**3.2. Profilin expression and characterization of the differential reactivity of birch, hazel, timothy-grass and maize profilins**

SDS-PAGE analysis of protein extracts of mature pollen from *Betula pendula*, *Corylus avellana, Phleum pratense* and *Zea mays* displayed distinctive protein profiles in the figure 3. Defined protein bands corresponding to profilin are clearly distinguishable in the interval of 14- 17kDa, which is the expected size for profilin isoforms.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 79

**Figure 4.** Immuno-reactivity analysis of crude protein extract from pollen of 5 plant species. Reactivity of proteins from 5 plant species were assayed against different maize profilin antisera, A) anti-PRA, B) anti-ZmPRO3, C) anti-ZmPRO5, D) anti-ZmPRO4, E) as well as against olive profilin antiserum anti-Ole e 2. It was appreciable up to 2 reactive bands about 13.7 and 14.2 kDa, corresponding to different isoforms of profilins for the species analyzed. The intensity of the reactive bands was quantitated by a densitometric analysis: pink color plot corresponded to 13.7kDa bands, and blue to 14.2 kDa. Red asterisks highlighted the differential reactivity (very high or very low) of defined species to different antisera, whereas blue asterisks highlighted the differential reactivity among cultivar to defined

antiserum.

**E**

**A**

**B**

**C**

**D**

**Figure 3.** Protein profile of crude protein extract from pollen of 5 individual plant species. 30μg of total protein was loaded in each line.

Profilin immunodetection was performed by using the same antisera described above for olive. In this case, different antisera are able to distinguish up to two reactive bands, with molecular weights of 13.7 and 14.2 kDa (Figure 4).

Clear differences can be appreciated when compared protein extracts reactivity of different species to individual antisera, as well as the reactivity of an individual protein extract to the different antisera assayed.

Cross-immune reactivity analysis between protein extracts from the five species with different antisera made against profilins from olive and maize pollen showed large differences both qualitative (intensity of bands) and quantitative (number of reactive bands) concerning profilins of MW around 13.7 and 14.2 kDa (Figure 4).

Statistical analysis of densitometric quantitations was performed. The variance analysis for the different antisera against different protein extracts showed significant differences among the 5 species (F-ratio= 8.13, p<0.05).

The reactivity values were inside of a normal distribution (test de Shapiro-Wilk: 0.95, p>0.05), while the Levene test indicated non-homogeneity of variances (Levene: 2.86, p<0.05).

protein was loaded in each line.

different antisera assayed.

the 5 species (F-ratio= 8.13, p<0.05).

molecular weights of 13.7 and 14.2 kDa (Figure 4).

concerning profilins of MW around 13.7 and 14.2 kDa (Figure 4).

**birch, hazel, timothy-grass and maize profilins**

17kDa, which is the expected size for profilin isoforms.

**3.2. Profilin expression and characterization of the differential reactivity of** 

SDS-PAGE analysis of protein extracts of mature pollen from *Betula pendula*, *Corylus avellana, Phleum pratense* and *Zea mays* displayed distinctive protein profiles in the figure 3. Defined protein bands corresponding to profilin are clearly distinguishable in the interval of 14-

**Figure 3.** Protein profile of crude protein extract from pollen of 5 individual plant species. 30μg of total

Profilin immunodetection was performed by using the same antisera described above for olive. In this case, different antisera are able to distinguish up to two reactive bands, with

Clear differences can be appreciated when compared protein extracts reactivity of different species to individual antisera, as well as the reactivity of an individual protein extract to the

Cross-immune reactivity analysis between protein extracts from the five species with different antisera made against profilins from olive and maize pollen showed large differences both qualitative (intensity of bands) and quantitative (number of reactive bands)

Statistical analysis of densitometric quantitations was performed. The variance analysis for the different antisera against different protein extracts showed significant differences among

The reactivity values were inside of a normal distribution (test de Shapiro-Wilk: 0.95, p>0.05),

while the Levene test indicated non-homogeneity of variances (Levene: 2.86, p<0.05).

**Figure 4.** Immuno-reactivity analysis of crude protein extract from pollen of 5 plant species. Reactivity of proteins from 5 plant species were assayed against different maize profilin antisera, A) anti-PRA, B) anti-ZmPRO3, C) anti-ZmPRO5, D) anti-ZmPRO4, E) as well as against olive profilin antiserum anti-Ole e 2. It was appreciable up to 2 reactive bands about 13.7 and 14.2 kDa, corresponding to different isoforms of profilins for the species analyzed. The intensity of the reactive bands was quantitated by a densitometric analysis: pink color plot corresponded to 13.7kDa bands, and blue to 14.2 kDa. Red asterisks highlighted the differential reactivity (very high or very low) of defined species to different antisera, whereas blue asterisks highlighted the differential reactivity among cultivar to defined antiserum.

Multiple comparison among the antisera showed statistically significant differences (p<0.05) between anti-ZmPRO3 and anti-PRA, anti-ZmPRO4, and anti-ZmPRO5, respectively, with Games-Howell test results of 65010.5, 64564.3 y 71150.2, respectively.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 81

**Figure 6.** Immune-reactivity analysis of crude protein extract of mature olive pollen (cv. Picual) before and after *in vitro* germination. Reactivity of proteins extracts from mature pollen, as well as different stages of *in vitro* germination were assayed against different maize profilin antisera, a) anti-PRA, b) anti-ZmPRO3, c) anti-ZmPRO5, d) anti-ZmPRO4, e) as well as against olive profilin antiserum anti-Ole e 2. It was appreciable up to 5 reactive bands about 13.0, 13.7, 14.2, 14.9 y 15.7 kDa, corresponding to different isoforms of profilins. The intensity of the reactive bands was quantitated by a densitometric analysis: blue color plot corresponded to 13.0kDa bands, pink, yellow, turquoise, and brown color to 13.7, 14.2,

14.9 and 15.7 kDa, respectively.

**D**

**C**

**B**

**A**

On the other hand, analysis of reactivity among species against individual antiserum showed statistical significant differences between *Olea europaea* L. and *Phleum pratense*, *Olea europaea* L. and *Corylus avellana*, *Zea mays* and *Phleum pratense*, and *Corylus avellana* and *Zea mays* for the antisera anti-PRA, anti-ZmPRO5, anti-ZmPRO4 and anti-Ole e 2, respectively.

Reversely, it is possible to observe clear differences between antisera for defined species, such as *Corylus avellana* and *Phleum pratense* for anti-ZmPRO3, and *Phleum pratense* for anti-Ole e 2.

#### **3.3. Analysis of olive pollen profilin during** *in vitro* **germination**

The study of the olive pollen germination was aimed to analyze the differential expression of profilin isoforms during the germination process. Figure 5 showed the protein profiles of olive pollen extracts (cv. Picual) obtained after hydration, and at different times of germination (5 min, 1h, 4h, 7h and 18h). No bands were distinguishable in the blue Coomassie blue stained gel around the molecular weight corresponding to profilins.

**Figure 5.** Protein profile of crude protein extract of mature olive pollen (cv. Picual) before and at different times of *in vitro* germination. 30μg of total protein was loaded in each line.

On the contrary, clear bands were obtained with immunoblotting experiments with the different antisera. 5 different bands can be distinguished corresponding to 5 different profilin isoforms (Figure 6), with calculated MW of 13.0, 13.7, 14.2, 14.9 and 15.7 kDa, respectively.

respectively.

respectively.

Ole e 2.

Multiple comparison among the antisera showed statistically significant differences (p<0.05) between anti-ZmPRO3 and anti-PRA, anti-ZmPRO4, and anti-ZmPRO5, respectively, with

On the other hand, analysis of reactivity among species against individual antiserum showed statistical significant differences between *Olea europaea* L. and *Phleum pratense*, *Olea europaea* L. and *Corylus avellana*, *Zea mays* and *Phleum pratense*, and *Corylus avellana* and *Zea mays* for the antisera anti-PRA, anti-ZmPRO5, anti-ZmPRO4 and anti-Ole e 2,

Reversely, it is possible to observe clear differences between antisera for defined species, such as *Corylus avellana* and *Phleum pratense* for anti-ZmPRO3, and *Phleum pratense* for anti-

The study of the olive pollen germination was aimed to analyze the differential expression of profilin isoforms during the germination process. Figure 5 showed the protein profiles of olive pollen extracts (cv. Picual) obtained after hydration, and at different times of germination (5 min, 1h, 4h, 7h and 18h). No bands were distinguishable in the blue

Coomassie blue stained gel around the molecular weight corresponding to profilins.

**Figure 5.** Protein profile of crude protein extract of mature olive pollen (cv. Picual) before and at

On the contrary, clear bands were obtained with immunoblotting experiments with the different antisera. 5 different bands can be distinguished corresponding to 5 different profilin isoforms (Figure 6), with calculated MW of 13.0, 13.7, 14.2, 14.9 and 15.7 kDa,

different times of *in vitro* germination. 30μg of total protein was loaded in each line.

Games-Howell test results of 65010.5, 64564.3 y 71150.2, respectively.

**3.3. Analysis of olive pollen profilin during** *in vitro* **germination** 

**Figure 6.** Immune-reactivity analysis of crude protein extract of mature olive pollen (cv. Picual) before and after *in vitro* germination. Reactivity of proteins extracts from mature pollen, as well as different stages of *in vitro* germination were assayed against different maize profilin antisera, a) anti-PRA, b) anti-ZmPRO3, c) anti-ZmPRO5, d) anti-ZmPRO4, e) as well as against olive profilin antiserum anti-Ole e 2. It was appreciable up to 5 reactive bands about 13.0, 13.7, 14.2, 14.9 y 15.7 kDa, corresponding to different isoforms of profilins. The intensity of the reactive bands was quantitated by a densitometric analysis: blue color plot corresponded to 13.0kDa bands, pink, yellow, turquoise, and brown color to 13.7, 14.2, 14.9 and 15.7 kDa, respectively.

The expression pattern of profilin is similar during *in vitro* germination, although conspicuous differences of the antisera reactivity can be pointed out for the protein extracts. Level of protein isoforms expression in mature pollen and hydration stage were quite similar. On the other hand, there was a notable decrease of protein expression level, equally for all the isoforms at the end of germination (7-18 hours). Proteins expression level between 5 min and 4 h of germination was variable for the different profilin isoforms, particularly for the variants of 13.7 and 14.2 KDa.

Differential Immune-Reactivity and Subcellular Distribution Reveal

**Pathway** 

**Others (Cytoplasm, Microsomes)** 

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 83

The immunoblotting analysis of seed proteins with anti-Ole e 2 showed at least two reactive bands located at 13.7 and 14.2 kDa (Figure 7B). In addition, there were quantitative differences in the expression level of profilin concerning both tissues (cotyledon and endosperm) and cultivar. Thus, profilin in endosperm of cultivar Picual showed a higher level of immune-reactivity in comparison with profilins of cotyledon from the same cultivar. Protein extracts from olive seed cultivars Acebuche and Picual were used in immunoblot experiments with antiserum anti-Ole e 2. A) SDS-PAGE of crude protein extract from olive seeds tissues (endosperm and cotyledon) of Acebuche and Picual cultivars. B) Inmunoblot of seeds protein samples from (A). Reactive bands were observed at 13.7 and 14.2 kDa, corresponding to vegetative profilins. C) Plot corresponding to the quantitation of reactive bands: blue lines were depicted for 14.2 kDa bands, whereas pink color was used for 13.7

**Chloroplast Mitochondria Secretory** 

*pendula* **- 14.4±1.1 8.6±0.5 11.7±0.3 65.3±1.0** 

*pratense* **- 6.6±0.2 5.0±0.3 27.9±0.7 61.5±1.4**  *Zea mays* **- 7.8±0.1 7.2±0.1 19.7±0.4 65.4±0.4** 

**Table 1.** Score calculated for the probability of finding a specific profilin in a particular cellular location. Bold numbers indicate that the score calculated for these sequences markedly differed from the average

**- 14.4±0.1 8.1±0.1 13.2±0.5 64.6±0.4**  DQ663545 8.4±0.0 6.8±0.0 22.3±0.0 62.5±0.0 DQ663547 6.5±0.0 4.2±0.0 27.7±0.0 61.6±0.0

X73280 2.5±0.0 9.9±0.0 27.1±0.0 60.5±0.0

**- 13.2±1.6 8.2±0.8 9.8±0.5 68.8±0.7**  DQ640909 5.9±0.0 4.4±0.0 39.9±0.0 49.8±0.0 DQ640910 7.6±0.0 7.0±0.0 20.0±0.0 65.4±0.0 DQ640906 5.8±0.0 5.3±0.0 32.2±0.0 56.7±0.0 DQ640903 9.7±0.0 7.4±0.0 14.2±0.0 68.7±0.0 DQ640908 7.9±0.0 7.8±0.0 17.0±0.0 67.3±0.0 DQ317580 6.7±0.0 4.8±0.0 28.4±0.0 60.1±0.0 DQ640904 8.2±0.0 6.8±0.0 15.2±0.0 69.8±0.0 DQ663553 6.7±0.0 4.8±0.0 28.4±0.0 60.1±0.0 DQ663554 6.7±0.0 4.8±0.0 28.5±0.0 60.0±0.0 DQ663555 6.9±0.0 4.4±0.0 28.2±0.0 60.5±0.0 DQ663556 6.7±0.0 4.8±0.0 28.5±0.0 60.0±0.0 DQ663558 8.7±0.0 6.8±0.0 20.0±0.0 64.5±0.0 DQ640905 7.1±0.0 8.4±0.0 16.5±0.0 68.0±0.0 DQ60907 8.2±0.0 6.8±0.0 15.3±0.0 69.7±0.0

kDa reactive bands.

**Sequence GeneBank Accession N°**

**Specie** 

*Olea europaea* L.

*Betula* 

*Corylus avellana* 

*Phleum* 

value.

#### **3.4. Analysis of cross-immunoreactivity between vegetative and reproductive profilins**

In order to determine the putative cross-reaction between reproductive profilins (see sections 3.1 to 3.3) and profilins from vegetative tissues, we have tested two of the antibodies used above (anti-ZmPRO4, and anti-ZmPRO5) against vegetative isoforms of maize profilins.

For this purpose, we have used protein extracts from olive seeds (cotyledon and endosperm) (cv Picual and Acebuche). The analysis of olive seed proteins Figure 7A showed a protein profile completely different to these of protein extracts from pollen. In this case, the mayor protein bands corresponded to different polypeptides of seed storage proteins, with MW ranging from 20 to 47kDa (Alché et al. 2006).

**Figure 7.** Immune-reactivity analysis of crude protein extract of vegetative profilins from olive seed tissues, cotyledon and endosperm.

The immunoblotting analysis of seed proteins with anti-Ole e 2 showed at least two reactive bands located at 13.7 and 14.2 kDa (Figure 7B). In addition, there were quantitative differences in the expression level of profilin concerning both tissues (cotyledon and endosperm) and cultivar. Thus, profilin in endosperm of cultivar Picual showed a higher level of immune-reactivity in comparison with profilins of cotyledon from the same cultivar.

82 Current Insights in Pollen Allergens

**profilins** 

particularly for the variants of 13.7 and 14.2 KDa.

ranging from 20 to 47kDa (Alché et al. 2006).

tissues, cotyledon and endosperm.

The expression pattern of profilin is similar during *in vitro* germination, although conspicuous differences of the antisera reactivity can be pointed out for the protein extracts. Level of protein isoforms expression in mature pollen and hydration stage were quite similar. On the other hand, there was a notable decrease of protein expression level, equally for all the isoforms at the end of germination (7-18 hours). Proteins expression level between 5 min and 4 h of germination was variable for the different profilin isoforms,

**3.4. Analysis of cross-immunoreactivity between vegetative and reproductive** 

In order to determine the putative cross-reaction between reproductive profilins (see sections 3.1 to 3.3) and profilins from vegetative tissues, we have tested two of the antibodies used above (anti-ZmPRO4, and anti-ZmPRO5) against vegetative isoforms of maize profilins.

For this purpose, we have used protein extracts from olive seeds (cotyledon and endosperm) (cv Picual and Acebuche). The analysis of olive seed proteins Figure 7A showed a protein profile completely different to these of protein extracts from pollen. In this case, the mayor protein bands corresponded to different polypeptides of seed storage proteins, with MW

**Figure 7.** Immune-reactivity analysis of crude protein extract of vegetative profilins from olive seed

Protein extracts from olive seed cultivars Acebuche and Picual were used in immunoblot experiments with antiserum anti-Ole e 2. A) SDS-PAGE of crude protein extract from olive seeds tissues (endosperm and cotyledon) of Acebuche and Picual cultivars. B) Inmunoblot of seeds protein samples from (A). Reactive bands were observed at 13.7 and 14.2 kDa, corresponding to vegetative profilins. C) Plot corresponding to the quantitation of reactive bands: blue lines were depicted for 14.2 kDa bands, whereas pink color was used for 13.7 kDa reactive bands.


**Table 1.** Score calculated for the probability of finding a specific profilin in a particular cellular location. Bold numbers indicate that the score calculated for these sequences markedly differed from the average value.

#### **3.5. Cellular localization of profilin**

#### *3.5.1. Predicting the cellular localization of profilin*

Predictions of the cellular location for profilins were carried out based in their primary sequence, and the putative presence of signal peptides responsible for targeting these proteins to specific cellular locations.

Differential Immune-Reactivity and Subcellular Distribution Reveal

**Sequence GeneBank Accession N°**

**Nuclear Targeting Motif** 

DQ663545 RGKKGAGGITVKKT

X77583 RGKKGAGGITIKKT

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 85

hazel and timothy-grass. These changes can represent differences in the affinity for

*avellana*

*Phleum pratense*

DQ640910 RGKKGAGGITVKKT DQ663547 RGKKGAGGITVKKT

DQ640906 RGKKGAGGITIKKT Y09546 RGKKGAGGITIKKT DQ640908 RGKKGAGGITVKKT Y09457 RGKKGAGGITIKKT DQ317574 RGKKGSGGITSKKT Y09458 RGKKGAGGITIKKT DQ663553 RGKKGAGGITIKKT DQ663535 RGKKGAGGITIKKT DQ663554 RGKKGAGGITIKKT DQ663536 RGKKGAGGITIKKT DQ663555 RGKKGAGGITIKKT DQ663537 RGKKGAGGITIKKT DQ663556 RGKKGAGGITIKKT DQ663538 RGKKGAGGITIKKT DQ663557 RGKKGAGGITIKKT DQ663539 RGKKGAGGITIKKT DQ640905 RGKKGAGGITVKKT DQ663540 RGKKGAGGITIKKT DQ138358 RGKKGTGGITIKKT DQ663541 RGKKGAGGITIKKT DQ138352 RGKKGSGGITIKET DQ663542 RGKKGAGGITIKKT

**Motif Specie**

localization of particular profilin isoforms in defined cellular locations.

**Nuclear Targeting** 

DQ640909 RGKKGAGGITIKKT *Corylus* 

DQ138336 RGKKGAGGITIKKT

DQ138354 RGKKGSGGITIKET

*3.5.2. Experimental approach for profilin cellular localization* 

**Table 3.** Changes in the motif targeting to a nuclear localization of profilin sequences.

Sequence of nuclear motif is characterized by the sequence RGKKGXGG(I/V)T(I/V)KKT, where X is the amino acid serine (**S**) in the majority of the analyzed sequences. The variable amino acids were

The experimental approach for profilin localization was performed to determine whether there is really a differential distribution of profilin isoforms. Immuno-localization assays were performed by transmission electron microscopy (TEM) in ultrathin sections of germinated pollen grains of olive (cv. Picual) using different antisera: anti-Ole e 2 and anti-PRA (Figures 8 and 9), anti-ZmPRO3 (Figure 10), anti-ZmPRO4 (Figure 11), anti-ZmPRO5

All immune-localizations showed that profilins (gold particles) were preferentially located in the cytoplasm (Figures 8A and 9-12A), in addition to both nuclei of vegetative/generative cell (Figures 8A and 9A). Moreover, abundant gold grains were located in the pollen apertures (Figures 8C, 10C and 11C), in along the pollen wall, pollen tube and the pollen tip (Figures 8A and 9-12B), as well as in the material associated with the pollen grain exine (pollen coat) (Figures 8B, 11B and 12C). No significant number of labeling were found in the negative controls, for which were omitted the primary antiserum (Figure 9C). The overall number of gold grains in the sections was variable, and depending on the antiserum used.

**Specie** 

*Olea europaea* L.

(Figure 12).

**Sequence GeneBank Accession N°**

highlighted in bold and red color.

Probability of profilins location was calculated by using the tools TargetP (www.cbs.dtu.dk) and v2.0 PSORT (psort.hgc.jp). Table 1 shows the average values of probability for profilins location in different cellular compartments.

Overall, profilins exhibited high probability for cytoplasm localization. However, some sequences had a significant score for being localized in mitochondria and chloroplasts, while others were targeted to the secretory pathway. These data were confirmed by the program SignalP 3.0 Server (www.cbs.dtu.dk) (results not shown).

In addition, it was calculated the average probability of nuclear localization of profilins in the table 2, where different sequences from species exhibited a higher or lower probability of localization in comparison with an average score. Based in the average score, profilins from *Olea europaea* L. are most likely localized in nucleus compared with the other species.


Plant profilins analyzed have a targeting motif for nuclear localization which sequence is (RGKKGXGG(I/V)T(I/V)KKT) (Yoneda 1997).

**Table 2.** Score of probability for nuclear distribution of profilins. Bold numbers indicate the average probability.

The polymorphism of the profilin sequences concerning this motif is depicted in the table 3. Overall, micro-heterogeneities in this motif were identified for several sequences of olive,


hazel and timothy-grass. These changes can represent differences in the affinity for localization of particular profilin isoforms in defined cellular locations.

84 Current Insights in Pollen Allergens

**3.5. Cellular localization of profilin** 

proteins to specific cellular locations.

location in different cellular compartments.

(RGKKGXGG(I/V)T(I/V)KKT) (Yoneda 1997).

*Olea europaea* L.

*Phleum pratense* 

*Zea mays* 

probability.

SignalP 3.0 Server (www.cbs.dtu.dk) (results not shown).

*3.5.1. Predicting the cellular localization of profilin* 

Predictions of the cellular location for profilins were carried out based in their primary sequence, and the putative presence of signal peptides responsible for targeting these

Probability of profilins location was calculated by using the tools TargetP (www.cbs.dtu.dk) and v2.0 PSORT (psort.hgc.jp). Table 1 shows the average values of probability for profilins

Overall, profilins exhibited high probability for cytoplasm localization. However, some sequences had a significant score for being localized in mitochondria and chloroplasts, while others were targeted to the secretory pathway. These data were confirmed by the program

In addition, it was calculated the average probability of nuclear localization of profilins in the table 2, where different sequences from species exhibited a higher or lower probability of localization in comparison with an average score. Based in the average score, profilins from

Plant profilins analyzed have a targeting motif for nuclear localization which sequence is

**Accession N°**

*Betula pendula* **- 25.5±0.7**  *Corylus avellana* **- 29.1±0.8** 

**Table 2.** Score of probability for nuclear distribution of profilins. Bold numbers indicate the average

The polymorphism of the profilin sequences concerning this motif is depicted in the table 3. Overall, micro-heterogeneities in this motif were identified for several sequences of olive,

**Probability of Nuclear Location (%)** 

**- 34.8±3.3**  DQ138337 19.0±0.1 DQ138325 18.0±0.1 DQ117904 19.0±0.1

DQ663545 17.0±0.1

**- 27.8±1.5**  X77583 33.0±0.1 Y09546 32.0±0.1 Y09457 31.0±0.1 Y09458 32.0±0.1

**- 26.9±2.0**  X73279 32.0±0.1 X73280 35.0±0.1

*Olea europaea* L. are most likely localized in nucleus compared with the other species.

**Specie Sequence GenBank** 

**Table 3.** Changes in the motif targeting to a nuclear localization of profilin sequences. Sequence of nuclear motif is characterized by the sequence RGKKGXGG(I/V)T(I/V)KKT, where X is the amino acid serine (**S**) in the majority of the analyzed sequences. The variable amino acids were highlighted in bold and red color.

#### *3.5.2. Experimental approach for profilin cellular localization*

The experimental approach for profilin localization was performed to determine whether there is really a differential distribution of profilin isoforms. Immuno-localization assays were performed by transmission electron microscopy (TEM) in ultrathin sections of germinated pollen grains of olive (cv. Picual) using different antisera: anti-Ole e 2 and anti-PRA (Figures 8 and 9), anti-ZmPRO3 (Figure 10), anti-ZmPRO4 (Figure 11), anti-ZmPRO5 (Figure 12).

All immune-localizations showed that profilins (gold particles) were preferentially located in the cytoplasm (Figures 8A and 9-12A), in addition to both nuclei of vegetative/generative cell (Figures 8A and 9A). Moreover, abundant gold grains were located in the pollen apertures (Figures 8C, 10C and 11C), in along the pollen wall, pollen tube and the pollen tip (Figures 8A and 9-12B), as well as in the material associated with the pollen grain exine (pollen coat) (Figures 8B, 11B and 12C). No significant number of labeling were found in the negative controls, for which were omitted the primary antiserum (Figure 9C). The overall number of gold grains in the sections was variable, and depending on the antiserum used.


Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 87

**Figure 8.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-Ole e 2 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls C) and the apertural region. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: material

adhered to the pollen walls (pollen coat). The bars represent 1 μm.

**Table 4.** Gold grains count for each antiserum used in this study. Measures corresponded to gold grains/μm2. Profilin counting values corresponding to the different subcellular localization were plotted. **C**= Cytoplasm; **PC**= Pollen Coat; **A**= Aperture; **NC**= Negative Control; **PGM**= Pollen Germination Media.

In order to determine differences in the gold grain distribution for the different antisera used as markers for profilin isoforms, we proceeded to count the gold particles present on each of the above mentioned compartments. The results of this quantitation are showed in the table 4. The most abundant immunolabeling was observed in sections incubated with individual antiserum following the next order: anti-ZmPRO3 > ZmPRO4 > ZmPRO5. Antisera anti-ZmPRO5 and ZmPRO3 showed a preferential cytoplasmic immunolocalization, whereas anti-PRA and anti-ZmPRO4 showed a preferential localization in the apertural regions of the pollen grain.

Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 87

86 Current Insights in Pollen Allergens

Germination Media.

**0**

**10**

**20**

**30**

**40**

**Number of gold grains /** 

**μ**

**m2**

**50**

**60**

**70**

**80**

regions of the pollen grain.

**Antiserum Cytoplasm Pollen** 

**Profilin distribution**

Anti-PRA 6.06±3.94 7.83±4.67 8.22±2.22 2.00±0.42 27.13±8.89 Anti-ZmPRO3 34.70±9.66 8.83±4.54 4.86±2.04 5.00±0.87 48.56±23.76 Anti-ZmPRO4 5.91±3.02 4.34±1.32 10.83±3.06 2.00±0.38 32.75±9.63 Anti-ZmPRO5 13.00±3.35 5.14±1.95 2.20±0.45 3.00±0.44 18.70±17.36

Anti-PRA

Anti-ZmPRO3 Anti-ZmPRO4 Anti-ZmPRO5

**Table 4.** Gold grains count for each antiserum used in this study. Measures corresponded to gold grains/μm2. Profilin counting values corresponding to the different subcellular localization were plotted. **C**= Cytoplasm; **PC**= Pollen Coat; **A**= Aperture; **NC**= Negative Control; **PGM**= Pollen

In order to determine differences in the gold grain distribution for the different antisera used as markers for profilin isoforms, we proceeded to count the gold particles present on each of the above mentioned compartments. The results of this quantitation are showed in the table 4. The most abundant immunolabeling was observed in sections incubated with individual antiserum following the next order: anti-ZmPRO3 > ZmPRO4 > ZmPRO5. Antisera anti-ZmPRO5 and ZmPRO3 showed a preferential cytoplasmic immunolocalization, whereas anti-PRA and anti-ZmPRO4 showed a preferential localization in the apertural

**C PC A NC PGM**

**Profilin cellular distribution**

**Coat Aperture Negative** 

**Control** 

**Pollen Germination Media** 

**Figure 8.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-Ole e 2 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls C) and the apertural region. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: material adhered to the pollen walls (pollen coat). The bars represent 1 μm.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 89

**Figure 10.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO3 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is

highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat.

The bars represent 1 μm.

**Figure 9.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-PRA antiserum. A) General view of the pollen tip, B) longitudinal section of a pollen tube, and C) negative control. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; Tp: pollen tube; V: vesicle, asterisks: pollen coat. The bars represent 1 μm.

**Figure 9.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-PRA antiserum. A) General view of the pollen tip, B) longitudinal section of a pollen tube, and C) negative control. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; Tp: pollen tube; V: vesicle,

asterisks: pollen coat. The bars represent 1 μm.

**Figure 10.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO3 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat. The bars represent 1 μm.

Differential Immune-Reactivity and Subcellular Distribution Reveal

the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 91

**Figure 12.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO5 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is

highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat.

The bars represent 1 μm.

**Figure 11.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO4 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat. The bars represent 1 μm.

Differential Immune-Reactivity and Subcellular Distribution Reveal the Multifunctional Character of Profilin in Pollen as Major Effect of Sequences Polymorphism 91

The bars represent 1 μm.

**Figure 11.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO4 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is

highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat.

**Figure 12.** TEM immune-localization of olive pollen profilin in sections of mature pollen (cv Picual) during in vitro germination by using anti-ZmPRO5 antiserum. A) General view of the vegetative cell cytoplasm, B) pollen grain walls, and C) apertural region. The location of the gold particles is highlighted with circles and arrows. Ap: apertures; CV: vegetative cell cytoplasm, Ex: exine, In: intine, M: mitochondria, N: nucleus, P: plastid, ER: endoplasmic reticulum; V: vesicle, asterisks: pollen coat. The bars represent 1 μm.

## **4. Discussion**
