**2. Materials and methods**

#### **2.1. Pollen samples**

*Olea europaea* L. pollen samples were obtained during May and June of 2000-2007 from cultivated trees of the cultivars: 'Arbequina', 'Blanqueta', 'Hojiblanca', 'Manzanilla de Sevilla', 'Picual', 'Verdial de Huévar' and 'Verdial de Vélez' situated at the IFAPA center "Alameda del Obispo" (Andalusian Regional Government, Córdoba, Spain). Pollen samples were collected from numerous branches of at least two trees of each cultivar by shaking flowering shoots inside paper bags. Prior to its storage in liquid nitrogen, the harvested pollen was sieved through a 150 µm mesh in order to eliminate fallen corollas, anthers and other rests. After light microscopy observation, foreign-species pollen was estimated to be <0.1% and other plant parts <0.5% for all the cultivars used.

#### **2.2. Preparation of crude protein extracts and SDS-PAGE**

Crude protein extracts were obtained by stirring 1 g of pollen for each cultivar in 10 ml extraction buffer (0.01 M ammonium bicarbonate, pH 8.0, and 2 mM phenylmethylsulfonyl fluoride) for 8 h at 4°C. After centrifugation (2 x 30 minutes at 14,000 rpm at 4°C), the supernatants were filtered through a 0.2 µm filter, and stored in aliquots at –20°C. Protein concentration in the different samples was measured using the Bio-Rad reagent (Bio-Rad, Hercules, CA, USA) and bovine serum albumin (BSA) as standard.

Proteins (30 µg per lane) and Mw1 (New England BioLabs, Ipswich, MA, USA) and Mw2 standards (MBI Fermentas, Vilnius, Lithuania) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in 15% gels in a MiniProtean II system (Bio-Rad). The resulting gels were stained with silver nitrate (Rabilloud et al., 1994).

#### **2.3. Immunoblotting**

Gels obtained as described above were transferred onto BioTrace® polyvinylidene difluoride (PVDF) membranes (Pall BioSupport, Port Washington, NY, USA) at 100 V for 1.5 hours using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Prior to the treatment with antibodies, the membranes were blocked with TBST buffer (Tris buffered saline: TBS + 0.3% v/v Tween 20) + 10% w/v dried skimmed milk. For membrane probing, a recently developed multiplex Western blotting system for the assessment of allergenic molecules (Morales et al. 2012) has been used. Briefly, the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit is carried out. We utilized unconjugated Fab antibody fragments for blocking rabbit primary antibodies, and fluorescence-based detection. These changes allowed an accurate and reliable comparative quantitation of these allergens among pollen protein samples.

#### **2.4. Absolute and relative quantitation of allergens**

Imaging was carried out with a Pharos FX Plus Molecular Imager (Bio-Rad) using the Quantity One v4.6.2 software (Bio-Rad). The intensity of each fluorescent band was calculated using the quantitation tools of the Quantity One v4.6.2 software.

#### **2.5. Climate parameters**

58 Current Insights in Pollen Allergens

**2. Materials and methods** 

**2.1. Pollen samples** 

**2.3. Immunoblotting** 

These results are discussed as regard to their putative incidence in the development of symptoms by patients, the requirements for medical assistance and the rates of admission into clinical centres and hospitals. Agronomical implications in olive sexual reproduction,

*Olea europaea* L. pollen samples were obtained during May and June of 2000-2007 from cultivated trees of the cultivars: 'Arbequina', 'Blanqueta', 'Hojiblanca', 'Manzanilla de Sevilla', 'Picual', 'Verdial de Huévar' and 'Verdial de Vélez' situated at the IFAPA center "Alameda del Obispo" (Andalusian Regional Government, Córdoba, Spain). Pollen samples were collected from numerous branches of at least two trees of each cultivar by shaking flowering shoots inside paper bags. Prior to its storage in liquid nitrogen, the harvested pollen was sieved through a 150 µm mesh in order to eliminate fallen corollas, anthers and other rests. After light microscopy observation, foreign-species pollen was estimated to be

Crude protein extracts were obtained by stirring 1 g of pollen for each cultivar in 10 ml extraction buffer (0.01 M ammonium bicarbonate, pH 8.0, and 2 mM phenylmethylsulfonyl fluoride) for 8 h at 4°C. After centrifugation (2 x 30 minutes at 14,000 rpm at 4°C), the supernatants were filtered through a 0.2 µm filter, and stored in aliquots at –20°C. Protein concentration in the different samples was measured using the Bio-Rad reagent (Bio-Rad,

Proteins (30 µg per lane) and Mw1 (New England BioLabs, Ipswich, MA, USA) and Mw2 standards (MBI Fermentas, Vilnius, Lithuania) were separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) in 15% gels in a MiniProtean II system (Bio-

Gels obtained as described above were transferred onto BioTrace® polyvinylidene difluoride (PVDF) membranes (Pall BioSupport, Port Washington, NY, USA) at 100 V for 1.5 hours using a Mini Trans-Blot Electrophoretic Transfer Cell (Bio-Rad). Prior to the treatment with antibodies, the membranes were blocked with TBST buffer (Tris buffered saline: TBS + 0.3% v/v Tween 20) + 10% w/v dried skimmed milk. For membrane probing, a recently developed multiplex Western blotting system for the assessment of allergenic molecules (Morales et al. 2012) has been used. Briefly, the simultaneous detection of four olive pollen allergens (Ole e 1, Ole e 2, Ole e 5 and Ole e 9) on a single blot using a monoclonal antibody from mouse and three polyclonal antibodies raised in rabbit is carried out. We utilized

Rad). The resulting gels were stained with silver nitrate (Rabilloud et al., 1994).

including fruit setting and fruit production, are also discussed.

<0.1% and other plant parts <0.5% for all the cultivars used.

**2.2. Preparation of crude protein extracts and SDS-PAGE** 

Hercules, CA, USA) and bovine serum albumin (BSA) as standard.

Climate parameters were obtained from the weather tracking station situated at Córdoba airport (Spain) during the years 2000 to 2007. Information was extracted from the Spanish long term ecological research network (REDOTE).

#### **2.6. Statistical analysis**

Continuous variables were subjected to a study of normality distribution by using the Kolmorov-Smirnov test. Associations between continuous variables (non-normal distribution) were described by analysis of bivariate Spearman correlation (two-tailed). Significant correlation was considered P<0.05.

## **3. Results**

#### **3.1. Multiplex determination of year-to-year allergen expression in seven olive cultivars**

Figure 1A shows the protein profiles of the extracts analyzed after SDS-PAGE and silver staining. The different panels correspond to the cultivars analyzed between the years 2000 and 2007. Bands were observed in the range of 15 to 75 kDa. Clear quantitative differences were distinguished, from which the most conspicuous were those in the bands of 20 kDa and 18.4 kDa among de different years, with the exception of cultivars 'Arbequina', 'Verdial de Huévar' and 'Hojiblanca', in which the Ole e 1 bands were not well identified after silver staining. The remaining allergens studied were not identified after silver staining.

Allergenic profiles were assessed by multiplex immunotransference (Figure 1B). The cultivars 'Arbequina' and 'Hojiblanca' displayed two Ole e 1 forms (20 kDa and 18.4 kDa) in contrast with the remaining cultivars, were the three major forms of Ole e 1 (22 kDa, 20 kDa and 18.4 kDa) were distinguished. Differences in the expression of this allergen depending on the year of pollen collection can be easily noticed.

Ole e 2 was visualized in all cultivars in the form of two bands with sizes of 15.1 kDa and 14 kDa, with the exception of the 'Verdial de Vélez' cultivar, in which only the 15.1 kDa band was present. As described for Ole e 1, the presence of noticeable differences in the

expression of the Ole e 2 allergen, depending the year of pollen collection can be observed in the corresponding multiplex immunoblots.

Involvement of Climatic Factors in the Allergen Expression in Olive Pollen 61

R .9\* 1\*\* .9\* P .037 <0.001 .037

R .9\* 1\*\* 0.9\* P .037 <0.001 .037

R ----- 1\*\* 1\*\* P <0.001 <0.001

R ----- 1\*\* 1\*\* P <0.001 <0.001

**Ole e 1 18.4 kDa**  **All Ole e 1 bands** 

**Ole e 1 20 kDa** 

R ----- .65 P .156

 **22 kDa** 

R .9\* P .037

R .9\* P .037

R ----- 1\*\*

P <0.001

R ----- 1\*\* P <0.001

P .119

P .021

P .049

P <0.001 <0.001 <0.001

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

P .05 .05 .05 .05

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

AT April (°C) R .643

mT April (°C) R ----- .829\*

MT April (°C) R ----- .757\*

No. days of rain in February R .949 .949 .949 .949

R 1\*\* P <0.001

**Vélez** No. days of rain in August R 1\*\* 1\*\* 1\*\*

**Cultivar Climatic factor Ole e 1**

Yearly AT accumulated (°C)

AT April (°C)

AT May (°C)

mT May (°C)

AT March (°C)

mT March (°C)

Accumulated precipitation winter (January-March) (mm)

precipitation February (mm)

AT February (°C)

Accumulated precipitation (January-May) (mm)

No. days of rain in April

Accumulated mRH (January-May) (%)

mRH January (%)

**Arbequina** Yearly AT accumulated (°C)

**Blanqueta** 

**Hojiblanca** 

**Picual** 

**Verdial de** 

**Verdial de Huévar** 

**Figure 1.** A) Silver staining of total protein extracts obtained from pollen corresponding to the seven cultivars analyzed trhoughout the different years. B) Multiplex detection of the allergens Ole e 1, Ole e 2, Ole e 5 and Ole e 9 in the same extracts described in panel A. Thirty micrograms of total protein were loaded in each lane. Lane numbers (0-7) correspond to the years analyzed (2000-2007). Molecular weight standards are displayed on the left.

Ole e 5 allergen can be observed in the cultivars analyzed. However, only a weak signal was detected in the cultivars 'Hojiblanca' and 'Verdial de Huévar'. Year-to-year differences in the expression of this allergen are clearly observed in 'Picual' and 'Blanqueta' cultivars.

Finally, Ole e 9 was expressed in all cultivars analyzed. Year-to-year differences in their expression are observed as well, with the sharpest differences in the cultivars 'Blanqueta' and 'Manzanilla de Sevilla'.

#### **3.2. Involvement of climatic factors in the expression of Ole e 1 allergen**

Correlation between the data corresponding to the major climatic factors and the expression of the allergens Ole e 1, Ole e 2, Ole e 5 y Ole e 9 was individually assessed for each cultivar by means of Spearman correlation analyses. The analysis of the correlation between the levels of the Ole e 1 and these climatic parameters is shown in Table 1. The presence of a statistically significant positive correlation between the expression of the different Ole e 1 forms (either individually or their added values) and the accumulated yearly average temperatures (the addition of the monthly average temperatures during those months prior to the blooming period -July of the prior year to June of the current year-) was determined for the cultivar 'Blanqueta'. Total levels of the Ole e 1 allergen correlated positively with the monthly average temperatures during the winter period (for the cultivar 'Verdial de Huévar') or the spring period (cultivars 'Blanqueta', 'Hojiblanca').


the corresponding multiplex immunoblots.

weight standards are displayed on the left.

and 'Manzanilla de Sevilla'.

expression of the Ole e 2 allergen, depending the year of pollen collection can be observed in

**Figure 1.** A) Silver staining of total protein extracts obtained from pollen corresponding to the seven cultivars analyzed trhoughout the different years. B) Multiplex detection of the allergens Ole e 1, Ole e 2, Ole e 5 and Ole e 9 in the same extracts described in panel A. Thirty micrograms of total protein were loaded in each lane. Lane numbers (0-7) correspond to the years analyzed (2000-2007). Molecular

Ole e 5 allergen can be observed in the cultivars analyzed. However, only a weak signal was detected in the cultivars 'Hojiblanca' and 'Verdial de Huévar'. Year-to-year differences in the expression of this allergen are clearly observed in 'Picual' and 'Blanqueta' cultivars.

Finally, Ole e 9 was expressed in all cultivars analyzed. Year-to-year differences in their expression are observed as well, with the sharpest differences in the cultivars 'Blanqueta'

Correlation between the data corresponding to the major climatic factors and the expression of the allergens Ole e 1, Ole e 2, Ole e 5 y Ole e 9 was individually assessed for each cultivar by means of Spearman correlation analyses. The analysis of the correlation between the levels of the Ole e 1 and these climatic parameters is shown in Table 1. The presence of a statistically significant positive correlation between the expression of the different Ole e 1 forms (either individually or their added values) and the accumulated yearly average temperatures (the addition of the monthly average temperatures during those months prior to the blooming period -July of the prior year to June of the current year-) was determined for the cultivar 'Blanqueta'. Total levels of the Ole e 1 allergen correlated positively with the monthly average temperatures during the winter period (for the cultivar 'Verdial de

**3.2. Involvement of climatic factors in the expression of Ole e 1 allergen** 

Huévar') or the spring period (cultivars 'Blanqueta', 'Hojiblanca').


Involvement of Climatic Factors in the Allergen Expression in Olive Pollen 63

Moreover, statistically significant positive correlations were observed between Ole e 1 expression and minimum temperatures (cultivars 'Blanqueta', 'Hojiblanca', 'Picual' and 'Manzanilla de Sevilla') or maximum temperatures (cultivars 'Picual' and 'Manzanilla de

Total expression of Ole e 1 correlated positively with the added monthly precipitation occurred during the winter and spring period (cultivars 'Hojiblanca' and 'Verdial de Huévar'), as well as with the total precipitation occurred during the months prior to flowering (cultivar 'Manzanilla de Sevilla'). Positive correlations are also observed between total levels of Ole e 1 allergen and precipitation, number of days of rain, and the monthly

average of relative humidity during the months prior to the blooming season.

**Cultivar Climatic factor Ole e 2** 

Accumulated precipitation winter (January-March) (mm)

Accumulated mRH (January-

mRH January (%) R 1\*\*

mRH May (%) R 1\*\*

**Verdial de Vélez** mRH December (%) R 1\*\*

AT January (°C) R 1\*\*

precipitation June (mm) R .775\*

MT June (°C)

AT February (°C)

May) (%)

**3.3. Involvement of climatic factors in the expression of Ole e 2 allergen**

Table 2 displays the observed correlations between Ole e 2 and different climatic factors.

precipitation February (mm) R .943\*\*

 **15.1 kDa** 

R .943\*\* P .005

P .005

AT June (°C) R .886\* .886\*

R 1\*\* P <0.001 mT April (°C) R .829\*

MT April (°C) R .757\*

R 1\*\* P <0.001

P <0.001

P <0.001

**Ole e 2 14 kDa** 

P .019 .019

R .886\* .886\* P .019 .019

P .021

P .049

P <0.001

P <0.001

P .041

**All Ole e 2 bands** 

Sevilla') during the spring period.

**Arbequina** 

**Hojiblanca** 

**Verdial de Huévar** 

AT: average temperature. mT: minimum temperature. MT: maximum temperature. mRH: monthly relative humidity

**Table 1.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of the different Ole e 1 forms both individually and jointly (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \* Statistically significant correlation P<0.05; \*\* P<0.01.

Moreover, statistically significant positive correlations were observed between Ole e 1 expression and minimum temperatures (cultivars 'Blanqueta', 'Hojiblanca', 'Picual' and 'Manzanilla de Sevilla') or maximum temperatures (cultivars 'Picual' and 'Manzanilla de Sevilla') during the spring period.

62 Current Insights in Pollen Allergens

**Manzanilla de Sevilla** 

**Cultivar Climatic factor Ole e 1**

mRH February (%)

mRH April (%)

mRH May (%)

AT March (°C)

AT March (°C)

mT March (°C)

MT May (°C)

Accumulated yearly precipitation (mm)

precipitation July (mm)

precipitation September (mm)

precipitation November (mm)

Accumulated yearly mRH (%)

mRH December (%) R .9\*

correlation coefficient. \* Statistically significant correlation P<0.05; \*\* P<0.01.

 **22 kDa** 

R 1\*\* P <0.001

**Ole e 1 20 kDa** 

R .8 P .104

R .7 P .108

R .9\* P .037

R .9\* P .037

R 1\*\* P <0.001

precipitation April (mm) R .9\* .9\*

No. days of rain in January R .9\* .9\*

No. days of rain in April R .9\* .9\*

mRH February (%) R .9\* .9\*

R 1\*\* P <0.001

P .037

AT: average temperature. mT: minimum temperature. MT: maximum temperature. mRH: monthly relative humidity **Table 1.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of the different Ole e 1 forms both individually and jointly (CNT\*mm2) and different climatic factors. R: Spearman

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

R 1\*\* 1\*\* 1\*\* P <0.001 <0.001 <0.001

R .9\* 1\*\* 0.9\* P .037 .001 .037

R .894 .894 P .04 .04

R .9\* .9\* .9\* P .03 .037 .037

P .037 .037

P .037 .037

P .037 .037

P .037 .037

**Ole e 1 18.4 kDa**  **All Ole e 1 bands** 

> Total expression of Ole e 1 correlated positively with the added monthly precipitation occurred during the winter and spring period (cultivars 'Hojiblanca' and 'Verdial de Huévar'), as well as with the total precipitation occurred during the months prior to flowering (cultivar 'Manzanilla de Sevilla'). Positive correlations are also observed between total levels of Ole e 1 allergen and precipitation, number of days of rain, and the monthly average of relative humidity during the months prior to the blooming season.

#### **3.3. Involvement of climatic factors in the expression of Ole e 2 allergen**


Table 2 displays the observed correlations between Ole e 2 and different climatic factors.


Involvement of Climatic Factors in the Allergen Expression in Olive Pollen 65

R .9\* P .0187

R .9 \* P .0037

R 1\*\* P <0.001

R 1\*\* P <0.001

R 1\*\* P <0.001

R 1\*\* P <0.001

de Vélez', Ole e 5 correlated to the number of days of rain and the relative humidity over the

precipitation February (mm)

No. days of rain in February

**Table 3.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of Ole e 5 (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \*Statistically significant correlation P<0.05; \*\* P<0.01. AT: average temperature. mT: minimum temperature. MT: maximum

**3.5. Involvement of climatic factors in the expression of Ole e 9 allergen**

As regard to the Ole e 9 allergen, its expression correlated positively (Table 4) with the average temperatures during the months of January (cultivars 'Arbequina', and 'Verdial de Huévar') and March ('Hojiblanca'). In the same way, a correlation was demonstrated between Ole e 9 expression and the number of days of rain during March in the cultivars 'Arbequina' and 'Verdial de Vélez'. The average relative humidity also showed a statistically relevant correlation during the months of winter, for the cultivar 'Picual', and specifically for January in the cultivar 'Verdial de Huévar'. In addition, and for this last cultivar 'Verdial de Huévar', the relative humidity presents a positive correlation during the

We describe next several examples of data distributions corresponding to climatic factors showing a statistically significant correlation with the expression level of the different allergens. Charts represent the allergen levels (represented as band intensities) plotted against climatic factors like temperature (Figure 2), precipitation, relative humidity and number of days of rain (Figure 3). A reference line showing a theoretical absolute linear

No. days of rain in April

mRH February (%)

mRH April (%)

mT May (°C)

**Cultivar Climatic factor Ole e 5** 

months of February and April.

**Blanqueta** 

**Verdial de Vélez** 

temperature. mRH: monthly relative humidity.

spring months of April and May.

**3.6. Several examples of data distributions**

correlation between the variables was added to the figures.

**Table 2.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of the different Ole e 2 forms both individually and jointly (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \* Statistically significant correlation P<0.05; \*\* P<0.01. AT: average temperature. mT: minimum temperature. MT: maximum temperature. mRH: monthly relative humidity.

The expression levels of the bands corresponding to the Ole e 2 allergen (Table 2) and/or their addition, correlated positively with the average temperatures for particular months during the winter period (cultivars 'Hojiblanca' and 'Verdial de Huévar') or spring (cultivar 'Arbequina'). Statistically significant correlations were also observed between the expression of Ole e 2 and the maximum/minimum temperatures over the months corresponding to the spring (cultivars 'Arbequina' and 'Hojiblanca').

The expression of one of the Ole e 2 form is positively correlated to the accumulated monthly precipitation occurred during the winter months for the cultivar 'Arbequina', as well as to the precipitation occurred in particular months during the winter and spring (cultivars 'Arbequina', 'Verdial de Huévar' and 'Manzanilla de Sevilla'). The Ole e 2 form with the higher molecular weight (15.1 kDa) also presented correlation to the accumulated monthly relative humidity during the months of winter and spring (cultivars 'Hojiblanca' and 'Verdial de Huévar'). Similarly, positive correlation was detected between the total levels of Ole e 2 and the total number of days of rain over the winter and spring months (cultivar 'Verdial de Huévar').

#### **3.4. Involvement of the climatic factors in the expression of Ole e 5 allergen**

As respects to Ole e 5 allergen (Table 3), the presence of a statistically significant positive correlation was determined between the minimum temperature during May, and the precipitation observed during February in the 'Blanqueta' cultivar. In the cultivar 'Verdial


de Vélez', Ole e 5 correlated to the number of days of rain and the relative humidity over the months of February and April.

**Table 3.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of Ole e 5 (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \*Statistically significant correlation P<0.05; \*\* P<0.01. AT: average temperature. mT: minimum temperature. MT: maximum temperature. mRH: monthly relative humidity.

#### **3.5. Involvement of climatic factors in the expression of Ole e 9 allergen**

As regard to the Ole e 9 allergen, its expression correlated positively (Table 4) with the average temperatures during the months of January (cultivars 'Arbequina', and 'Verdial de Huévar') and March ('Hojiblanca'). In the same way, a correlation was demonstrated between Ole e 9 expression and the number of days of rain during March in the cultivars 'Arbequina' and 'Verdial de Vélez'. The average relative humidity also showed a statistically relevant correlation during the months of winter, for the cultivar 'Picual', and specifically for January in the cultivar 'Verdial de Huévar'. In addition, and for this last cultivar 'Verdial de Huévar', the relative humidity presents a positive correlation during the spring months of April and May.

#### **3.6. Several examples of data distributions**

64 Current Insights in Pollen Allergens

**Manzanilla de Sevilla** 

Accumulated No. of raining

mRH April (%) R 1\*\*

mRH May (%) R 1\*\*

mT: minimum temperature. MT: maximum temperature. mRH: monthly relative humidity.

R 1\*\* P <0.001

R 1\*\* P <0.001

P .05 .05 .05

P <0.001 <0.001

P <0.001 <0.001

P .014 .014 .014

P .05 .05 .05

No. days of rain in February R .949 .949 .949

No. days of rain in April R 1\*\* 1\*\*

mRH February (%) R 1\*\* 1\*\*

P <0.001

P <0.001

precipitation June (mm) R .949\* .949\* .949\*

No. days of rain in June R .872 .872 .872

**Table 2.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of the different Ole e 2 forms both individually and jointly (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \* Statistically significant correlation P<0.05; \*\* P<0.01. AT: average temperature.

The expression levels of the bands corresponding to the Ole e 2 allergen (Table 2) and/or their addition, correlated positively with the average temperatures for particular months during the winter period (cultivars 'Hojiblanca' and 'Verdial de Huévar') or spring (cultivar 'Arbequina'). Statistically significant correlations were also observed between the expression of Ole e 2 and the maximum/minimum temperatures over the months corresponding to the

The expression of one of the Ole e 2 form is positively correlated to the accumulated monthly precipitation occurred during the winter months for the cultivar 'Arbequina', as well as to the precipitation occurred in particular months during the winter and spring (cultivars 'Arbequina', 'Verdial de Huévar' and 'Manzanilla de Sevilla'). The Ole e 2 form with the higher molecular weight (15.1 kDa) also presented correlation to the accumulated monthly relative humidity during the months of winter and spring (cultivars 'Hojiblanca' and 'Verdial de Huévar'). Similarly, positive correlation was detected between the total levels of Ole e 2 and the total number of days of rain over the winter and spring months

**3.4. Involvement of the climatic factors in the expression of Ole e 5 allergen**

As respects to Ole e 5 allergen (Table 3), the presence of a statistically significant positive correlation was determined between the minimum temperature during May, and the precipitation observed during February in the 'Blanqueta' cultivar. In the cultivar 'Verdial

days (January-May)

Accumulated mRH (January-May) (%)

spring (cultivars 'Arbequina' and 'Hojiblanca').

(cultivar 'Verdial de Huévar').

We describe next several examples of data distributions corresponding to climatic factors showing a statistically significant correlation with the expression level of the different allergens. Charts represent the allergen levels (represented as band intensities) plotted against climatic factors like temperature (Figure 2), precipitation, relative humidity and number of days of rain (Figure 3). A reference line showing a theoretical absolute linear correlation between the variables was added to the figures.


Involvement of Climatic Factors in the Allergen Expression in Olive Pollen 67

**Figure 3.** Examples of charts displaying data distribution referring to precipitation, relative humidity and number of days of rain plotted against the expression levels of the following allergens: A) Ole e 1,

The allergen content of the olive pollen is clearly influenced by the genetic origin of the cultivar analyzed (Hamman Khalifa et al. 2008). Diverse authors have also indicated that climatic factors may have different effects over olive flowering and pollen development (Galan et al. 2001; Orlandi et al. 2010), as well as over olive pollen allergenicity (Fernández-

The results presented here demonstrate that changes occur in the expression of four relevant allergens in seven cultivars, and throughout consecutive years. However, results are considered heterogeneous between the different cultivars. The climatic parameters taken into account were temperature, precipitation, number of days of rain or relative humidity during the months prior to the flowering period, which normally corresponds to the months

As regard to the temperature, correlations were not observed for a particular month or for the accumulated yearly temperature in all cultivars in a simultaneous way. However, the point correlations detected always corresponded to the winter and summer months (January-June) prior to the flowering period. This can be due to the intrinsic characteristics and requirements of each cultivar, or to the low number of cultivars and years studied,

This results are in good agreement with those described for the birch major pollen allergen Bet v 1 (Buters et al. 2008), where an increase of this allergen was observed in higher temperatures, determined by growth of sampling trees in different climatic regions, over

B) Ole e 2, C) Ole e 5 and D) Ole e 9.

Caldas et al. 2007).

of May and June.

**4. Discussion and conclusions** 

which may limit the statistical power of the analysis.

**Figure 2.** Examples of charts displaying data distribution referring to temperatures plotted against the expression levels of the following allergens: A) Ole e 1, B) Ole e 2, C) Ole e 5 and D) Ole e 9.

**Figure 3.** Examples of charts displaying data distribution referring to precipitation, relative humidity and number of days of rain plotted against the expression levels of the following allergens: A) Ole e 1, B) Ole e 2, C) Ole e 5 and D) Ole e 9.

#### **4. Discussion and conclusions**

66 Current Insights in Pollen Allergens

**Arbequina** 

**Hojiblanca** 

**Verdial de Huévar** 

temperature. mRH: monthly relative humidity.

**Cultivar Climatic factor Ole e 9** 

**Picual** mRH December (%) R .857\*

**Verdial de Vélez** No. days of rain in March R .949

**Table 4.** Analysis of bivariate Spearman correlation (two-tailed) between the expression of Ole e 9 (CNT\*mm2) and different climatic factors. R: Spearman correlation coefficient. \*Statistically significant correlation P<0.05; \*\* P<0.01. AT: average temperature. mT: minimum temperature. MT: maximum

**Figure 2.** Examples of charts displaying data distribution referring to temperatures plotted against the

expression levels of the following allergens: A) Ole e 1, B) Ole e 2, C) Ole e 5 and D) Ole e 9.

AT January (°C) R .829\*

No of days of rain in March R .088\*

AT March (°C) R 1\*\*

mT March (°C) R 1\*\*

AT January (°C) R 1\*\*

No. days of rain in February R .949

mRH January (%) R 1\*\*

mRH April (%) R 1\*\*

mRH May (%) R 1\*\*

P .042

P .02

P <0.001

P <0.001

P .014

P .05

P <0.001

P .05

P <0.001

P <0.001

P <0.001

The allergen content of the olive pollen is clearly influenced by the genetic origin of the cultivar analyzed (Hamman Khalifa et al. 2008). Diverse authors have also indicated that climatic factors may have different effects over olive flowering and pollen development (Galan et al. 2001; Orlandi et al. 2010), as well as over olive pollen allergenicity (Fernández-Caldas et al. 2007).

The results presented here demonstrate that changes occur in the expression of four relevant allergens in seven cultivars, and throughout consecutive years. However, results are considered heterogeneous between the different cultivars. The climatic parameters taken into account were temperature, precipitation, number of days of rain or relative humidity during the months prior to the flowering period, which normally corresponds to the months of May and June.

As regard to the temperature, correlations were not observed for a particular month or for the accumulated yearly temperature in all cultivars in a simultaneous way. However, the point correlations detected always corresponded to the winter and summer months (January-June) prior to the flowering period. This can be due to the intrinsic characteristics and requirements of each cultivar, or to the low number of cultivars and years studied, which may limit the statistical power of the analysis.

This results are in good agreement with those described for the birch major pollen allergen Bet v 1 (Buters et al. 2008), where an increase of this allergen was observed in higher temperatures, determined by growth of sampling trees in different climatic regions, over

two consecutive years. Another study suggests a higher allergenic response to the Bet v 1 allergen present in birch pollen extracts grown in high average daily temperatures, in comparison to trees cultivated at lower temperatures (Ahlholm et al. 1998). This assay was carried out by using sera from patients sensitive to this pollen by using immunoblotting experiments. Our results indicate that high temperatures also seem to exert an increase in the expression of allergens in certain olive cultivars.

Involvement of Climatic Factors in the Allergen Expression in Olive Pollen 69

correlated to increased rates of pollen germination and lower degree of olive selfincompatibility. This hypothesis is also in good agreement with experimental observations made by several authors as regard to the effects of climatic factor on self-pollination and

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucía (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274). The authors acknowledge the availability of plant material and the collaboration of the staff of the IFAPA center "Venta del Llano" (Mengíbar, Spain) depending from the Andalusian

Ahlholm, J.U., Helander, M.L., & Savolainen, J. (1998). Genetic and environmental factors affecting the allergenicity of birch (Betula pubescens ssp. czerepanovii [Orl.] Hametahti) pollen. Clinical and Experimental Allergy, Vol. 28, No. 11, pp. 1384-1388, ISSN

Androulakis, I.I., & Loupassaki, M.H. (1990). Studies on the self-fertility of some cultivars in

Buters, J.T., Kasche, A., Weichenmeiera, I., Schober, W., Klaus, S., Traidl-Hoffmann, C., Menzel, A., Huss-Marp, J., Krämer, U., & Behrendt, H. (2008). Year-to-year variation in release of Bet v 1 allergen from birch pollen: evidence for geographical differences between West and South Germany. *International Archives of Allergy & Immunology*, Vol.

Cecchi, L., D'Amato, G., Ayres, J.G., Galan, C., Forastiere, F., Forsberg, B., Gerritsen, J., Nunes, C., Behrendt, H., Akdis, C., Dahl, R., & Annesi-Maesano, I. (2010). Projections of the effects of climate change on allergic asthma: the contribution of aerobiology. *Allergy*,

Fernández-Caldas, E., Carnés, J., Iraola, V., & Casanovas, M. (2007). Comparison of the allergenicity and Ole e 1 content of 6 varieties of Olea euroapea pollen collected during 5 consecutive years. *Annals of Allergy, Asthma & Immunology*, Vol. 98, No. 5, pp. 464-470,

the area of Crete. *Acta Horticulturae*, Vol. 286, pp. 159-162, ISSN 0567-7572

productivity in the olive (Androulakis and Loupassaki 1990, Lavee et al. 2002).

*Proteomic Research Service, Hospital Universitario San Cecilio, Granada, Spain* 

Antonio Jesús Castro, María Isabel Rodríguez-García and Juan de Dios Alché

*Estación Experimental del Zaidín (CSIC), Granada, Spain* 

*Estación Experimental del Zaidín (CSIC), Granada, Spain* 

145, No. 2, pp. 122-130, ISSN 1018-2438

Vol. 65, No. 9, pp. 1073-1081, ISSN 0105-4538

**Author details** 

**Acknowledgement** 

Regional Government.

**5. References** 

1365-2222

ISSN 1081-1206

Sonia Morales

We may extrapolate that an increase in the average temperatures would bring a parallel increase in pollen allergenic content and allergenicity as a consequence. This could be therefore one of the consequences of the so-called global warming produced by the greenhouse effect (Ahlholm et al. 1998; Cecchi et al. 2010). Moreover, we may speculate that the fact that urban areas are often exposed to temperatures 2 to 8 °C higher that rural areas (Oke 1987) may result in increased olive pollen allergenicity and allergy prevalence in and around the cities, where the olive tree is also present, as it is widely used as an ornamental plant.

Fernández-Caldas et al. (2007) performed a similar study using six olive cultivars, over a period of five consecutive years. This study analyzed the content in the olive pollen major allergen (Ole e 1) only, which did not show statistically significant correlations to the precipitation average, nor to the average temperatures over the winter months prior to flowering. These authors described, however, a positive correlation between overall allergenicity and the average precipitation occurred during the winter months, for all the cultivars analyzed.

Several relevant differences are noticeable between the present study and that of Fernández-Caldas et al. (2007). First, this work combines the information available on four allergens, Ole e 1, Ole e 2, Ole e 5 and Ole e 9, instead of the olive pollen major allergen only. Therefore, the conclusions obtained are more similar to those described by Fernández-Caldas et al. referring to whole olive pollen allergenicity than those referring to Ole e 1 only. On the other hand, the present study uses accumulated data instead of averages for parameters like precipitation and number of monthly/seasonal/yearly days of rain. Moreover, although in some cases, monthly averages of temperature and relative humidity were considered, specific correlations were calculated upon the accumulated data for seasonal/yearly periods.

From an allergenic point of view, the present results suggest that those years with high levels of precipitation and high temperatures during the winter and the spring periods, contribute an enhanced allergenic content of the pollen grains at the end of the spring. We also may postulate that those areas of olive culture which are supplemented by artificial watering may yield a higher allergenicity for olive pollen-sensitized patients. The same would apply to urban areas, where ornamental olive trees are usually watered.

From an agronomic point of view, the described increase in the expression of the allergen Ole e 1 which takes place under increased temperature and precipitation might result in important differences in the reproductive behavior of the plant. Thus, preliminary observations (Morales et al. unpublished), indicated that the enhanced levels of Ole e 1 correlated to increased rates of pollen germination and lower degree of olive selfincompatibility. This hypothesis is also in good agreement with experimental observations made by several authors as regard to the effects of climatic factor on self-pollination and productivity in the olive (Androulakis and Loupassaki 1990, Lavee et al. 2002).

## **Author details**

68 Current Insights in Pollen Allergens

plant.

cultivars analyzed.

seasonal/yearly periods.

the expression of allergens in certain olive cultivars.

two consecutive years. Another study suggests a higher allergenic response to the Bet v 1 allergen present in birch pollen extracts grown in high average daily temperatures, in comparison to trees cultivated at lower temperatures (Ahlholm et al. 1998). This assay was carried out by using sera from patients sensitive to this pollen by using immunoblotting experiments. Our results indicate that high temperatures also seem to exert an increase in

We may extrapolate that an increase in the average temperatures would bring a parallel increase in pollen allergenic content and allergenicity as a consequence. This could be therefore one of the consequences of the so-called global warming produced by the greenhouse effect (Ahlholm et al. 1998; Cecchi et al. 2010). Moreover, we may speculate that the fact that urban areas are often exposed to temperatures 2 to 8 °C higher that rural areas (Oke 1987) may result in increased olive pollen allergenicity and allergy prevalence in and around the cities, where the olive tree is also present, as it is widely used as an ornamental

Fernández-Caldas et al. (2007) performed a similar study using six olive cultivars, over a period of five consecutive years. This study analyzed the content in the olive pollen major allergen (Ole e 1) only, which did not show statistically significant correlations to the precipitation average, nor to the average temperatures over the winter months prior to flowering. These authors described, however, a positive correlation between overall allergenicity and the average precipitation occurred during the winter months, for all the

Several relevant differences are noticeable between the present study and that of Fernández-Caldas et al. (2007). First, this work combines the information available on four allergens, Ole e 1, Ole e 2, Ole e 5 and Ole e 9, instead of the olive pollen major allergen only. Therefore, the conclusions obtained are more similar to those described by Fernández-Caldas et al. referring to whole olive pollen allergenicity than those referring to Ole e 1 only. On the other hand, the present study uses accumulated data instead of averages for parameters like precipitation and number of monthly/seasonal/yearly days of rain. Moreover, although in some cases, monthly averages of temperature and relative humidity were considered, specific correlations were calculated upon the accumulated data for

From an allergenic point of view, the present results suggest that those years with high levels of precipitation and high temperatures during the winter and the spring periods, contribute an enhanced allergenic content of the pollen grains at the end of the spring. We also may postulate that those areas of olive culture which are supplemented by artificial watering may yield a higher allergenicity for olive pollen-sensitized patients. The same

From an agronomic point of view, the described increase in the expression of the allergen Ole e 1 which takes place under increased temperature and precipitation might result in important differences in the reproductive behavior of the plant. Thus, preliminary observations (Morales et al. unpublished), indicated that the enhanced levels of Ole e 1

would apply to urban areas, where ornamental olive trees are usually watered.

Sonia Morales *Proteomic Research Service, Hospital Universitario San Cecilio, Granada, Spain Estación Experimental del Zaidín (CSIC), Granada, Spain* 

Antonio Jesús Castro, María Isabel Rodríguez-García and Juan de Dios Alché *Estación Experimental del Zaidín (CSIC), Granada, Spain* 

## **Acknowledgement**

This work was supported by the Spanish Ministry of Science and Innovation (MICINN) (ERDF-cofinanced projects AGL2008-00517, BFU2011-22779 and PIE-200840I186) and the Junta de Andalucía (ERDF-cofinanced projects P2010-CVI5767 and P2010-AGR6274). The authors acknowledge the availability of plant material and the collaboration of the staff of the IFAPA center "Venta del Llano" (Mengíbar, Spain) depending from the Andalusian Regional Government.

#### **5. References**


Galan, C., Cariñanos, P., Garcia-Mazo, H., Alcazar, P., & Dominguez-Vilches, E. (2001). Model for forecasting *Olea europaea* L. airborne pollen in South-West Andalusia, Spain. *International Journal of Biometeorology*, Vol. 45, No. 2, pp. 59-63, ISSN 0020-7128

**Chapter 5** 

© 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.

**Differential Immune-Reactivity and Subcellular** 

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;

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

**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** 

Kovar et al 2000).

