Towards an in vitro bioassay for the self-incompatibility response in Brassica oleracea

Summary Eluates of stigmas of Brassica oleracea that were known to contain S locus-specific glycoproteins (SLSG) discriminated between self and cross pollen in vitro in three different media. Discrimination was equally evident in experiments that were the in vitro equivalents of reciprocal pollinations. In a TAPS-buffered medium, self eluates depressed pollen germination in a dose-dependent manner. TAPS medium allowed a bioassay of the effects of SLSG in eluates because it optimized germination in a way that eliminated the complicating features of the stimulatory substances in the eluates. Stigma eluates affected percentage pollen germination and optimum calcium concentrations in vitro whether or not SLSG were present in the eluates, but differently in different media, and depending on whether the eluates were cross or self with respect to the pollen tested. Thus, the effect of stigma eluates on the in vitro germination of pollen in Brassica depends on the balance of stimulatory versus inhibitory substances in the eluates.     

Transformation of Brassica oleracea with an S-locus gene from B. campestris changes the self-incompatibility phenotype

Summary An SLG gene derived from the S-locus and encoding and S-locus-specific glycoprotein of Brassica campestris L. was introduced via Agrobacterium-mediated transformation into B. oleracea L. A self-incompatible hybrid and another with partial self-compatibility were used as recipients. The transgenic plants were altered in their pollen-stigma interaction and were fully compatible upon self-pollination. Reciprocal crosses between the transgenic plants and untransformed control plants indicated that the stigma reaction was changed in one recipient strain while the pollen reaction was altered in the other. Due to interspecific incompatibility, we could not demonstrate whether or not the introduced SLG gene confers a new allelic specificity in the transgenic plants. Our results show that the introduced SLG gene perturbs the self-incompatibility phenotype of stigma and pollen.     

Esterases in pollen and stigma of Brassica

The dry type stigma of Brassica is covered with a continuous layer of cuticle. Cutinase and non-specific esterases may be involved in breakdown of this cuticle barrier during pollen-stigma interaction, but only a little is known about their nature and characteristics. We report here the presence of two distinct esterases from stigma and pollen of Brassica. A 33 kD esterase assayed using MU-butyrate substrate shows high activity in stigma papillae. A similar esterase from Tropaeolum pollen has been shown to possess active cutinase activity. The esterase activity in anther tissue is due to a 24 kD enzyme with substrate specificity toward acetate esters. Both enzymes require sulfhydryl groups for their catalytic activity. Immunogold labelling of antibodies raised against these esterases localised the proteins at the subcellular level. Antibodies for MU-butyrate hydrolase gave a positive signal in the cell walls of mature stigma papillae and in the tapetum and microspores during early stages of anther development. In the mature anther, a positive signal in the cytoplasm of pollen grains with some detectable localisation in the exine layer of the pollen wall was obtained. Similar results were obtained with acetate hydrolase antibodies. These esterases are thus spatially and temporally regulated in stigma and anther tissues.Abbreviations MU methyl umbelliferyl - pAbC anti-butyrate hydrolase polyclonal antibodies - pAbE anti-acetate hydrolase polyclonal antibodies     

A cDNA clone encoding an IgE-binding protein from Brassica anther has significant sequence similarity to Ca2+-binding proteins

Thirteen cDNA clones encoding IgE-binding proteins were isolated from expression libraries of anthers of Brassica rapa L. and B. napus L. using serum IgE from a patient who was specifically allergic to Brassica pollen. These clones were divided into two groups, I and II, based on the sequence similarity. All the group I cDNAs predicted the same protein of 79 amino acids, while the group II predicted a protein of 83 amino acids with microheterogeneity. Both of the deduced amino acid sequences contained two regions with sequence similarity to Ca²⁺-binding sites of Ca²⁺-binding proteins such as calmodulin. However flanking sequences were distinct from that of calmodulin or other Ca²⁺-binding proteins. RNA-gel blot analysis showed the genes of group I and II were preferentially expressed in anthers at the later developmental stage and in mature pollen. The recombinant proteins produced in Escherichia coli was recognized in immunoblot analysis by the IgE of a Brassica pollen allergic patient, but not by the IgE of a non-allergic patient. The cDNA clones reported here, therefore, represent pollen allergens of Brassica species.     

Cold pretreatment enhances microspore embryogenesis in oilseed rape (Brassica napus L.)

Stress is an essential component during embryogenesis induction in microspore culture. Cold pretreatment has been used in cereal microspore culture but very seldom attempted in Brassica microspore culture. The effect of cold pretreatment of flower buds subjected to a liquid medium on microspore embryogenesis was investigated in spring and winter Brassica napus, as well as in B. rapa and B. oleracea. Cold pretreatment significantly enhanced microspore embryogenesis (by 1–7 fold) compared to commonly used microspore culture protocol in B. napus, while it was less effective in B. rapa or even negative in B. oleracea. The appropriate duration of cold pretreatment was found to be 2–4 days, which stimulated the best microspore embryogenesis. Cold pretreatment was also able to promote embryo development including the improvement of embryo quality and acceleration of embryogenesis. When incorporating with medium refreshing, cold pretreatment could initiate the most microspore embryogenesis than any other treatment used. With further improvement cold pretreatment method may have a positive potential in Brassica breeding programmes.     

Self-incompatibility in a distylous species of Rubiaceae: is there a single incompatibility response of the morphs?

Heterostyly is a genetically controlled floral polymorphism usually associated with an incompatibility system. This set of features is known to occur in several angiosperm families, but some aspects of its biology has not been well studied. The present study investigates cellular aspects of the pollen–pistil interaction after compatible and incompatible pollinations of Psychotria nuda, to increase our knowledge of heteromorphic self-incompatibility (HetSI). The use of bright field, fluorescence and transmission electron microscopy methods allowed us to demonstrate that pollen tubes behave differently after incompatible and compatible pollinations. Pollen tubes were particularly distinct after incompatible pollinations of L- and S-morph flowers. Relative to compatible pollen tubes, incompatible L-morph tubes had a drastic reduction in cellular contents, but no cell rupture. Incompatible S-morph tubes exhibited dense cytoplasm in apical regions, as well as in other regions, accompanied by a rupture of the apex. These results support the hypothesis that L- and S-morph flowers have different incompatibility mechanisms during HetSI.     

INTERCELLULAR ADHESIONS IN THE POLLEN-STIGMA SYSTEM: POLLEN CAPTURE,GRAIN BINDING,AND TUBE ATTACHMENTS

Four independent pollen-stigma binding forces are differentiated after pollen contacts stigmas of Brassica oleracea var. capitata. Identification is based on their different rates and sequence of appearance during gametophyte development; on their differential occurrence after compatible and incompatible pollinations; and on their different stabilities to NaOH and hexane. The first binding force develops most rapidly, begins seconds after pollen-stigma contact, is complete within minutes, occurs on compatible or incompatible papillae, and dissociates in methanol. A second slower binding reaction begins about 15 min after contact, continues for at least 90 min (when pollen tubes emerge), results in a binding structure termed the ocreatine, develops only on compatible papillae, and is dissociated by NaOH. A third attractive mechanism binds the tip of the emerging tube to a cuticle, is detected only on incompatible papillae, and is not dissociated by NaOH or methanol. Ocreatine formation and tube development beyond the emergence stage are prevented by the incompatibility response. A fourth attraction mechanism occurs between the surfaces of papilla and elongating tubes. Reviews of physical and biochemical evidence indicate that van der Waals forces and enzymatically mediated lipid polymerization are alternatives to agglutination as mechanisms for binding male gametophyte to papilla.     

Assessment of the degree of restriction fragment length polymorphism in Brassica

Summary The feasibility of creating a restriction fragment length polymorphism (RFLP) linkage map in Brassica species was assessed by screening EcoRI-, HindIII-, or EcoRV-digested total genomic DNA from several accessions of B. campestris, B. oleracea, and B. napus using random genomic DNA clones from three Brassica libraries as hybridization probes. Differences in restriction fragment hybridization patterns occurred at frequencies of 95% for comparisons of accessions among species, 79% for comparisons of accessions among subspecies within species, and 70% for comparisons among accessions within subspecies. In addition, species differences in the level of hybridization were noted for some clones. The high degree of polymorphism found even among closely related Brassica accessions indicates that RFLP analysis will be a very useful tool in genetic, taxonomic, and evolutionary studies of the Brassica genus. Development of RFLP linkage maps is now in progress.     

Loss of callose in the stigma papillae does not affect the Brassica self-incompatibility phenotype

As part of the Brassicaceae self-incompatibility response, callose is deposited in the stigma papillar cells. To determine if callose plays an important role in the rejection of incompatible pollen by the stigma, transgenic Brassica napus. L. plants were produced which express the tobacco β-1,3-glucanase cDNA (the enzyme which degrades callose) in the stigma papillae. Using aniline blue fluorescence, little or no callose was detected in the papillar cells of transgenic stigmas. However, the self-incompatibility system appeared to be unaffected based on the lack of pollen tube growth and the subsequent lack of seed set. The transgene had no effect on compatible pollinations. Thus, while callose deposition is associated with the B. napus self-incompatibility response, it is not required for the rejection of incompatible pollen. Received: 14 March 1997 / Accepted: 15 April 1997     

Commonality of self-recognition specificity of S haplotypes between Brassica oleracea and Brassica rapa

We have identified several interspecific pairs of S haplotypes having highly similar SRK and SP11/SCR sequences between Brassica oleracea and Brassica rapa. The recognition specificities of S haplotypes in these pairs were examined with three different methods. Stigmas of interspecific hybrids between an S-32 homozygote in B. oleracea and an S-60 homozygote in B. rapa, which were produced to avoid the interspecific incompatibility between the two species, showed incompatibility to the pollen of an S-8 homozygote in B. rapa and to the pollen of an S-15 homozygote in B. oleracea, while it showed compatibility to the pollen of other S haplotypes, suggesting B. oleracea S-32 and B. rapa S-60 have the same recognition specificity as B. rapa S-8 and B. oleracea S-15. Pollen grains of transgenic S-60 homozygous plants in B. rapa carrying a transgene of SP11-24 from B. oleracea were incompatible to B. rapa S-36 stigma, indicating that B. oleracea S-24 and B. rapa S-36 have the same recognition specificity. Application of the SP11 protein of B. rapa S-41 and S-47 onto the surface of B. oleracea S-64 stigmas and S-12 stigmas, respectively, resulted in the incompatibility reaction to pollen grains of another S haplotype, but application onto the stigmas of other S haplotypes did not, suggesting that B. oleracea S-64 stigmas and S-12 stigmas recognized the B. rapa SP11-41 and SP11-47 proteins as self SP11 proteins, respectively. Besides having evolutionary implications, finding of many interspecific pairs of S haplotypes can provide insight into the molecular mechanism of self-recognition. Comparing deduced amino-acid sequences of SP11 proteins and SRK proteins in the pairs, regions of SP11 and SRK important for self-recognition are discussed.     

Pollen stigma interactions in Brassica oleracea

Summary Recent studies on the mechanism of self-incompatibility in Brassica indicate the location, nature and mode of action of the molecules involved. Characteristics of the pollen surface and the stigma surface are described in detail, together with new information pertaining to the recognition molecules located therein. A sequence of events is outlined leading from pollination, through adhesion, hydration, germination, and tube growth to acceptance and ultimate compatibility. The characteristics of rejection of incompatible grains are described for each stage of the pollen-stigma interaction. It is proposed that recognition of proteins from the coating of self-pollen by the molecules in the pellicle results in the formation of a biologically-active complex which inhibits water supply to the incompatible grain, and that all other manifestations of incompatibility are a consequence of this initial response.     

The interrelationship between the accumulation of lipids,protein and the level of acyl carrier protein during the development of Brassica napus L. pollen

Lipid accumulation during pollen and tapetal development was studied using cryostat sections of unfixed anthers from Brassica napus (rapeseed). Diamidino-2-henylindole (DAPI), a DNA fluorochrome, was used to stain the pollen nuclei in order to identify ten stages of pollen development in Brassica. Storage lipids (i.e. triacylglycerides) were stained using the fluorochrome Nile red. Pollen coat lipids are formed in tapetal plastids between the mid-vacuolate and early maturation pollen stages. The pollen coat components, including lipids and a proportion of the proteins, are derived from the remnants of the tapetum, after its rupture, during the second pollen mitosis. Quantitative microfluorometric analyses demonstrated four phases of lipid body accumulation or depletion in the developing pollen cytoplasm. The majority of storage lipids found in the cytoplasm of the mature pollen grain accumulated during the late vacuolate and early maturation stages when the pollen is bicellular. The level of acyl carrier protein, a protein integrally involved in lipid synthesis, was also found to be maximal in the developing pollen during the bicellular pollen stages of development. This coincided with the most active period of lipid accumulation. These data could indicate that the lipids of the pollen are synthesized in situ, by metabolic processes regulated by expression of genes in the haploid genome.To whom correspondence should be addressed     

The Brassica MIP-MOD gene encodes a functional water channel that is expressed in the stigma epidermis

In crucifers, the ability of the stigma to differentially modulate hydration of pollen grains, depending on whether the pollen is recognized to be compatible or incompatible, represents a crucial stage in pollination. Our recent analysis of the mod mutation of Brassica, which results in a breakdown of the self-incompatibility response, led to the isolation of a gene linked to the MOD locus which is expressed at low levels in mod mutants. The gene is predicted to encode a plasma membrane-localized aquaporin-like protein and has been designated MIP-MOD. We utilized reporter gene analysis to demonstrate that the MIP-MOD promoter is active in Brassica papillar cells as well as in some vegetative tissues. The encoded protein is also likely to be plasma membrane-localized based on the observation that all plasma membrane-intrinsic aquaporin-like proteins in Brassica leaves are enriched in plasma membrane fractions. The MIP-MOD protein results in a low but measurable enhancement in osmotic water permeability of Xenopus oocytes and hence represents a functional aquaporin. The results are consistent with the notion that MIP-MOD is involved in the regulation of water transport across the stigma epidermal cell membrane.     

Intergeneric hybridization between Diplotaxis siettiana and crop brassicas for the production of alloplasmic lines

Summary Intergeneric hybrids were produced between Diplotaxis siettiana and Brassica campestris through embryo rescue. The hybrids were completely pollen sterile and backcrosses with pollen of B. campestris did not yield any seeds. Induction of colchiploidy restored pollen fertility and backcross pollinations yielded viable seeds. Cytological details of the hybrid, amphidiploid and backcross progenies were studied. Both pollen-sterile and pollen-fertile plants have been obtained in backcross 2 progeny. This hybrid (D. siettiana x B. campestris) was used as a bridge cross to transfer the cytoplasm of D. Siettiana to two other incompatible cultivars of Brassica — B. juncea and B. napus. Pollinations of the amphidiploid (D. siettiana x B. campestris, 2n = 36) with pollen of B. juncea/B. napus readily produced seeds without embryo rescue. These hybrids were grown to flowering and their cytological details were studied. Seeds have been produced from backcross pollinations of both these hybrids with the pollen of the respective cultivars. The results clearly show the feasibility of producing alloplasmic lines in all the three oilseed brassicas.     

Genetic variation within and among different cultivars and landraces of Brassica campestris L. and B. oleracea L. based on isozymes

Genetic variation based on isozymes was studied in 43 landraces and cultivars of Brassica campestris from China, 4 cultivars of B. campestris from Sweden and 1 from India, and 5 cultivars of B. oleracea from Sweden and 1 from China (B. alboglabra). A total of 17 isozyme loci was studied, 10 of these were polymorphic in B. campestris and 6 were polymorphic in B. oleracea. The level of heterozygosity seemed to be reduced in the Swedish cultivars compared to the Chinese landraces and cultivars of B. campestris. The level of heterozygosity in B. oleracea was even lower than that in the Swedish cultivars of B. campestris. A phylogeny of the cultivars and landraces of B. campestris showed that the B. campestris var yellow sarson cultivar, originating from India, deviated significantly from the other cultivars of B. campestris. A phylogeny of the cultivars of B. oleracea confirmed the expectations that the cultivar B. alboglabra was not closely related to the cultivated forms of B. oleracea.     

Heat shock response during anther culture of broccoli (Brassica oleracea var italica)

Embryo formation from microspores of Brassica oleracea var Italica (Broccoli) and other Brassica species is greatly enhanced by an initial incubation at elevated temperatures (eg 35°C) followed by continued incubation of 25°C. In the present study we observed that a three hour high temperature treatment induced the formation of heat shock proteins in cultured anthers. These were identified in two dimensional gels by silver staining, and labelled heat shock proteins were synthesised in vitro from isolated anther RNA. The appearance of heat shock proteins in anthers followed a similar pattern and displayed similar characteristics to that from leaves. Comparison of the heat shock proteins induced in isolated cultured anthers of known highly embryogenic and less embryogenic plans did not reveal obvious qualitative differences.     

An efficient method for flow cytometric analysis of pollen and detection of 2n nuclei in Brassica napus pollen

A simple and reliable method was developed for isolating pollen nuclei from Brassica napus and Triticum aestivum for DNA analysis using flow cytometry. The nuclei were released from pollen by ultrasonic treatment. The isolated nuclei following filtration through nylon mesh and a purification procedure were suitable for flow cytometric analysis as well as for isolating genomic DNA. Ultrasonic treatment time was optimized for B. napus pollen at different developmental stages. The method is effective and suitable for the preparation of many samples. We analyzed the nuclear DNA levels in pollen of B. napus at three major developmental stages as well as in mature wheat pollen. Only a single 1C peak representing the haploid DNA level was detected in the nuclei isolated from Brassica uninucleate microspores as well as in mature Triticum pollen. Interestingly, diploid nuclei were detected in both binucleate and mature pollen of B. napus. The possible origins of the diploid nuclei are discussed.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - NIB Nuclear isolation buffer     

Conservation of S-locus for self-incompatibility in Brassica napus (L.) and Brassica oleracea (L.)

 Self-incompatibility (SI) in Brassica is a sporophytic system, genetically determined by alleles at the S-locus, which prevents self-fertilization and encourages outbreeding. This system occurs naturally in diploid Brassica species but is introduced into amphidiploid Brassica species by interspecific breeding, so that in both cases there is a potential for yield increase due to heterosis and the combination of desirable characteristics from both parental lines. Using a polymerase chain reaction (PCR) based analysis specific for the alleles of the SLG (S-locus glycoprotein gene) located on the S-locus, we genetically mapped the S-locus of B. oleracea for SI using a F₂ population from a cross between a rapid-cycling B. oleracea line (CrGC-85) and a cabbage line (86-16-5). The linkage map contained both RFLP (restriction fragment length polymorphism) and RAPD (random amplified polymorphic DNA) markers. Similarly, the S-loci were mapped in B. napus using two different crosses (91-SN-5263×87-DHS-002; 90-DHW-1855-4×87-DHS-002) where the common male parent was self-compatible, while the S-alleles introgressed in the two different SI female parents had not been characterized. The linkage group with the S-locus in B. oleracea showed remarkable homology to the corresponding linkage group in B. napus except that in the latter there was an additional locus present, which might have been introgressed from B. rapa. The S-allele in the rapid-cycling Brassica was identified as the S₂₉ allele, the S-allele of the cabbage was the S ₅ allele. These same alleles were present in our two B. napus SI lines, but there was evidence that it might not be the active or major SI allele that caused self-incompatibility in these two B. napus crosses. Received: 7 June 1996/Accepted: 6 September 1996