The mutated S1-haplotype in sour cherry has an altered S-haplotype-specific F-box protein gene

Gametophytic self-incompatibility (GSI) is an outcrossing mechanism in flowering plants that is genetically controlled by 2 separate genes located at the highly polymorphic S-locus, termed S-haplotype. This study characterizes a pollen part mutant of the S(1)-haplotype present in sour cherry (Rosaceae, Prunus cerasus L.) that contributes to the loss of GSI. Inheritance of S-haplotypes from reciprocal interspecific crosses between the self-compatible sour cherry cultivar Ujfehértói Fürt?s carrying the mutated S(1)-haplotype (S(1)'S(4)S(d)S(null)) and the self-incompatible sweet cherry (Prunus avium L.) cultivars carrying the wild-type S(1)-haplotype revealed that the mutated S(1)-haplotype confers unilateral incompatibility with a functional pistil component and a nonfunctional pollen component. The altered sour cherry S(1)-haplotype pollen part mutant, termed S(1)', contains a 615-bp Ds-like element within the S(1)-haplotype-specific F-box protein gene (SFB(1)'). This insertion generates a premature in-frame stop codon that would result in a putative truncated SFB(1) containing only 75 of the 375 amino acids present in the wild-type SFB(1). S(1)' along with 2 other previously characterized Prunus S-haplotype mutants, S(f) and S(6m), illustrate that mobile element insertion is an evolutionary force contributing to the breakdown of GSI.     

Genetic and molecular characterization of three novel S-haplotypes in sour cherry (Prunus cerasus L.)

Tetraploid sour cherry (Prunus cerasus L.) exhibits gametophytic self-incompatibility (GSI) whereby the specificity of self-pollen rejection is controlled by alleles of the stylar and pollen specificity genes, S-RNase and SFB (S haplotype-specific F-box protein gene), respectively. As sour cherry selections can be either self-compatible (SC) or self-incompatible (SI), polyploidy per se does not result in SC. Instead the genotype-dependent loss of SI in sour cherry is due to the accumulation of non-functional S-haplotypes. The presence of two or more non-functional S-haplotypes within sour cherry 2x pollen renders that pollen SC. Two new S-haplotypes from sour cherry, S(33) and S(34), that are presumed to be contributed by the P. fruticosa species parent, the complete S-RNase and SFB sequences of a third S-haplotype, S(35), plus the presence of two previously identified sweet cherry S-haplotypes, S(14) and S(16) are described here. Genetic segregation data demonstrated that the S(16)-, S(33)-, S(34)-, and S(35)-haplotypes present in sour cherry are fully functional. This result is consistent with our previous finding that 'hetero-allelic' pollen is incompatible in sour cherry. Phylogenetic analyses of the SFB and S-RNase sequences from available Prunus species reveal that the relationships among S-haplotypes show no correspondence to known organismal relationships at any taxonomic level within Prunus, indicating that polymorphisms at the S-locus have been maintained throughout the evolution of the genus. Furthermore, the phylogenetic relationships among SFB sequences are generally incongruent with those among S-RNase sequences for the same S-haplotypes. Hypotheses compatible with these results are discussed.     

Inheritance of S(f)-RNase in Japanese apricot ( Prunus mume) and its relation to self-compatibility

Self-compatible cultivars of Japanese apricot ( Prunus mume Shieb. et Zucc.), a tree species that normally shows S-RNase-based self-incompatiblity, have a horticultural advantage over self-incompatible cultivars. Inheritance of self-compatibility and a common S(f)-RNase allele that is observed in self-compatible cultivars was investigated using progenies from controlled crosses. Total DNAs were isolated from the parents and progenies of seven crosses that included at least one self-compatible cultivar as a parent. These DNAs were PCR-amplified with the Pru-C2 and PCE-R primer pair to determine S-haplotypes of the parents and progenies. A novel S-haplotype, S(8), was found. In all crosses examined, the S(f)-RNase gene was inherited from either the seed or pollen parent as a pistil S-allele in a non-functional S-haplotype. Self-compatibility of about 20 trees each from reciprocal crosses of 'Benisashi ( S(7) S(f))' and 'Shinpeidayu ( S(3) S(f))', and 26 selections from 16 different crosses was tested by pollination and pollen-tube growth studies. Cosegregation of the S(f)-RNase allele and self-compatibility was confirmed with all but selection 1K0-26 ( S(3) S(7)). Selection 1K0-26 ( S(3) S(7)) that originated from 'Benisashi ( S(7) S(f))' x 'Koshinoume ( S(3) S(f))' appeared to be self-compatible even without the S(f)-RNase allele. The possible role of pollen- S, a presumably existing pollen component of gametophytic self-incompatibility, is discussed.     

Stylar trimorphism in four functionally andromonoeciousProsopis species (Mimosaceae)

The reproductive system ofProsopis chilensis, P. pugionata, P. flexuosa, andP. torquata is described. Observations were made of floral morphology, stigmatic receptivity, pollen grain viability, and the nature of pollen reserves. Scanning electrone microscopy and statistical analysis were carried out. The results allow us to conclude that: 1) The studied species have three distinct floral morphs characterized by significantly different style lengths and, to a lesser extent, different stamen lengths; 2) style precocity found in all species does not indicate protogyny; 3) flowers with shortest styles are not receptive; 4) the pollen grains of all floral morphs showed high viability; 5) peroxidase is present in both the pollen and pistil; 6) viable pollen grains invariably have both starch and pollenkitt; 7) the studied species are andromonoecious.     

Analysis of the S-locus structure in Prunus armeniaca L. Identification of S-haplotype specific S-RNase and F-box genes

The gametophytic self-incompatibility (GSI) system in Rosaceae has been proposed to be controlled by two genes located in the S-locusan S-RNase and a recently described pollen expressed S-haplotype specific F-box gene (SFB). However, in apricot (Prunus armeniaca L.) these genes had not been identified yet. We have sequenced 21kb in total of the S-locus region in 3 different apricot S-haplotypes. These fragments contain genes homologous to the S-RNase and F-box genes found in other Prunusspecies, preserving their basic gene structure features and defined amino acid domains. The physical distance between the F-boxand the S-RNase genes was determined exactly in the S ₂-haplotype (2.9kb) and inferred approximately in the S ₁-haplotype (< 49kb) confirming that these genes are linked. Sequence analysis of the 5 flanking regions indicates the presence of a conserved region upstream of the putative TATA box in the S-RNase gene. The three identified S-RNase alleles (S ₁, S ₂ and S ₄) had a high allelic sequence diversity (75.3 amino acid identity), and the apricot F-box allelic variants (SFB₁, SFB₂ and SFB₄) were also highly haplotype-specific (79.4 amino acid identity). Organ specific-expression was also studied, revealing that S ₁- and S ₂-RNases are expressed in style tissues, but not in pollen or leaves. In contrast, SFB ₁ and SFB ₂ are only expressed in pollen, but not in styles or leaves. Taken together, these results support these genes as candidates for the pistil and pollen S-determinants of GSI in apricot.     

Identification of <Emphasis Type="Italic">S</Emphasis>-haplotype-specific F-box gene in Japanese plum (<Emphasis Type="Italic">Prunus salicina</Emphasis> Lindl.)

Self-incompatibility has been studied extensively at the molecular level in Solanaceae, Rosaceae and Scrophulariaceae, all of which exhibit gametophytic self-incompatibility controlled by a single polymorphic locus containing at least two linked genes, i.e., the S-RNase gene and the pollen-expressed SFB/SLF (S-haplotype-specific F-box/S-locus F-box) gene. However, the SFB gene in Japanese plum (Prunus salicina Lindl.) has not yet been identified. We determined eight novel sequences homologous to the SFB genes of other Prunus species and named these sequences PsSFB. The gene structure of the SFB genes and the characteristic domains in deduced amino acid sequences were conserved. Three sequences from 410 to 2,800 bp of the intergenic region between the PsSFB sequences and the S-RNase alleles were obtained. The eight identified PsSFB sequences showed S-haplotype-specific polymorphism, with 74–83% amino acid identity. These alleles were exclusively expressed in the pollen. These results suggest that the PsSFB alleles are the pollen S-determinants of GSI in Japanese plum. Nucleotide sequence data reported are available in the NCBI database under the accession numbers DQ849084–DQ849090 and DQ849118.     

Characterization of SLFL1, a pollen-expressed F-box gene located in the Prunus S locus

The S locus and its flanking regions in the genus Prunus (Rosaceae) contain four pollen-expressed F-box genes. These genes contain the S locus F-box genes with low allelic sequence polymorphism genes 1, 2, and 3 (SLFL1, SLFL2, and SLFL3) as well as the putative pollen S gene, named the S haplotype-specific F-box protein gene (SFB). As much less information is available on the function of SLFLs than that of SFB, we analyzed the SLFLs of six S haplotypes of sweet cherry (Prunus avium) in this study. Genomic DNA blot analysis and the isolation of SLFL1 showed that the SLFL1 gene in a functional self-incompatible S ³ haplotype is deleted and only a partial sequence resembling SLFL1 is left in the S ³ locus region, suggesting that SLFL1 by itself is not directly involved in either the GSI reaction or pollen-tube growth. Genomic DNA blot analysis showed that there was no substantial modification or mutation in SLFL2 and SLFL3. A phylogenic analysis of F-box genes in the rosaceous S locus and its border regions showed that Prunus SLFLs were more closely related to maloid S locus F-box brothers than to Prunus SFBs. The functions of SLFLs and the evolution of self-incompatibility in Prunus are discussed based on these results. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The nucleotide sequence data reported appear in the DDBJ, EMBL, and GenBank Nucleotide Sequence Databases under the accession numbers, AB360339, AB360340, AB360341, and AB360342, for SLFL1-S ¹ , SLFL1-S ² , SLFL1-S ⁵ , and SLFL1-S ⁶ , respectively.     

Primary structural features of the<Emphasis Type="Italic"> S</Emphasis> haplotype-specific F-box protein,SFB, in<Emphasis Type="Italic"> Prunus</Emphasis>

The gene SFB encodes an F-box protein that has appropriate S-haplotype-specific variation to be the pollen determinant in the S-RNase-based gametophytic self-incompatibility (GSI) reaction in Prunus (Rosaceae). To further characterize Prunus SFB, we cloned and sequenced four additional alleles from sweet cherry (P. avium), SFB ¹ , SFB ² , SFB ⁴ , and SFB ⁵ . These four alleles showed haplotype-specific sequence diversity similar to the other nine SFB alleles that have been cloned. In an amino acid alignment of Prunus SFBs, including the four newly cloned alleles, 121 out of the 384 sites were conserved and an additional 65 sites had only conservative replacements. Amino acid identity among the SFBs ranged from 66.0% to 82.5%. Based on normed variability indices (NVI), 34 of the non-conserved sites were considered to be highly variable. Most of the variable sites were located at the C-terminal region. A window-averaged plot of NVI indicated that there were two variable and two hypervariable regions. These variable and hypervariable regions appeared to be hydrophilic or at least not strongly hydrophobic, which suggests that these regions may be exposed on the surface and function in the allele specificity of the GSI reaction. Evidence of positive selection was detected using maximum likelihood methods with sites under positive selection concentrated in the variable and hypervariable regions.K. Ikeda and B. Igic contributed equally to this paperNucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the accession numbers AB111518, AB111519, AB111520, and AB111521, for SFB ¹, SFB ², SFB ⁵, and SFB ⁴, respectively     

异叶苦竹花粉管生长及双受精过程

以异叶苦竹为材料,采用扫描电镜、荧光显微镜技术及传统的石蜡制片技术,解剖观察其花粉管生长途径及双受精过程。结果表明:(1)授粉后,花粉在柱头上吸水膨胀,约30 min即可萌发。(2)授粉1～2 h后花粉管可达到花粉长度的5～10倍,花粉管在柱头分支中进一步伸长,并开始伸入花柱中生长。(3)授粉后5 h,大量花粉管沿引导组织进入花柱基部与子房顶部之间的子房壁,有少量花粉管在子房壁与外珠被之间的缝隙中生长。(4)授粉后8 h,少量花粉管到达珠孔端。(5)授粉后15～18 h,精核与极核融合,形成初生胚乳核;精、卵核融合,形成合子。(6)授粉后20～30 h,仍可在花柱中见到大量呈束状的花粉管。(7)授粉后48 h,子房内的大部分花粉管出现解体,大多数花粉死亡。研究认为,精细胞到达胚珠的时间为8 h。     

The effect of isolated components of Prunus avium L. styles on in vitro growth of pollen tubes

A number of components isolated from styles of P. avium cv. Napoleon (S ₃ S ₄) have been tested for their capacity to influence in vitro growth of pollen tubes from fresh and stored pollen (cv. Napoleon (S ₃ S ₄)). An antigenic glycoprotein (Antigen S) is a potent inhibitor of in-vitro pollen tube growth, causing a 65% reduction in tube length at a concentration of 20 g/ml. None of the other style components were effective inhibitors of pollen tube growth; neither were proteins of animal origin such as histone, serum albumin, cytochrome C, and the glycoproteins ovalbumin and thyroglobulin, effective inhibitors.     

A study on pollen morphology of the genus Vitis L.

The pollen grains of 32 species, 1 subspecies and 4 varieties of Vitis and 1 species of Muscadinia were scanned with Hitachi S-800 scanning electron microscope and described in the present paper. In Vitis, the pollen morphology is found to be valuable for the identification of species, though it is of little significance in supraspecific classification and systematic consideration. The ornamentation of Vitis could be divided into four types: ( 1 ) The pollen grains are oblate or rarely spheroidal (P/E =0.97 ～ 1.08) in shape, with colpi wide enough to show dense granulae inside. The pollen grains of V. coignetiae Pull. Ex Planch. belong to this type. (2) The pollen grains are prolate to subprolate (P/E = 1.25 ～ 1.76 ), with sparse granulae within the colpi. This type includes l0 species, 1 subspecies and 3 varieties, such as V. hui Cheng etc. (3) The pollen grains of V. romanetii Roman du Caill. ex Planch. and V. davidii (Roman du Caill. ) Foex. var. ferruginea Merr. et Chun are prolate (P/E= 1.87 ～ 2.1), with colpi narrow and linear-shaped, but rhombic in the equational region. (4) The pollen grains are prolate or perprolate (P/E= 1.30 ～ 2. 0), with narrow and linear-shaped colpi. The species with this type of pollen grains are Muscadiniarotundifolia (Michx.) Small and 20 species of Vitis.     

Rhizobium nepotum sp. nov. isolated from tumors on different plant species

Five Gram-negative, rod-shaped, non-spore-forming bacteria were isolated from galls on different plant species in Hungary: strain 39/7(T) from Prunus cerasifera Myrobalan, strain 0 from grapevine var. Ezerjó, strain 7/1 from raspberry var. Findus and in Poland, strain C3.4.1 from Colt rootstock (Prunus avium × Prunus pseudocerasus) and strain CP17.2.2 from Prunus avium. Only one of these isolates, strain 0, is able to cause crown gall on different plant species. On the basis of 16S rRNA gene sequence similarity, the strains cluster together and belong to the genus Rhizobium and their closest relative is Rhizobium radiobacter (99.1%). Phylogenetic analysis of the novel strains using housekeeping genes atpD, glnA, gyrB, recA and rpoB revealed their distinct position separate from other known Rhizobium species and confirmed their relation to Rhizobium radiobacter. The major cellular fatty acids are 18:1 w7c, 16:0, 16:0 3OH, summed feature 2 (comprising 12:0 aldehyde, 16:1 iso I and/or 14:0 3OH) and summed feature 3 (comprising 16:1 w7c and/or 15 iso 2OH). DNA-DNA hybridization of strain 39/7(T) with the type strain of R. radiobacter LMG 140(T) revealed 45% DNA-DNA hybridization. Phenotypic and physiological properties differentiate the novel isolates from other closely related species. On the basis of the results obtained, the five isolates are considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium nepotum sp. nov. (type strain 39/7(T)=LMG 26435(T)=CFBP 7436(T)) is proposed.     

Ambrosia pollen in the air of Lublin, Poland

Studies on Ambrosia pollen concentrations were carried out in Lublin in the period 1995–2004. The effects of a number of meteorological factors were analysed. In the first period of the study, the gravimetric method was used (1995–1999), while in the second period, the volumetric method was applied. The results show an increasing trend in the amount of airborne pollen. The Ambrosia pollen season in Lublin lasts from August to October. Over a period of 5 years, the highest number of pollen grains was recorded in September (53%), followed by August (44%) and October (3%). There were wide variations in annual totals. The annual total pollen counts was 167–1180 grains, with the peak value in 2002. Maximum daily pollen concentrations (56–312 pollen grains m⁻³) were recorded in the first half of August and in the first half of September. On the days when high Ambrosia pollen concentrations occurred, the temperature was above 21°C and the winds were mainly from the southeast, south and east. Maximum intradiurnal concentrations of pollen grains occurred in the afternoon hours. These results indicate, to some degree, that Ambrosia pollen is transported for long distances before descent.     

Morphological and functional characterization of BcMF13 in the antisense-silenced plants of Brassica campestris ssp. chinensis var. parachinensis

The gene Brassica campestris male fertility 13 (BcMF13, GenBank accession number EF158459) was isolated as a reproductive organ-specific gene from Chinese cabbage (Brassica campestris L. ssp. chinensis Makino, syn. B. rapa ssp. chinensis). It is exclusively expressed in stage four and five flower buds of fertile lines and is most strongly expressed in stamens. Here, we report a functional characterization of this BcMF13 gene in the antisense-silenced plants. The inflorescence of the BcMF13 mutant was compacted with anthers curved outside. The fertility of this mutant was greatly reduced with less than 5 seeds per silique. Under scanning electron microscopy, the mutant demonstrated numerous shriveled pollen grains with deep invaginations. The frequency of normal pollen grains was just 45.34%. The pollen mother cell, the tetrad, and the mature pollen of the BcMF13 mutant were abnormal resulting in the poor pollen vitality. Germination test in vivo suggested BcMF13 delayed the pollen tubes’ extension in the style. All these indicated BcMF13 had a vital role in pollen development of Chinese cabbage.     

Viability of Cururbita pepo pollen: biophysical and structural data

During ageing of the short-lived pollen grains of Cucurbita pepo L., water loss was examined in relation to viability using biophysical (¹H-nuclear magnetic resonance, NMR) and cytological methods (fluorochromatic reaction test, freezefracture and scanning electron microscopy). A semi-logarithmic representation of the pollen weight loss demonstrated the complexity of the dehydration process. A the study of proton loss using ¹H-NMR indicated that two major releases water of had taken place, each with different flux rates. Pulse ¹H-NMR experiments showed the occurrene of non-exponential signal decay as a function of time, indicating the existence of different fractions of water in a pollen grain sample. These fractions leave the pollen grain at different times during pollen dehydration, and one of them (that of the so-called vital water) can be related to pollen viability. The quantity of protons giving a signal during pulse ¹H-NMR experiments was very low when the pollen grains were judged to be dead according to the fluorochromatic test. Freeze-fracture replicas of these dead pollen grains (less than 25% water content) showed that the plasma membrane had become detached from the intine surface; this ultrastructural feature might therefore be involved in the loss of pollen viability.Abbreviations A initial amplitude of the NMR signal - A₂ quantity of water charcterized by T_2-2 - A₅ quantity of water characterized by T_2–5 - FCR fluorochromatic reaction - NMR nuclear magnetic resonance - T₂ transverse relaxation time - T_2-2 T₂ measured with 2 ms between each pulse of radiofrequency - T_2–5 T₂ measured with 5 ms between each pulse of radiofrequency