Involvement of extracellular calcium influx in the self-incompatibility response of Papaver rhoeas

We have previously demonstrated that increases in cytosolic free Ca2+ are triggered by the self-incompatibility (SI) response in incompatible Papaver rhoeas (the field poppy) pollen. However, one key question that has not been answered is whether extracellular Ca2+ may be involved. To address this question, we have used an ion-selective vibrating probe to measure changes in extracellular Ca2+ fluxes around poppy pollen tubes. Our data reveal several findings. First, we confirm that there is an oscillating Ca2+ influx directed at the apex of the pollen tube; we also provide evidence that Ca2+ influx also occurs at the shanks of pollen tubes. Second, upon challenge with self-incompatibility (S) proteins, there is a stimulation of Ca2+ influx along the shank of incompatible pollen tubes, approximately 50 microm behind the pollen tube tip. This demonstration of SI-induced Ca2+ influx suggests a role for influx of extracellular Ca2+ in the SI response.     

Evidence for DNA fragmentation triggered in the self-incompatibility response in pollen of Papaver rhoeas

Studies of the molecular and biochemical basis of self-incompatibility (SI) in Papaver rhoeas have revealed much about the signalling pathways triggered in pollen early in this response. The aim of the current investigation was to begin to study downstream events in order to elucidate some of the later cellular responses involved in the SI response and identification of the mechanisms controlling the irreversible inhibition of pollen tube growth. We have used the FragEL assay to investigate if there is any evidence for DNA fragmentation stimulated in pollen of P. rhoeas in an S-specific manner. Our data clearly demonstrate that S proteins are responsible for triggering this, specifically in incompatible, and not compatible, pollen. DNA fragmentation was first detected in incompatible pollen tubes 4 h after challenge with S proteins, and continued to increase for a further 10 h. This provides the first evidence, to our knowledge, that this phenomenon is associated with the SI response. We also demonstrate that mastoparan, which increases [Ca2+]i, also triggers DNA fragmentation in these pollen tubes, thereby implicating an involvement of Ca2+ signalling in this process. Together, our data represent a significant breakthrough in understanding of the SI response in Papaver pollen.     

Self-incompatibility in Papaver rhoeas: there is no evidence for the involvement of stigmatic ribonuclease activity

Abstract. Ribonuclease assays have revealed, in contrast to the self-incompatibility (SI) system of Nicotiana alata , there is no detectable ribonuclease activity that correlates with the presence of the functional stigmatic S-gene product in Papaver rhoeas . Thus, we have shown that the inhibition of incompatible pollen tube growth in P. rhoeas is not associated with ribonuclease activity. Furthermore, the finding that pollen from P. rhoeas , unlike that from N. alata , is insensitive to purified bovine pancreatic ribonuclease A at very high concentrations, suggests that the involvement of ribonucleases in the inhibition reaction of the SI response in P. rhoeas is unlikely. In addition, the level of ribonuclease activity in mature stigmas of P. rhoeas is very much lower than that in N. alata and significantly, the level of ribonuclease activity did not rise in conjunction with the developmental expression of SI. Therefore, as a result of these studies, we believe that SI in P. rhoeas does not involve ribonuclease activity.     

Identification of a membrane glycoprotein in pollen of Papaver rhoeas which binds stigmatic self-incompatibility (S-) proteins

Recombinant stigmatic self-incompatibility (S-) proteins from Papaver rhoeas have previously been shown to be biologically functional, inhibiting only pollen of the same S -genotype. In an attempt to identify molecules in pollen which interact with these proteins, Western ligand blotting was used, with the recombinant S-proteins as probes followed by immunodetection of the bound S-protein. This revealed that pollen of all S -genotypes tested contained a 70–120 kDa protein which bound the S₁, S₃ and S₈ proteins in an indistinguishable manner. Binding was destroyed by pretreatment of blots with periodate, implicating a glycoprotein with activity being dependent on the glycan moiety. The activity completely partitioned into the detergent phase on condensation with Triton X-114, indicating an integral membrane protein. On aqueous two-phase partition of microsomal membrane preparations, the majority of the binding activity partitioned into the upper phase, suggesting that the molecule is located in the plasma membrane.
No equivalent binding could be detected in extracts of leaves, stems, roots or stigmas of P. rhoeas , nor in immature anthers. Identical activity was detected in pollen of some other Papaver species, but not in pollen of Brassica oleracea, Nicotiana tabacum or Petunia hybrida . The presence in mature pollen of P. rhoeas of a plasma membrane glycoprotein which binds S-proteins from the stigma of the same species, albeit in a non S -allele-specific manner, strongly suggests that this molecule has a role somewhere in the interaction of the stigma proteins with pollen. The activity is not that expected of an S-specific receptor, but by analogy with certain mammalian systems the molecule may act as an accessory receptor, or co-receptor, the presence of which may be essential for a functional interaction with an S-specific receptor.     

Molecular analysis of two functional homologues of the S 3 allele of the Papaver rhoeas self-incompatibility gene isolated from different populations

The S ₃ allele of the S gene has been cloned from Papaver rhoeas cv. Shirley. The sequence predicts a hydrophilic protein of 14.0 kDa, showing 55.8% identity with the previously cloned S ₁ allele, preceded by an 18 amino acid signal sequence. Expression of the S ₃ coding region in Escherichia coli produced a form of the protein, denoted S₃e, which specifically inhibited S₃ pollen in an in vitro bioassay. The recombinant protein was ca. 0.8 kDa larger than the native stigmatic form, indicating post-translational modifications in planta, as was previously suggested for the S₁ protein. In contrast to other S proteins identified to date, S₃ protein does not appear to be glycosylated. Of particular significance is the finding that despite exhibiting a high degree of sequence polymorphism, secondary structure predictions indicate that the S₁ and S₃ proteins may adopt a virtually identical conformation. Sequence analysis also indicates that the P. rhoeas S alleles share some limited homology with the SLG and SRK genes from Brassica oleracea. Previously, cross-classification of different populations of P. rhoeas had revealed a number of functionally identical alleles. Probing of western blots of stigma proteins from plants derived from a wild Spanish population which contained an allele functionally identical to the Shirley S ₃ allele with antiserum raised to S₃e, revealed a protein (S ₃ s) which was indistinguishable in pI and M _r from that in the Shirley population. A cDNA encoding S ₃ s was isolated, nucleotide sequencing revealing a coding region with 99.4% homology with the Shirley-derived clone at the DNA level, and 100% homology at the amino acid level.     

Variability patterns and positively selected sites at the gametophytic self-incompatibility pollen SFB gene in a wild self-incompatible Prunus spinosa (Rosaceae) population

Current models for the generation of new gametophytic self-incompatibility specificities require that neutral variability segregates within specificity classes. Furthermore, one of the models predicts greater ratios of nonsynonymous to synonymous substitutions in pollen than in pistil specificity genes. All models assume that new specificities arise by mutation only. To test these models, 21 SFB (the pollen S-locus) alleles from a wild Prunus spinosa (Rosaceae) population were obtained. For seven of these, the corresponding S-haplotype was also characterized. The SFB data set was also used to identify positively selected sites. Those sites are likely to be the ones responsible for defining pollen specificities. Of the 23 sites identified as being positively selected, 21 are located in the variable (including a new region described here) and hypervariable regions. Little variability is found within specificity classes. There is no evidence for selective sweeps being more frequent in pollen than in pistil specificity genes. The S-RNase and the SFB genes have only partially correlated evolutionary histories. None of the models is compatible with the variability patterns found in the SFB and the S-haplotype data.     

A tale of two continents: Baker's rule and the maintenance of self-incompatibility in Lycium (Solanaceae)

Over 50 years ago, Baker (1955, 1967) suggested that self-compatible species were more likely than self-incompatible species to establish new populations on oceanic islands. His logic was straightforward and rested on the assumption that colonization was infrequent; thus, mate limitation favored the establishment of self-fertilizing individuals. In support of Baker's rule, many authors have documented high frequencies of self-compatibility on islands, and recent work has solidified the generality of Baker's ideas. The genus Lycium (Solanaceae) has ca. 80 species distributed worldwide, and phylogenetic studies suggest that Lycium originated in South America and dispersed to the Old World a single time. Previous analyses of the S-RNase gene, which controls the stylar component of self-incompatibility, have shown that gametophytically controlled self-incompatibility is ancestral within the genus, making Lycium a good model for investigating Baker's assertions concerning reproductive assurance following oceanic dispersal. Lycium is also useful for investigations of reproductive evolution, given that species vary both in sexual expression and the presence of self-incompatibility. A model for the evolution of gender dimorphism suggests that polyploidy breaks down self-incompatibility, leading to the evolution of gender dimorphism, which arises as an alternative outcrossing mechanism. There is a perfect association of dimorphic gender expression, polyploidy, and self-compatibility (vs. cosexuality, diploidy, and self-incompatibility) among North American Lycium. Although the association between ploidy level and gender expression also holds for African Lycium, to date no studies of mating systems have been initiated in Old World species. Here, using controlled pollinations, we document strong self-incompatibility in two cosexual, diploid species of African Lycium. Further, we sequence the S-RNase gene in 15 individuals from five cosexual, diploid species of African Lycium and recover 24 putative alleles. Genealogical analyses indicate reduced trans-generic diversity of S-RNases in the Old World compared to the New World. We suggest that genetic diversity at this locus was reduced as a result of a founder event, but, despite the bottleneck, self-incompatibility was maintained in the Old World. Maximum-likelihood analyses of codon substitution patterns indicate that positive Darwinian selection has been relatively strong in the Old World, suggesting the rediversification of S-RNases following a bottleneck. The present data thus provide a dramatic exception to Baker's rule, in addition to supporting a key assumption of the Miller and Venable (2000) model, namely that self-incompatibility is associated with diploidy and cosexuality.     

Visualization of a self-incompatibility gene in Brassica campestris L. by multicolor FISH

 The physical localization of the S-glycoprotein (SLG) locus in the chromosome of Brassica campestris L. ‘pekinensis’ cv ‘Kukai’ was visualized by multi-color fluorescent in situ hybridization (McFISH). ‘Kukai’, which is an F₁ hybrid between two parental lines, T-17 and T-18, has two SLG genes from both T-17 and T-18. In this study, a 1.3-kb DNA fragment was amplified from the genomic DNA of T-17 by PCR using a set of primers specific to the class-I SLG. From the genomic DNA of T-18, no DNA fragment was amplified using these primers. In the genomic Southern hybridization, a cloned PCR product hybridized with the genomic DNA of T-17 or F₁ but not with that of T-18. The PCR product had a sequence homology of approximately, 85% to another class-I SLG gene, SLG-9. Therefore, the PCR product from T-17 was named SLG-17, as it is thought to be a member of the class-I SLG. Using SLG-17 as the probe, FISH was carried out to visualize the position of the SLG locus. McFISH was also carried out simultaneously using the SLG-17 and SLG-9 genes as probes. The SLG-17 gene was detected as a doublet signal at the interstitial region close to the end of a small chromosome, with the signal site being identical to that of SLG-9. Therefore, it is concluded that the SLG-17 gene is localized at the interstitial region close to the end of the chromosome derived from T-17 in Brassica campestris L. ‘pekinensis’ cv ‘Kukai’. Received: 18 September 1997 / Accepted: 6 October 1997     

Identification of random amplified polymorphic DNA (RAPD) markers for self-incompatibility alleles in Corylus avellana L.

 Random amplified polymorphic DNA (RAPD) markers were identified for self-incompatibility (SI) alleles that will allow marker-assisted selection of desired S-alleles in hazelnut (Corylus avellana L.). DNA was extracted from young leaves collected from field-planted parents and 26 progeny of the cross OSU 23.017 (S₁S₁₂)×VR6-28 (S₂S₂₆) (OSU23×VR6). Screening of 10-base oligonucleotide RAPD primers was performed using bulked segregant analysis. DNA samples from 6 trees each were pooled into four ‘bulks’, one for each of the following: S₁ S₂, S₁ S₂₆ ^, S₂ S₁₂, and S₁₂ S₂₆. ‘Super bulks’ of 12 trees each for S₁, S₂, S₁₂, and S₂₆ were then created for each allele by combining the appropriate bulks. The DNA from these four super bulks and from the parents was used as a template in the PCR assays. A total of 250 primers were screened, and one RAPD marker each was identified for alleles S₂ (OPI07₇₅₀) and S₁ (OPJ14₁₇₀₀). OPJ14₁₇₀₀ was identified in 13 of 14 S₁ individuals of the cross OSU23×VR6 used in bulking and yielded a false positive in 1 non-S₁ individual. This same marker was not effective outside the original cross, identifying 4 of 5 S₁ progeny in another cross, ‘Willamette’×VR6-28 (‘Will’×VR6), but yielded false positives in 4 of 9 non-S₁ individuals from the cross ‘Casina’×VR6-28 (‘Cas’×VR6). OPI07₇₅₀ served as an excellent marker for the S₂ allele and was linked closely to this allele, identifying 12 of 13 S₂ individuals in the OSU23×VR6 population with no false positives. OPI07₇₅₀ was found in 4 of 4 S₂ individuals from ‘Will’×VR and 7 of 7 S₂ individuals of ‘Cas’×VR6 with no false positives, as well as 10 of 10 S₂ individuals of the cross OSU 296.082 (S₁S₈)×VR8-32 (S₂S₂₆), with only 1 false positive individual out of 21 progeny. OPI07₇₅₀ was also present in 5 of 5 cultivars carrying the S₂ allele, with no false-positive bands in non-S₂ cultivars, and correctly identified all but 2 S₂ individuals in 57 additional selections in the breeding program. In the OSU23×VR6 population, the recombination rate between the marker OPJ14₁₇₀₀ and the S₁ allele was 7.6% and between the OPI07₇₅₀ marker and the S₂ allele was 3.8%. RAPD marker bands were excised from gels, cloned, and sequenced to enable the production of longer primers (18 or 24 bp) that were used to obtain sequence characterized amplified regions (SCARs). Both the S₁ and S₂ markers were successfully cloned and 18 bp primers yielded the sole OPJ14₁₇₀₀ product, while 24-bp primers yielded OPI07₇₅₀ as well as an additional smaller product (700 bp) that was not polymorphic but was present in all of the S-genotypes examined. Received: 10 January 1998 / Accepted: 26 January 1998     

Genetic mapping and protein product diversity of the self-incompatibility locus in wild tomato (Lycopersicon peruvianum)

Phenotypic diversity of self-incompatibility (S) alleles within nine natural populations ofLycopersicon peruvianum was investigated. Only 7 incompatible responses were observed of a total of 276 unique combinations tested, on the basis of controlled pollinations, indicating the large number of alleles that exist within these populations. Molecular weight polymorphism for specific major stylar proteins observed on SDS-PAGE was also evident in two of the populations examined. Five proteins were shown to map to theS locus and to be associated with differentS alleles through controlled pollinations and segregation of the proteins. Two of theseS related proteins had been described previously in terms of spatial and temporal expression consistent with their involvement in self-incompatibility (Mauet al., Planta 169, 184–191, 1986). A mapping population derived from a fully compatible cross was used to establish linkage of theS locus to two DNA markers,CD15 andTG184, that lie on chromosome 1. The order of the markers and estimates of map distances are given.     

Characterization and promoter activity of chromoplast specific carotenoid associated gene (CHRC) from Oncidium Gower Ramsey

Tissue-specific promoters are required for plant molecular breeding to drive a target gene in the appropriate location in plants. A chromoplast-specific, carotenoid-associated gene (OgCHRC) and its promoter (Pchrc) were isolated from Oncidium orchid and characterized. Northern blot analysis revealed that OgCHRC is specifically expressed in flowers, not in roots and leaves. Transient expression assay of Pchrc by bombardment transformation confirmed its differential expression pattern in floral tissues of different horticulture plants and cell-type location in conical papillate cells of adaxial epidermis of flower. These results suggest that Pchrc could serve as a useful tool in ornamental plant biotechnology to modify flower color.     

Analysis of the Tissue-SpecificS RNase gene promoter associated with self-incompatibility in tomato (Lycopersicon peruvianum L.)

To understand the expression pattern of theS RNase gene in the floral tissues associated with self-incompatibility (SI), promoter region of S₁₁ RNase gene was serially deleted and fused GUS. Five chimeric constructs containing a deleted promoter region of the S₁₁ RNase gene were constructed, and introduced intoNicotiana tabacum using Agrobacterium-mediated transformation. Northern blot analysis revealed that the GUS gene was expressed in the style, anther, and developing pollen of all stages in each transgenic tobacco plant The developing pollen expressed the same amount of GUS mRNA in all stages in transgenic tobacco plants. In addition, histochemical analysis showed GUS gene expression in vascular bundle, endothecium, stomium, and tapetum cells during pollen development in transgenic plants. From these results, it is speculated that SI ofLycopersicon peruvianum may occur through the interaction ofS RNase expressed in both style and pollen tissues.     

Characterization of an endo-1,3(4)-β-d-glucanase gene from Cellvibrio mixtus

Abstract An endo-1,3(4)-β- d -glucanase gene ( cwd2 ) of Cellvibrio mixtus encoding laminarinase activity was cloned on a 3.9-kb Pst I fragment. The Cwd2 enzyme, extracted from recombinant Escherichia coli , degraded both β-1,3 glucans and β-1,3–1,4 mixed-linkage glucans, was entohydrolytic and so conformed to the enzyme class 3.2.1.6. The pH and temperature optima of the enzyme were approximately 7 and 40°C respectively. The M _r of specifically labelled Cwd2 was approximately 34 000. This gene was quite distinct from two other C. mixtus β-1,3 glucanases previously described.     

‘Neuron-specific’ protein gene product 9.5 (PGP 9.5) is also expressed in glioma cell lines and its expression depends on cellular growth state

Protein gene product 9.5 (PGP 9.5), which in the normal nervous system is restricted to certain neurons, has been detected in two glioma cell lines, rat C6 and human GL15, by immunoblotting and immunocytochemistry. Its expression in these cells depends on the cellular growth state, being maximal between the first and second post-plating day. Only a faint PGP 9.5 immunoreactivity can be observed in glioma cells after the eleventh post-plating day, i.e. about one week after confluency has been reached. The present results suggest that PGP 9.5 in cultured glial cells is maximally expressed during the growth phase and that the protein could play a role during brain development in glial cells, in reactive gliosis, or in tumorigenesis of the glial lineage.     

山葡萄几丁质酶基因VCH3启动子的分离及鉴定

利用接头PCR技术首次分离了长度为1 216bp的"双优"山葡萄(Vitis amurensis Rupr.)classⅢ几丁质酶基因VCH3上游启动子序列(GenBank登录号AF441123),并用引物延伸方法鉴定了该启动子的转录起始位点,为5'-ATCAAGCAC-31序列中的第二个A.序列分析结果表明,代表真核基因启动子特征的CAAT盒和TATA盒分别位于VCH3启动子转录起始位点上游-122和-29处.另外,在转录起始位点上游-1 181 bp和-293 bp处各有一个水杨酸(SA)响应的顺式作用元件TGACG.为了鉴定该启动子的功能,将该启动子连接到β-葡糖苷酸酶基因(GUS)编码区的上游构建了VCH3启动子-GUS融合基因,并用农杆菌介导叶盘转化法将该融合基因转入烟草栽培品种NC89中.SA处理的转基因烟草根系和叶片GUS酶活性的荧光和组织化学检测结果表明VCH3启动子的驱动作用被SA诱导,因而该启动子在基因工程中将具有潜在的应用价值.