Callose plug deposition patterns vary in pollen tubes of Arabidopsis thalianaecotypes and tomato species
© Qin et al.; licensee BioMed Central Ltd. 2012
Received: 10 May 2012
Accepted: 26 September 2012
Published: 3 October 2012
The pollen grain contains the male gametophyte that extends a pollen tube that grows through female tissues in order to deliver sperm to the embryo sac for double fertilization. Growing pollen tubes form periodic callose plugs that are thought to block off the older parts of the tube and maintain the cytoplasm near the growing tip. The morphology of callose plugs and the patterns of their deposition were previously shown to vary among species, but variation within a species had not been examined. We therefore systematically examined callose plug deposition in Arabidopsis thaliana ecotypes, tested for heritability using reciprocal crosses between ecotypes that had differing deposition patterns, and investigated the relationship between callose plugs and pollen tube growth rate. We also surveyed callose plug deposition patterns in different species of tomato.
We used in vitro grown pollen tubes of 14 different A. thaliana ecotypes and measured the distance from the pollen grain pore to the first callose plug (termed first interval). This distance varied among Arabidopsis ecotypes and in some cases even within an ecotype. Pollen tubes without a callose plug were shorter than those with a callose plug, and tubes with a callose plug near the grain were, on average, longer than those with the first callose plug farther from the grain. Variations in the first callose plug position were also observed between different species of tomato.
We showed that the position of the first callose plug varied among Arabidopsis ecotypes and in tomato species, and that callose plug deposition patterns were heritable. These findings lay a foundation for mapping genes that regulate callose plug deposition or that determine pollen tube length or growth rate.
KeywordsCallose plugs Pollen tube growth Sperm
In angiosperms, the pollen grain contains the male gametophyte. The male gametophyte extends a pollen tube that grows through female tissues in order to deliver sperm to the embryo sac for double fertilization. Callose, a β 1,3-glucan, is the major component of the pollen tube cell wall . Callose can be visualized by staining with decolorized aniline blue . Growing pollen tubes form periodic callose plugs that are thought to block off the older parts of the tube and maintain the cytoplasm near the growing tip . Callose plugs exist in pollen tubes of all flowering plants, although their morphology and the pattern of callose plug deposition varies among species . Callose plugs were proposed as a critical novelty for accelerated pollen tube growth during angiosperm evolution , as angiosperm pollen tubes have callosic walls and callose plugs, whereas callose plugs are absent in pollen tubes of gymnosperms.
Callose plug deposition has been correlated with pollen tube growth rate. For example, the number of callose plugs was used as an indicator of pollen tube growth rate in Hibiscus moscheutos[6, 7]. In tomato, antisense lines for the pollen receptor kinase LePRK2 showed abnormal callose plug deposition and slower pollen tube growth than in wild type . LeSHY is a protein that interacts with LePRK2; in Petunia, antisense lines for SHY showed no callose plugs in pollen tubes, and nearly all the pollen tubes failed to reach ovules . However, in Arabidopsis, one report  questioned a need for callose plugs, as pollen tubes carrying a mutant allele of callose synthase 5, cals5-3, had no callose plugs, but grew normally in vitro and in vivo. Seed set on cals5-3 homozygous plants was normal, although transmission of the cal5-3 allele was slightly reduced in heterozygotes (when in competition with WT pollen tubes).
Arabidopsis has been used as a model plant for genetic studies. Many ecotypes have been used to investigate genetic variation in numerous physiological processes , and recombinant inbred lines and doubled haploid populations have been used to map genes underlying such variation [12, 13]. Although callose plug morphology differs in different species , to our knowledge variation within a species has not been examined. During the course of other experiments, we noticed that pollen tubes of the Arabidopsis thaliana ecotypes Ler and Columbia (Col) appeared to vary in the position of the first callose plug; Columbia had the callose plug near the grain, while in Ler the callose plug was farther away. We therefore decided to systematically examine callose plug position in many Arabidopsis ecotypes, and in tomato species. Here we document variation of callose plug deposition in Arabidopsis ecotypes and in tomato species  and demonstrate that there is a relationship between callose plug deposition and pollen tube growth.
Results and discussion
In vitro pollen germination is robust and reproducible in many ecotypes
The first callose plug position varies among ecotypes in Arabidopsis
After 6 hours of germination, we measured the first interval length (distance from the pollen grain pore to the first callose plug) of 50 randomly selected pollen tubes in 14 ecotypes. We used the 6 hour time point in order to ensure that all the pollen grains had had ample time to grow a tube, as germination initiation is not synchronous . Within an ecotype, the average lengths of the first interval in two independent experiments were reproducible (Figure 1B) but the average lengths were different in different ecotypes. In about 50% of the ecotypes, the average length of the first interval was around 50μm, indicating that the first callose plug was consistently close to the pollen grain, but in the Ws, C24, Est, and Van ecotypes, the average first interval length was longer, and the standard deviations were extremely large. Large standard deviations were also observed in Cvi, Bay, Nd and Nok.
To further investigate the differences of the first callose plug position in all ecotypes, we pooled together 100 first intervals from two independent experiments and grouped them according to the length of first interval (Figure 1C). There was a wide distribution of first interval lengths among the 14 ecotypes, ranging from 11μm to 394μm. We arbitrarily divided pollen tubes into two groups. In the first group the first interval was <150μm, i.e. the first callose plugs were close to the grain; in the second group the first interval was ≥150μm, i.e. the first callose plugs were farther away from the grain. All ecotypes had some pollen tubes with the first interval <150μm, but some ecotypes also had pollen tubes with the first interval ≥150μm. The portion of pollen tubes with the first interval ≥150μm varied from 0% (e.g. Col) to 66% (e.g. Est) among these 14 ecotypes. We further subdivided this group; if the portion of pollen tubes with the first interval ≥150μm in one ecotype was lower than 20%, we treated that ecotype as having one pattern of first callose plug deposition, i.e. with the first callose plug close to the pollen grain. However, if the portion of pollen tubes with the first interval ≥150μm in one ecotype was higher than 20%, we defined that ecotype as having two deposition patterns. After applying this criterion, Ws, C24, Est and Van had two patterns for first callose plug position (Figure 1C). Thus the first callose plug position not only varied between different ecotypes, but also varied within certain ecotypes.
Patterns of callose plug deposition are heritable
Pollen tube length variability in parents and F1 hybrids
Callose plugs continue to elongate in most ecotypes
Callose plug deposition is associated with pollen tube length
Average length of pollen tubes with and without callose plugs in C24
tube lengths with callose plug
tube length without callose plug
tube lengths with first interval <150um
tube lengths with first interval ≥150um
Time course analysis to determine the relationship between sperm cell position and callose plugs
Pollen germination hours
Additional file 1: Movement of sperm in pollen tube with a forming callose plug.(MOV 245 KB)
As many pollen tubes of the C24 ecotype did not have callose plugs after 2 or 3 hours germination, we were able to use C24 to investigate whether callose plug deposition correlated with pollen tube length, without any ecotype effect. The time course analysis (Table 1) was used to compare the lengths of pollen tubes with or without callose plugs. The average tube length of pollen tubes without a callose plug was significantly shorter than those with a callose plug. Additionally, after 4 hours of germination, the average length of tubes with the first callose plug close to the grain (i.e. first interval <150μm) was significantly longer than tubes with the first callose plug far away from the grain (first interval ≥150μm). We carried out correlation analyses with the 4, 5 and 6 hour time points; the first interval length and pollen tube length were significantly correlated at 5 hours (r = −0.235, P<0.05) and at 6 hours (r = −0.242, P<0.05), but not at 4 hours. Perhaps at the 4 hour time point there was still residual variability due to different times of pollen tube initiation.
Variation of callose plug deposition in tomato species
Our studies show that all but one of the Arabidopsis ecotypes tested formed pollen tubes during in vitro germination. We showed that the position of the first callose plug varies among Arabidopsis ecotypes and in some cases within one ecotype. The callose plug deposition patterns were heritable; having two patterns of callose plug within an ecotype is dominant. Callose plug deposition correlated with pollen tube length and pollen tube lengths in F1 hybrids sometimes exceeded those of the parents. Variation in callose plug deposition was also seen in species of tomato. These assays were all carried out in vitro, and it is important to acknowledge that there might be differences in the parameters we measured when pollen tubes grow in the pistil, because of potential influences of the female tissue. Although the significance of these callose plug deposition differences are not known, these findings lay a foundation for mapping genes that regulate callose plug deposition or those that determine pollen tube lengths or growth rate.
Plant materials and growth conditions
Fourteen ecotypes of Arabidopsis thaliana (Col-0, Ws-0, C24, Est-1, Shahdara, Van-1, Bay-0, Tsu-1, Cvi-0, Nok-0, Ei-2, An-1, Nd-1, Wa-1, and Got-22) were used. All of the ecotypes except Col-0 were provided by Brian Staskawicz’s lab, UC Berkeley; they were originally obtained from NASC and the accession numbers are available there (http://arabidopsis.info/). All Arabidopsis plants were grown in the greenhouse in a 4:1:1 mix of Fafard 4P: perlite: vermiculite under an 18-h light/6-h dark cycle at 21°C. Flowers of seven tomato species (Solanum lycopersicum, Solanum pimpinellifolium, Solanum chilense, Solanum peruvianum Solanum sitiens, Solanum pennellii, Solanum habrochaites) were obtained from plants grown in UC-Davis greenhouses.
Pollen germination and aniline blue staining
Arabidopsis in vitro pollen tube germination was carried out as described in . After 6h or 30h pollen germination, decolorized aniline blue was added and then the slides were immediately transferred to 4°C. A DAPI filter was used to observe callose plug and sperm cells. To observe the movement of sperm cells in the presence of aniline blue, we captured images, using a 40× objective, immediately after adding decolorized aniline blue (without transferring slides to 4°C). Tomato pollen germination followed the protocol in  . Decolorized aniline blue staining was as described in .
Microscopic imaging and software for measuring pollen tube lengths and statistical analyses
Microscopic imaging was performed using an Axiovert microscope (Zeiss). Images were captured using a Spot digital camera (Diagnostic Instruments; http://www.diaginc.com/). Distances between a callose plug and the pollen grain pore were measured using Image J software (http://rsbweb.nih.gov/ij/) . The unaired t test, correlation analysis, analysis of variance (ANOVA) and multiple comparisons with least significant difference (LSD) were performed using SPSS 10.0 software (SPSS Inc., Chicago, IL, USA).
We thank Brian Staskawicz’s lab for seed of the Arabidopsis ecotypes and Roger T. Chetelat for the flowers from the species of tomato. We thank Binglian Zheng, Guang Wu and Hua Jiang for comments and discussion, and Jorge Muschietti and Weihua Tang for comments on the manuscript. This work was supported by the U.S. Department of Agriculture-Agricultural Research Service Current Research Information System (grant no. 5335–21000–030–00D). PQ was partially supported by a fellowship from the China Scholarship Council. DT was a participant in the SPUR (sponsored projects for undergraduate research) program of the College of Natural Resources, UC-Berkeley and AS was a participant in UC-Berkeley URAP (undergraduate research apprentice program).
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