplasm collection of the Estación Experimental del Zaidín, CSIC, Granada, Spain. Pollen samples were collected in large paper bags by vigorously shaking the inflorescences sequentially sieved through 150 and 50 μm mesh

Mature seeds from Acebuche (wild olive) and Picual cultivars were obtained from the same collection of well-characterized olive trees growing in the "Estación Experimental del

Olive mature pollen from the Picual cultivar was *in vitro* germinated. Pre-hydration was performed by incubation in a humid chamber at 30°C for 30 min. The grains (0.02 g/plate) were then transferred to Petri dishes containing 10mL of the germination medium [10% (w/v) sucrose, 0.03% (w/v) Ca(NO3)2, 0.01% (w/v) KNO3, 0.02% (w/v) MgSO4 and 0.03% (w/v) boric acid], as described by M'rani-Alaoui (2000). The Petri dishes were maintained at 25°C in the dark, and the pollen samples were taken at 5 min, 1, 4, 7, and 18h after the onset of the culture, pollen tube growth was monitoring by light microscopy. Finally, the pollen

Mature pollen or germinated samples were re-suspended in an extraction buffer (PBS), pH 7.4 [140 mM NaCl, 2.7 mM KCl, 8.15 mM Na2HPO4 and 1.8 mM KH2PO4] added with 10

to determine the localization of each profilin isoform individually and carefully.

high sequence polymorphism, which also drives profilins the subcellular locations.

**2. Material and methods** 

**2.2. In vitro pollen germination** 

**2.3. Protein extraction** 

filters to eliminate debris and maintained at -80°C.

was pelleted by centrifugation (1000 × g for 20 s).

Zaidín" (Granada, Spain), 210 days after anthesis (DAA).

**2.1. Plant material** 

Profilin is a major regulator of actin dynamics and is crucial for cellular growth, morphogenesis and cytokinesis (Jockusch et al. 2007). In addition to binding to actin, profilins bind to other partners like stretch of poly-proline (PLP) and proline-rich proteins, and phospholipids. The proline-binding ability could be a major function, being different among profilin isoforms, affecting actin-based structures (Kovar et al. 2001). The importance of the binding of profilin to PLP is supported by the finding of the differential preference for profilin isoforms of formin (essential actin-binding and nucleator protein) (Neidt et al. 2009), together with the evidence that *Arabidopsis* formins have preference for different profilin isoforms (Deeks et al. 2005).

Another binding ligand of profilin is phospholipids. The binding of profilin to phospholipids links to its potential role in vesicle trafficking (Janssen and Schleicher 2001). Profilins have been revealed as key mediators of the membrane–cytoskeleton communication, acting at critical points of signaling pathways initiated by events in the plasma membrane and transmitted by transduction cascades to promote cytoskeletal rearrangements (Baluska et al. 2002). This functionality arises from their capacity of interaction with phosphatidylinositides (PIP2), as well as with poly-L-proline-rich proteins (Kovar et al. 2001).

Several locations have been attributed to profilin. They have been localized in different plant cells and tissues, including the microspores, pollen grains and pollen tubes (Grote et al. 1993, 1995; Hess et al. 1995; Fischer et al. 1996; Vidali & Hepler 1997; Kandasamy 2002). Plant profilin was reported to be localized in the cytoplasm of pollen tube uniformly (Hess et al. 1995; Vidali & Hepler 1997). However, no clear picture has yet been established about the precise location of profilin in the pollen tubes. In consideration of the existence of calcium gradient in pollen tube and the regulation of profilin's sequestering activity by calcium (Kovar et al. 2000), the existence of a gradient of total sequestering activity of profilin in the pollen tube is expected.

Upon pollen hydration and pollen germination, profilin was detected close to the site of pollen tube emergence, forming a ring-like structure around the apertural region. Profilin was also detected in the pollen exine of the germinating pollen grains and in the germination medium. Profilin was also localized in the cytoplasm of the pollen tube, particularly at both the proximal and apical ends (Morales et al. 2008).

Depending on the fixation and extraction protocol used, nuclear localization has been also observed (Buss et al. 1992). Profilin has also been found in generative and vegetative nuclei of *Ledebouria socialis* pollen (Hess and Valenta 1997), the nuclei of *Phaseolus vulgaris* cells (Vidali et al. 1995) and *Arabidopsis thaliana* and maize root hairs (Braun et al. 1999, Baluska et al. 2001). Microinjection of a fluorescently labeled birch profilin in *Micrasterias denticulata*  also shows an accumulation of profilin in the nucleus (Holzinger et al. 1997).

Other different profilin localizations have been described, like the chloroplast outer membrane, which interacts with CHUP1 protein during chloroplast movement in response to light (Schmidt von Braun and Schleiff 2008), and in amyloplasts (Fischer et al. 1996), in addition to a preferential localization in association with the plasma membrane. Moreover, a differential expression of different profilin isoforms has been reported in microspores and maize pollen (von Witsch et al. 1998). Profilin isoforms PRF1 and PRF2 were localized differentially in *Arabidopsis* epidermal cell (Wang et al. 2009), which emphasizes the existence of differentially regulated and localized profilin isoforms, as well as the necessity to determine the localization of each profilin isoform individually and carefully.

In the present study, we have used two experimental approaches: immune-reactivity in blotting experiments, and immunogold experiments for profilin cellular localization at TEM, with the aim of analyzing the differential immune-reactivity of profilins as result of their high sequence polymorphism, which also drives profilins the subcellular locations.
