ORIGINAL ARTICLE

Two-dimensional patterns of soluble proteins including threehydrolytic enzymes of mature pollen of tristylous Lythrumsalicaria

A. Kalinowski Æ A. Bocian Æ A. Kosmala ÆK. Winiarczyk

Received: 18 July 2006 / Accepted: 9 December 2006 / Published online: 15 March 2007

� Springer-Verlag 2007

Abstract Lythrum salicaria, now a widespread invasive

species, exhibits tristyly, a form of heteromorphic selfin-

compatibility. In tristyly, each plant exhibits one (and only

one) of three morphologically different floral forms.

Moreover, each flower produces two types of stamens, and

these two exhibit different incompatibility reactions. Dif-

ferences between stamens of a single flower must be the

result of epigenetic phenomena and for that reason, we

performed two-dimensional gel electrophoresis (2-DE) to

analyze fractions of soluble proteins derived from the pollen

coat and protoplast including three hydrolytic enzymes

from the six different stamen types (two from each of three

floral forms). There were significant differences in the 2-D

protein profiles both between pollen from the same flower

and between the same type of pollen from two different

flowers, in the pollen coat as well as in the protoplast ex-

tracts. In five of the six samples of pollen fractions, char-

acteristic peptides were found. Quantitative differences

between pollen from the same flower were observed in case

of esterases. Furthermore, analysis of proteases and acid

phosphatases revealed also qualitative differences between

these enzymes in pollen from the same flower.

Keywords Lythrum salicaria � Pollen � Protein/enzymes �Two-dimensional gel electrophoresis

Introduction

Classically, (see Darwin 1877) tristylous populations con-

tain approximately equal proportion of three genetically

determined floral forms. Individuals of each form are

moderately self incompatible and incompatible also with

other individuals showing the same form. This kind of

species has long been studied because it exhibits a fasci-

nating syndrome of characteristics. Plants of each form

exhibit one, and only one, of three different lengths of

styles. Flowers of the long-styled form contain also two

types of stamens: mid- and short length. Flowers of the

mid-styled form contain both long- and short stamens, and

those of the short-styled form contain mid- and long sta-

mens. Long stamens, in every case, produce pollen that is

larger than that of the mid-length stamens, pollen of which

is larger than that of the short stamens. The inheritance of

the three floral forms is determined by two diallelic loci,

termed S and M. Long-styled plants are homozygous

recessive at both loci (ssmm), mid-styled plants are ssM-

(meaning ssMM or ssMm), and any plant carrying the

dominant S allele will produce short-styled flowers (that is,

S is epistatic to M). It should be noted that the S locus

referred to here presumably bears no relation to the S locus

known to influence homomorphic selfincompatibility. For

more recent references, see Barrett (1992, 2002, 2004),

Barrett et al. (2000), de Nettancourt (2001) and references

Communicated by Celestina Mariani.

A. Kalinowski � A. Bocian � A. Kosmala (&)

Institute of Plant Genetics, Polish Academy of Sciences,

Strzeszynska 34, 60-479 Poznan, Poland

e-mail: akos@igr.poznan.pl

A. Kalinowski

e-mail: akal@igr.poznan.pl

A. Bocian

e-mail: aboc@igr.poznan.pl

K. Winiarczyk

Department of Anatomy and Cytology of Plants,

Marie Curie-Skłodowska University,

Akademicka 19, 20-033 Lublin, Poland

e-mail: winiark@biotop.umcs.lublin.pl

123

Sex Plant Reprod (2007) 20:51–62

DOI 10.1007/s00497-006-0042-4

therein. The present paper is an investigation into the

differences between genetically identical, but behaviorally

dissimilar, pollen from a single form of Lythrum salicaria

(purple loosestrife) plant.

Diversity in forms and functions of flowers within an-

giosperms is favored by natural selection. The spatial and

temporal diversity systems of plant reproductive organs, in

most cases, are associated with the promotion of cross-

fertilization reducing negative effects of self-fertilization.

Floral heteromorphism, like heterostyly and enathiostyly,

is active promoting of a directional pollen dispersal be-

tween plants, while homomorphic self-incompatibility is

passive in character. In heterostylic plants, different polli-

nation and mating systems, even between closely related

taxa, indicate that the traits related with reproduction are

evolutionarily labile (Ganders 1979). The paper by Hus-

band and Barrett (1993) is a good example presenting the

evolutionary instability of tristylous Eichhornia paniculata.

The 24 enzymatic loci from flower buds (11 enzyme sys-

tems) in 167 populations in north-eastern Brazil were

investigated using the starch gel electrophoresis method.

The authors showed that 118 (70.7%) populations were

composed from trimorphic-flower plants, which were poly-

morphic in 20 enzymatic loci. A total of 42 (21.1%) popu-

lations were composed of long- and mid-styled flowers,

which were polymorphic in 16 loci, and the remaining 7

(4.2%) populations were composed of mid-styled flowers

only. No data regarding the analysis of generative parts of

flowers on the molecular level have been found so far.

Different lengths of stamens and styles were observed in

tristylous L. salicaria in relationship with different stigma

and pollen morphology (Hermann et al. 1999). The num-

bers of papillae are the same for each stigma type; how-

ever, the stigma of the long style possesses relatively

bigger, longer and more scarcely distributed papillae,

compared to the stigma of the short style and intermediate

stigma of the mid style (Hermann et al. 1999). The dif-

ferences in profiles of stigma proteins, generated using

two-dimensional gel electrophoresis, revealed that each of

the three stigma types possesses characteristic peptides

(Kalinowski et al., to be published).

Lythrum salicaria shows also differences both in pollen

morphology and color (Mulcahy and Caporello 1970).

Pollen from the long stamen is green, while that from the

mid- and short stamens is yellow. Differences in pollen

diameters were also observed. The biggest pollen comes

from the long stamen and the smallest one from the short

stamen. The diameters were correlated with the amount of

pollen production in the anthers (Mulcahy and Caporello

1970); however, the other results showed no differences in

the pollen diameters (O’Neil 1992). Mal and Hermann

(2000) obtained similar results as Mulcahy and Caporello

(1970) and they pointed out that in bigger populations the

plants produced relatively bigger pollen compared to plants

from small populations. Thus, both the yield of pollen

production and its diameter are influenced by environ-

mental conditions. No references concerning molecular

markers for different pollen types of L. salicaria have been

published so far.

One of the most important phenomena of heteromorphic

incompatibility is a relation between pollen morphology

and directional pollen flow between different morphs. It is

obvious that compatible pollination occurs between the

stigma and anther located on the same level; incompatible

pollination is not as strongly restricted as homomorphic

self-incompatibility. Mulcahy and Caporello (1970) found a

significant amount of self-pollination within the same

flowers after artificial pollination of emasculated flowers of

L. salicaria; however, this pollination was predominant

between the stamen and styles of the same length. These

authors, just like Ottenbreit and Staniforth (1994), con-

cluded that most events of incompatible pollination were

found in mid-styled flowers. Furthermore, the plants with

this type of flower produced the highest set of seed. In

purple loosestrife, asymmetric pollen flow is based on a

limited deposition of pollen on the short style and its

unlimited deposition on long- and mid styles (O’Neil 1992).

The characteristics of possible S loci in determined

heterostyly are still unknown. Barrett et al. (2000) wrote:

‘‘Protein differences between morphs may have nothing

to do with incompatibility per se but instead may simply

be associated with differential organs growth’’, and:

‘‘...ignorance of the molecular genetics of heterostyly will

continue’’.

Some results regarding organ-specific proteins were

obtained for distylous plants. Using one- and two-dimen-

sional gel electrophoresis methods in Averrhoa caram-

bola, Wong et al. (1994) detected 72 kDa protein in the

pin style and 45 kDa protein in the stamens of the thrum

morph. The specific 70 kDa peptide was also found in

vegetative organs. In the stigmas and styles of three spe-

cies of distylous Turnera three characteristic protein bands

were identified. Two of them were only found in the

pollen of short-styled plants. It was evident that genes

controlling the production of these proteins were closely

linked with the S-alleles. The functions of these proteins

have not been recognized, but it is supposed that small

amounts of these proteins are also present in vegetative

organs (Athanasiou and Shore 1997).

Since no data concerning floral-morph-specific molec-

ular markers are available, particularly for pollen and

pistils in tristylous L. salicaria, we started with separate

analyses of the pollen coat and pollen protoplast proteins.

Tristylous species are particularly valuable for such

investigations because each allows comparison of two

pollen types within a single type of flower. That is,

52 Sex Plant Reprod (2007) 20:51–62

123

comparison of the pollen from long- and short stamens

within a single mid-styled plant should be more informa-

tive than comparing pollen from long- and short stamens of

a distylous species, such as Primula sp. In Primula, the

differences may relate to heterostyly, but more likely they

represent irrelevant differences between two individual

plants. With the tristylous species, the comparison can be

made within a single plant, thus eliminating many extra-

neous differences. Furthermore, proteins common to, for

example, long stamens from mid- and short-styled Lythrum

plants can be selected for further investigation if they are

not found in other pollen types of the same plants.

Different proteins (some of them possess enzymatic

activity), lipid-like substances, carbohydrates and second-

ary chemicals, mainly flavones and carotenes, could be

found both in the pollen coat and in the protoplast. Proteins

in the protoplast and in the inner layers of the intine are

under control of microgametophyte genes. Proteins pro-

duced in the tapetum and deposited with other substances

in cavities and on the surface of exine formed a pollen coat

(Heslop-Harrison et al. 1973; Vithanage and Knox 1979;

Murphy and Ross 1998; Doughty et al. 2000). The pollen

coat may be easily washed off with a water solution of

mannitol (Howlett et al. 1975), cyclohexane (Doughty et al.

1993) and with diethyl ether (Suen et al. 2003). We pre-

ferred to use an isotonic solution of sucrose with dithio-

treitol and other chemicals—it allowed us to analyze

soluble proteins including enzymes. The use of the solution

without dithiotreitol allowed us to perform in vivo exper-

iments (Kalinowski et al. 2002b).

The current work presents the results of the qualitative

analyses of sporophytically and gametophytically ex-

pressed pollen proteins of L. salicaria together with the

analysis of pollen enzymes without separation into pollen

coat and protoplast fractions, with the use of two-dimen-

sional gel electrophoresis with SDS. The aim of the study

was to find: (1) differences between pollen from the same

morph, (2) differences between the same type of pollen

from different morphs, (3) pollen coat and protoplast

characteristic peptides for each type of pollen.

Materials and methods

Three plants—one with long-, one with mid- and one with

short-styled flowers—were transplanted from their natural

habitat to big flowerpots and then were kept in the garden

to provide the same conditions.

Early morning, before opening, the flowers were col-

lected and kept in Petri dishes with wet filter paper at 4�C

to prevent the dehiscence of the anthers. Indehiscent an-

thers were transferred to sieves. Flowers with opening

anthers were discarded. After 20–30 min. under bubble

light, the anthers were broken and then pollen was sifted

using a little stiff-haired brush. Mature pollen was kept in

Eppendorf tubes at –70�C.

Six samples of pollen were taken for analysis. The

samples were designated as follows: (L, M and S—long-,

mid- and short style, respectively; l, m, and s—long-, mid-

and short stamen, respectively) according to Mulcahy and

Caporello (1970). Thus, Lm indicates a sample from a

long-styled flower, mid-length stamens; Ls—long-styled

flower, short stamens, etc. Samples were as follows: (1)

Lm, (2) Ls, (3) Ml, (4) Ms, (5) Sl and (6) Sm. For con-

venience the following marks are used: pc—pollen coat,

pt—pollen protoplast.

Soluble proteins: pollen coat elution, extraction

of proteins and two-dimensional gel electrophoresis

Pollen coat elution and protein extraction were performed

as in Kalinowski et al. (2002b). Samples of 10 mg of each

type of pollen were shaken gently at room temperature for

three times with 300 ll of isotonic extraction buffer (0.7 M

sucrose, 0.5 M Tris, 30 mM HCl, 0.1 M KCl) according to

Hurkman and Tanaka (1986). During the first wash, pollen

samples were eluted for 20 min. and during the other two

washes for 5 min. Each of the three fractions was then

shaken successively with phenol dissolved in water

(400 ll) to ensure the concentration of proteins from three

eluates in one volume of phenol solution. Pollen devoid of

pollen coat proteins was homogenized with 400 ll of the

extraction buffer and then shaken with phenol. Proteins

were precipitated from the phenol phase by a solution of

0.1 M ammonium acetate in methanol. After drying, the

proteins were dissolved in an appropriate volume of

resolving buffer (9 M urea, 4% Nonidet NP-40, 2% 2-

mercaptoethanol, 2% ampholytes pH 2–4)—20 ll in case

of pollen coat and 30 ll in case of protoplast fraction,

according to Hurkman and Tanaka (1986). On the column

gels, 15 ll of pollen coat and 10 ll of pollen protoplast

extracts were applied.

Two-dimensional gel electrophoresis was performed

according to Hochstrasser et al. (1988). In the first

dimension, isoelectrofocusing (IEF), a 0.8 · 150 mm

capillary was used for polymerization of polyacrylamide

column gel with Servalyte pH range 3–10. In the second

dimension (SDS-PAGE), the proteins were separated in

13% polyacrylamide slabs (1 · 140 · 140 mm). After

electrophoresis, the peptides were stained with silver ni-

trate according to Heukeshoven and Dernick (1985).

Enzymes: extraction of proteins and 2-DE conditions

The 20 mg of each type of pollen (six samples) was

homogenized with 80 ll solution of 15% sucrose with 2%

Sex Plant Reprod (2007) 20:51–62 53

123

dithiotreitol (DTT) and then centrifuged at 14,000 rpm

(Hettich—Universal 32R; 1620A) at 4�C. The volume of

20 ll of extract was applied on column gels for separation

in the first dimension (IEF).

Analyses of esterases (EST) and acid phosphatases

(ACPH) using two-dimensional electrophoresis in dena-

turing conditions (with SDS) and the renaturing of their

activities were conducted according to a previously

developed methodology (Kalinowski et al. 2002a). After

the separation of proteases (PRO) in the first dimension

(IEF), the column gels were transferred into the same

buffer as used for EST and ACPH, containing 0.1% SDS.

In the second dimension, the proteins were separated in 7%

polyacrylamide slabs containing 0.4% SDS and 0.4% gel-

atin from porcine skin as a substrate for protease activity.

Undigested gelatin in the gel was stained with Coomasie

Brillant Blue G-250 (Michaud et al. 1993).

Gel analysis

Each extraction procedure and electrophoretic separation

was performed twice. Total separated protein/enzyme

spots on the gels were scanned (SHARP JX-330) and

subjected to LabScan program processing. The data were

further processed with Image Master 2-D Elite LKB

software. Enzymes were compared also quantitatively on

the basis of light absorption of their particular isoforms

revealed after electrophoresis (using the densitometry

option in Image Master 2-D Elite software). The obtained

data were then used to estimate approximate enzymatic

activities.

The Pharmacia 2-DE calibration kit (17-0582) and

Sigma silver stain SDS molecular weight mixture (M-

6539) were used to standardize the gels and to determine

the molecular weights (MW) and isoelectric points (iP) of

the analyzed proteins.

Results

All the 2-D patterns were highly repeatable. The 2-D

profiles of proteins from the pollen coat were different

from those of the protoplast. Furthermore, differences be-

tween two types of pollen from the same flower and also

between the same type of pollen produced in two morphs

were found. ‘‘Characteristic peptides’’ refer to the peptides

present only in one of the six samples of pollen coat

fractions or pollen protoplast fractions, or the isozymes

present only in one of the six types of pollen. Excluding the

pollen coat of the Sl, characteristic peptides were observed

for the remaining samples both from the pollen coat and

from the protoplast.

Pollen coat proteins

Proteins from six samples of the pollen coat (pc) after two-

dimensional gel electrophoresis separated at a pH range of

4.0–9.8 and their molecular weights were from ~9 to

~80 kDa. In each pc fraction, excluding the Sl, character-

istic peptides were found and their numbers were as fol-

lows: Lm—9, Ls—3, Ml—5, Ms—5 and Sm—19. These

peptides were marked on Figs. 1, 2, and 3.

Taking into account two electrophoretic parameters

(molecular weights and isoelectric points), clear differ-

ences between protein profiles of six pollen coat fractions

were observed. Comparing pollen coat fractions of the two

pollen types found within a single flower morph, we found

that in the pc of the Lm 53 peptides were present, which

were absent in the pc of the Ls. Inversely, 68 peptides were

identified in the pc of the Ls and undetected in the pc of the

Lm. In case of mid-styled flowers, 26 pc proteins charac-

teristic for the Ml were undetected in the pc profiles of the

Ms. Moreover, 85 pc peptides found in the Ms were not

detected in the Ml. The pc of the Sl pollen contained 12

Fig. 1 The 2-D protein patterns

from pollen coat of mid- (Lm)

and short (Ls) stamen of a long-

styled flower. Characteristic

peptides were marked with

arrows. Their molecular

weights in kDa and isoelectric

points at pH (in brackets) were

for the Lm: 11.5 (5.1), 12.0

(5.2), 23.0 (8.8), 27.0 (7.8), 39.0

(5.5 and 5.6) and 56.0 (8.9, 9.2

and 9.4) and for the Ls: 19.0

(5.0), 20.5 (8.7) and 39.0 (9.0)

54 Sex Plant Reprod (2007) 20:51–62

123

peptides, which were absent in the pc of the Sm, and in-

versely, 177 pc peptides from the Sm were not detected in

the pc of the Sl.

Differences were also detected for the same type of

pollen found in different floral morphs. In the pc of the Lm,

34 characteristic peptides were detected, which were absent

in the pc of the Sm. The profile of the pc of the Sm showed

the occurrence of 172 peptides undetected in the profile of

the pc of the Lm. In the coat of the Ls, 13 peptides were

detected, while these were simultaneously undetected in

the pc of the Ms. Inversely, 76 peptides present in the Ms

were absent in the Ls. The comparisons of 2-D protein

profiles within the pc of long-stamen pollen showed that in

the Ml 23 peptides present were undetected in the Sl and 37

peptides were observed in the Sl and simultaneously not

observed in the Ml. Thus, comparing the pollen coat pep-

tide profiles from a single type of pollen from different

forms of flowers, we found that differences were highest

for mid-stamen pollen and lowest for long-stamen pollen.

One of the most interesting results was the identification

of common pollen coat peptides for the same type of pollen

produced in two different types of flowers (the peptides

which were simultaneously absent in the two other pollen

types). For the pc of long-stamen pollen eight (omitting

two weakly stained), for mid stamen—nine and for short

stamen—five such peptides were found. The MWs and iPs

(at pH) of these peptides are given in Table 1.

Fig. 2 The 2-D protein patterns

from pollen coat of long- (Ml)and short (Ms) stamen of a mid-

styled flower. Characteristic

peptides were marked with

arrows. Their molecular

weights in kDa and isoelectric

points at pH (in brackets) were

for the Ml: 11.0 (4.5), 14.2

(4.5), 18.4 (8.5), 29.0 (5.8) and

44.0 (5.9) and for the Ms: 13.0

(7.0), 14.5 (4.4), 26.0 (5.6) 28.5

(7.9) and over 66.0 kDa marker

at pH 6.2

Fig. 3 The 2-D protein patterns

from pollen coat of long- (Sl)and mid (Sm) stamen of a short-

styled flower. Characteristic

peptides were marked with

arrows. Their molecular

weights in kDa and isoelectric

points at pH (in brackets) were

for the Sm as follows: 11.0

(5.9), 11.5 (5.6 and 7.6), 12.0

(5.0, 7.6 and 7.8), 23.0 (7.4),

28.5 (5.4 and 5.5). 32.0 (8.7),

three 33.0–34.0 (5.6–5.7), 36.0

(8.2), 39.0 (4.9), 43.0 (8.3), 46.0

(8.5) and 61.0 (7.4 and 7.6)

Table 1 List of pollen coat and protoplast peptides present only in

one of the long-, mid- or short-stamen pollen

Lm Ls Ml

10.4/4.9 10.5/6.1 11.0/6.8

12.0/7.5 21.5/6.8 18.0/8.7

12.5/5.1 22.0/6.9 21.5/5.9

13.0/5.0 31.0/8.5 23.0/6.9

14.0/4.9 35.0/7.9 23.5/7.1

14.2/4.8 44.0/7.5

15.0/4.7 49.0/5.6

31.0/7.6 62.0/7.3

32.0/7.5

In the first place the MW in kDa and in the second place iP at pH are

given (kDa/pH)

Sex Plant Reprod (2007) 20:51–62 55

123

Pollen protoplast proteins

Proteins from six samples of pollen protoplast (pt) after

two-dimensional gel electrophoresis, as was the case with

pollen coat proteins, were separated at pH 3.5–9.8 range

and their molecular weight differed from ~9 to ~80 kDa. In

each of six pt fractions, characteristic peptides were found

and their numbers were as follows: Lm—7, Ls—6, Ml—8,

Ms—6, Sl—3 and Sm—3. These peptides were marked on

Figs. 4, 5, and 6.

Similar to the 2-D patterns of pollen coat proteins, the 2-

D patterns of protoplast protein fractions between two types

of pollen from the same kind of flower were also different.

In the pt of the Lm, 74 characteristic peptides were present;

simultaneously, these peptides were absent in the pt of the

Ls. Inversely, 47 peptides typical for the pt of the Ls were

undetected in the pt of the Lm. In the case of the mid-style

flowers, 81 proteins identified in the pt of the Ml were not

observed in the pt of the Ms, whereas 39 characteristic

peptides found in the pt of the Ms were not detected in the

Ml. The pt of the Sl pollen contained 39 peptides, which

were absent in the pt of the Sm, while the pt of the Sm

contained 32 peptides not detected in the pt of the Sl.

Comparing pc and pt protein profiles between the same

type of pollen showed that in the pt of the Lm, 156 peptides

were detected—they were absent in the pt of the Sm. In-

versely, 89 peptides in pc of the Sm (undetected in pt of the

Lm) were found. In the pollen coat of Ls, 99 peptides were

detected and simultaneously undetected in the pt of the Ms,

while 73 peptides characteristic for the Ms were not ob-

served in the Ls. Comparisons between the 2-D profiles of

the pt of long-stamen pollen showed that 36 Ml peptides

were undetected in the Sl, while 75 Sl peptides were absent

in the Ml. These comparisons showed that the pollen pro-

toplast fraction profiles were most varied in the case of

mid-stamen pollen and least varied in the case of long-

stamen pollen, similar to the results obtained for pollen

coat protein profiles. We identified also common pollen

protoplast peptides for the same type of pollen produced in

two different types of flowers (the peptides that were

simultaneously absent in two remaining pollen types). Only

two such peptides in the pt of the Ml were found. Their

Fig. 4 The 2-D protein patterns

from pollen protoplast of mid-

(Lm) and short (Ls) stamen of a

long-styled flower.

Characteristic peptides were

marked with arrows. Their

molecular weights in kDa and

isoelectric points at pH (in

brackets) were for the Lm: 20.5

(9.2), 21.0 (8.9), 26.5 (9.2 and

9.3), 29.0 (8.4 and 8.6) and 42.0

(4.6) and for the Ls: 111.5 (9.1),

23.0 (9.1), 24.0 (9.3), 41.0 (9.2),

43.0 (9.1) and 46.0 (9.2)

Fig. 5 The 2-D protein patterns

from pollen protoplast of long-

(Ml) and short (Ms) stamen

of a mid-styled flower.

Characteristic peptides were

marked with arrows. Their

molecular weights in kDa and

isoelectric points at pH (in

brackets) were for the Ml: 12.0

(7.6), 12.5 (7.2), 13.0 (7.7), 15.0

(3.9), 15.5 (8.2), 18.4 (3.7), 40.0

(8.7) and 47.0 (8.3) and for the

Ms: 10.5 (7.8), 15.0 (9.1 and

9.3), 22.5 (4.6), 25.0 (3.9) and

26.0 (3.9)

56 Sex Plant Reprod (2007) 20:51–62

123

electrophoretic parameters (MW and iP) were as follows:

19 kDa at pH 9.6 and 64 kDa at pH 7.8.

Enzymes

Esterases

Four isoesterases were separated using two-dimensional

gel electrophoresis in denaturing conditions (with SDS).

Their MWs ranged from 17.5 to 25 kDa, at pH 5.1–6.3

(Fig. 7). No qualitative differences were observed be-

tween the two types of pollen from the same morph.

Only quantitative differences were found between the Ml

and the Sl, and also between the Lm and the Sm. Dif-

ferences in the activities of some isoesterases from the

same type of pollen from two morphs were observed

after electrophoresis. The isophorm designated as no. 2

from the Lm was twice as active as the isophorm from

the Ls pollen, while three remaining pollen isoesterases

revealed similar, high activities after electrophoresis.

Isophorm no. 4 was significantly less active in the Sl

than in the Sm. Four isoesterases in the Ml and the Ms

pollen were clearly less active compared to the pollen

isoesterases from the L and the S morphs, while these

enzymes from the Ml were slightly more active than

those from the Ms.

Fig. 6 The 2-D protein patterns

from pollen protoplast of long-

(Sl) and mid (Sm) stamen of a

short-styled flower.

Characteristic peptides were

marked with arrows. Their

molecular weights in kDa and

isoelectric points at pH (in

brackets) for Sl were as follows:

38.0 (4.1), 40.0 (3.9) and 45.0

(4.6) and for Sm: 49 (4.3), 50.0

(4.1) and 54.0 (4.0)

Fig. 7 Fragments of gels

showed 2-D profiles of esterases

from six samples of pollen of

purple loosestrife. Activities of

particular isoesterases were

based on light absorption

revealed after electrophoresis

using the densitometry option in

Image Master 2-D Elite

software

Sex Plant Reprod (2007) 20:51–62 57

123

Acid phosphatases

Eight isophosphatases were detected in six samples of

pollen. Their MWs ranged from 66 to ~100 kDa and their

isoelectric points were at pH range 5.8–6.4. As was shown

in Fig. 8, only quantitative differences between the Lm and

the Ls pollen were observed. Isophorm no. 1 from the Ls

and the Lm revealed the same low activities after electro-

phoresis, while isophorm no. 2 was four times more active.

The other two (nos. 3 and 4) forms were over two times

more active in the Lm than in the Ls pollen. Pollen from

the M-styled flowers was different with respect to the

occurrence of isophorms nos. 5 and 6, but the activities of

all the isozymes were similar after electrophoresis. The

highest, both qualitative and quantitative, differences were

found in pollen from short-style flowers (Fig. 8). Three

isophorms (nos. 1, 2 and 8) present in the Sl and the Sm

were more active in the first type of pollen.

The same types of pollen from two morphs were also

compared. Two isozymes (no. 1 and no. 2) were more

intensively stained in the Lm and in the Sl than in the Sm.

In the Ls and the Ms, these isozymes were stained with an

almost identical intensity.

Proteases

Several proteases were observed in six samples of pollen of

purple loosestrife. They were separated at the pH range of

4.1–6.0 and their molecular weights ranged from 26 to

~180 kDa (Fig. 9). One of the biggest spots, designated as

no.1, was probably an effect of the activities of six prote-

ases, which had very close isoelectric points and similar

MWs. The other five proteases showed low activities;

however, they showed qualitative differences between the

Lm and the Ls and between the Sl and the Sm pollen. No

differences between the Ml and the Ms pollen were iden-

tified. Identical 2-D profiles of proteases were obtained for

the Ls and the Sl and also for the Ml and the Ms. After

comparisons between the same type of pollen derived from

two morphs, two different protease spots were observed.

Discussion

Excluding general information with regard to starch, lipids

and flavonoids contents in the pollen of L. salicaria, no

data about molecular markers (proteins and DNA, for

example RAPD or AFLP) of finding genes governing

heterostyly have been published. It was suggested that

differences in proteins may have nothing with the deter-

mination of incompatibility and that they may only be

associated with the morphological development of sexual

organs in heterostylous flowers (Barrett et al. 2000). That

hypothesis is in conflict with our results. Moreover, the

results described herein could suggest that the pollen coat

and protoplast protein/enzymes may have an important

Fig. 8 Fragments of gels

showed 2-D profiles of acid

phosphatases from six samples

of pollen of purple loosestrife.

Activities of particular

isoesterases were based on light

absorption revealed after

electrophoresis using the

densitometry option in Image

Master 2-D Elite software

58 Sex Plant Reprod (2007) 20:51–62

123

influence on the prezygotic events in L. salicaria. The

investigations presented in this paper represent the first

analysis of L. salicaria pollen on the molecular level and

could be the starting point for wider investigations

regarding the probable role of pollen and style proteins in

compatible and incompatible pollination in purple loose-

strife.

In tristylous Narcisus triandrus, a significantly lower

seed set was observed after self-pollination than after

cross-pollination (Sage et al. 1999). In this species, self-

sterility operates prezygotically as the ovarian self-

incompatibility. Authors hypothesized that the degradation

of the embryo sac was due to the absence of a stimulus.

Similar results have been obtained for L. salicaria by

O’Neil (1994). Pollination induces a cascade of changes in

relation with chemical signals (Du et al. 1996; Cheung and

Wu 1999; Cheung et al. 2000; Lindsey et al. 2002). Thus,

different proteins including probably also some enzymes in

different types of pollen coat may induce different changes

in the style of L. salicaria. Different chemical signals de-

rived from pollen coat chemicals, mainly proteins, induce

changes in the pathway of pollen tube growth that can

inhibit or stimulate this growth. For each of the six pollen

coat samples the characteristic peptides were found. Spe-

cific pollen coat proteins may induce some kinds of

changes in the pistil after incompatible pollination, which

are different from changes started after compatible polli-

nation.

Fig. 9 Fragments of gels

showed 2-D profiles of

proteases from six samples of

pollen of purple loosestrife

Sex Plant Reprod (2007) 20:51–62 59

123

Scribailo and Barrett (1991) showed that in L. salicaria

and L. junceum after incompatible pollination the pollen

tubes growth is inhibited in the stigma or in the upper part

of the style, while in tristylous Pontederia sagittata and P.

cordata that event takes place in the style and in the ovary.

Interactions between specifies are under control of S-genes

present both in the pollen and in the pistil (heteromorphic

incompatibility), (Richards 1986, cited after Scribailo and

Barrett 1991). Thus, it may suggest that some pollen coat

and protoplast characteristic peptides are also present in the

stigmas (Kalinowski, unpublished data) and they may be

involved in this interaction. However, Scribailo and Barrett

(1991) excluded an active rejection by molecular specifies

in P. sagittata. The authors suggested that incompatibility

is rather governed by interactions of heteromorphic traits.

In L. salicaria, except for compatible pollination, the

incompatible pollination was also observed. The lack of

physiological barriers to self-fertilization makes possible

the composition of pollen load on the stigma (Eckert and

Barrett 1994). The authors pollinated stigmas of Decodon

verticillatus using the mixture of self- and out-crossed

pollen and pointed out the possible signification of pollen

competition. The consequences of pollen competition was

seed set and seed germination. One of the requirements for

pollen competition is the existence of pollen polymorphism

(different pollen loads on the stigma); the distance between

the place of pollen arresting to the ovule location and the

number of ovules are also important (Mulcahy 1974;

Mulcahy and Mulcahy 1975; Winsor et al. 2000). Our re-

sults revealed protein polymorphism between six types of

pollen (between two pollen types from each of the three

morphs). Furthermore, characteristic peptides both in the

pollen coat and in the protoplast were found. Additionally,

2-D protein profiles from the stigmas of the three morphs

of L. salicaria (unpublished data) showed significant dif-

ferences.

This paper focuses on the differences in proteins. Two-

dimensional gel electrophoresis of pollen coat and protoplast

proteins as well as enzymes from unwashed pollen showed

differences between pollen from the same morph. Despite

the variations in the degree of incompatibility among pop-

ulations and even among individuals, differences between

pollen of L. salicaria presented in our work, together with

the results obtained by O’Neil (1994), suggest the existence

of preferences for one of the two types of pollen from dif-

ferent morphs in the case of compatible pollinations. These

preferences are probably genetically determined among the

populations because experiments were performed on plants

growing under identical conditions in the greenhouse. The

plants from which pollen was collected for our experiments

were also grown in the same garden conditions.

Lythrum salicaria produces pollen of different sizes

(Mulcahy and Caporello 1970). Moreover, Mal and

Hermann (2000) suggested differences between three types

of pollen also between populations. While the morphology

of exine and the composition of the pollen coat are under

the control of sporophyte genes, pollen size is under the

control of both sporophyte and microgametophyte genes

(Ottaviano and Mulcahy 1989). Common peptides for the

same type of pollen were mainly found among proteins

derived from the sporophyte; only two peptides were

identified in the protoplast of the Ml pollen. So far, there

are no answers to questions concerning the probable role of

these peptides in the legitimate/illegitimate pollination on

the basis of the presented results.

The 2-DE with SDS analysis of esterases showed no

qualitative differences between two types of pollen in each

of the three morphs. Only quantitative differences between

pollen from the same morph and between the same type of

pollen were observed. In Nicotiana alata, the esterases

were not associated with the expression of self-incompat-

ibility, but they were important in the pollen-pistil

interaction (Blaas and Bredemeijer 1978). Esterases-

hydrolyzing substances present in the stigma and style

facilitated the growth of the pollen tube in Brassica

(Hiscock et al. 2002). The results presented in this paper

may only suggest that pollen esterase activities are corre-

lated with differences in the anatomy of L. salicaria stig-

mas. Thus, pollen esterases could facilitate penetration of

the stigma by the pollen tube in compatible pollination and

this process could be quantitative in character.

The 2-D protein patterns of acid phosphatases showed

both qualitative (with the exception of the Lm and the Ls)

and quantitative differences between pollen from one

morph and between the same type of pollen derived from

different morphs. Roggen and Stanley (1969) stated that

phosphatases, proteinases and a-amylases inhibit pollen

tube growth. Phosphatases through hydrolysis of phosphate

residues act antagonistically to protein kinases, which play

an essential role in signal transductions (Kunz et al. 1996;

Cheung et al. 2000). Thus, the proportion of phosphory-

lation (catalyzed by protein kinases) and dephosphoryla-

tion (catalyzed by phosphatase) of proteins may induce

different signals in the pistil after legitimate pollination

when compared to the signals after illegitimate pollination.

Pollen proteases showed high activities after electro-

phoresis. Protease spot designated as no. 1 was probably an

effect of the activities of six proteases, which had very

close isoelectric points and similar MWs (clearly seen as a

negative picture). Different 2-D profiles of proteases were

observed both between the same type of pollen as well as

pollen from the same morphs, leaving out of account dif-

ferences between types of pollen. Hiscock and Dickinson

(1993) suggested that some proteases caused the death of

the stigma papillary cells. Pollen proteases facilitate pollen

tube growth through the hydrolysis of stigma proteins in

60 Sex Plant Reprod (2007) 20:51–62

123

the transmission tissue of the style (Radłowski et al. 1996).

However, some of the proteases can inhibit the growth of

the pollen tube as well (Roggen and Stanley 1969). Thus,

proteases may play an important role in the pollen–pistil

interaction in tristylous L. salicaria.

The differences between 2-D protein patterns of pollen

within the same morph as well as between the same type of

pollen from two morphs may be partially explained by

post-translational modifications of the observed proteins

(Zannis and Breslow 1981).

Acknowledgment We wish to thank Prof. David L. Mulcahy,

University of Massachusetts, for his inspiration to start the investi-

gation of pollen molecular markers of purple loosestrife, for his

extensive and valuable advice before the performance of the experi-

ments and for his critical reading of the manuscript.

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