Recognition specificity of self-incompatibility maintained after the divergence of Brassica oleracea and Brassica rapa

The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen, respectively. In the pair of S haplotypes BrS46 (S46 in B. rapa) and BoS7 (S7 in B. oleracea), which have highly similar SRK alleles, the SP11 alleles were found to be similar, with 96.1% identity in the deduced amino acid sequence. Two other pairs of S haplotypes, BrS47 and BoS12, and BrS8 and BoS32, having highly similar SRK and SP11 alleles between the two species were also found. The haplotypes in each pair are considered to have been derived from a single S haplotype in the ancestral species. The allotetraploid produced by interspecific hybridization between homozygotes of BrS46 and BoS15 showed incompatibility with a BoS7 homozygote and compatibility with other B. oleracea S haplotypes in reciprocal crossings. This result indicates that BrS46 and BoS7 have maintained the same recognition specificity after the divergence of the two species and that amino acid substitutions found in such cases in both SRK alleles and SP11 alleles do not alter the recognition specificity. DNA blot analysis of SRK, SP11, SLG and other S-locus genes showed different DNA fragment sizes between the interspecific pairs of S haplotypes. A much lower level of sequence similarity was observed outside the genes of SRK and SP11 between BrS46 and BoS7. These results suggest that the DNA sequences of the regions intervening between the S-locus genes were diversified after or at the time of speciation. This is the first report demonstrating the presence of common S haplotypes in different plant species and presenting definite evidence of the trans-specific evolution of self-incompatibility genes.     

Expression level of the SLG gene is not correlated with the self-incompatibility phenotype in the class II S haplotypes of Brassica oleracea

In Brassica, the S-locus glycoprotein (SLG) gene has been strongly implicated in the self-incompatibility reaction. Several alleles of this locus have been sequenced, and accordingly grouped as class I (corresponding to dominant S-alleles) and class II (recessive). We recently showed that a self-compatible (Sc) line of Brassica oleracea expressed a class II-like SLG (SLG-Sc) gene. Here, we report that the SLG-Sc glycoprotein is electrophoretically and immunochemically very similar to the recessive SLG-S15 glycoprotein, and is similarly expressed in stigmatic papillae. Moreover, by seed yield analysis, we observe that both alleles are associated with a self-compatibility response, in contrast with the other known recessive S haplotypes (S2 and S5). By genomic DNA blot analysis, we show the existence of molecular homologies between the Sc and S15 haplotypes, but demonstrate that they are not identical. On the other hand, we also report that the S2 haplotype expresses very low amounts of SLG glycoproteins, although it exhibits a self-incompatible phenotype. These results strongly question the precise role of the SLG gene in the molecular mechanisms that control the self-incompatibility reaction of Brassica.     

Interspecific pairs of class II S haplotypes having different recognition specificities between Brassica oleracea and Brassica rapa

There are several pairs of similar class I S haplotypes between Brassica oleracea and Brassica rapa. The similar S halotypes in these interspecific pairs have been reported to have the same recognition specificities. In the present study, three interspecific pairs showing a high sequence similarity were found in class II S haplotypes, i.e. between BoS-2b (B. oleracea S-2b) and BrS-44 (B. rapa S-44), between BoS-5 and BrS-40, and between BoS-15 and BrS-60. By pollination tests using interspecific hybrids between B. oleracea and B. rapa, BoS-5 and BoS-2b were revealed to have slightly and completely different recognition specificities from those of BrS-40 and BrS-44, respectively. The recognition reaction between SP11 and SRK of BoS-15 was suggested to be incomplete. The regions of class II SP11 and SRK important for self-recognition specificity and the diversification of class II S haplotypes are discussed herein.     

Introduction of SLG (S locus glycoprotein) alters the phenotype of endogenous S haplotype,but confers no new S haplotype specificity in Brassica rapa L.

Self-incompatibility (SI) in Brassicaceae is genetically controlled by the S locus complex in which S locus glycoprotein (SLG) and S receptor kinase (SRK) genes have been identified, and these two genes encoding stigma proteins are believed to play important roles in SI recognition reaction. Here we introduced the SLG⁴³ gene of Brassica rapa into a self-incompatible cultivar, Osome, of B. rapa, and examined the effect of this transgene on the SI behavior of the transgenic plants. Preliminary pollination experiments demonstrated that Osome carried S⁵² and S⁶⁰, and both were codominant in stigma, but S⁵² was dominant to S⁶⁰ in pollen. S⁴³ was found to be recessive to S⁵² and codominant with S⁶⁰ in stigma. The nucleotide sequence of SLG⁴³ was more similar to that of SLG⁵² (87.8% identity) than to that of SLG⁶⁰ (74.8% identity). Three of the ten primary transformants (designated No. 1 to No. 10) were either completely (No. 9) or partially (No. 6 and No. 7) self-compatible; the SI phenotype of the stigma was changed from S⁵²S⁶⁰ to S⁶⁰, but the SI phenotype of the pollen was not altered. In these three plants, the mRNA and protein levels of both SLG⁴³ and SLG⁵² were reduced, whereas those of SLG⁶⁰ were not. All the plants in the selfed progeny of No. 9 and No. 6 regained SI and they produced a normal level of SLG⁵². These results suggest that the alteration of the SI phenotype of the stigma in the transformants Nos. 6, 7, and 9 was the result of specific co-suppression between the SLG⁴³ transgene and the endogenous SLG⁵² gene. Three of the transformants (Nos. 5, 8 and 10) produced SLG⁴³ protein, but their SI phenotype was not altered. The S⁶⁰ homozygotes in the selfed progeny of No. 10 which produced the highest level of SLG⁴³ were studied because S⁴³ was codominant with S⁶⁰ in the stigma. They produced SLG⁴³ at approximately the same level as did S⁴³S⁶⁰ heterozygotes, but did not show S⁴³ haplotype specificity at the stigma side. We conclude that SLG is necessary for the expression of the S haplotype specificity in the stigma but the introduction of SLG alone is not sufficient for conferring a novel S haplotype specificity to the stigma.     

Physical distances between S-locus genes in various S haplotypes of Brassica rapa and B. oleracea

In Brassica, self-incompatibility genes SLG (for S-locus glycoprotein) and SRK (for S-receptor kinase) are located in the S-locus complex region with several other S-linked genes. The S locus is a highly polymorphic region: polymorphism has been observed not only in sequences of SLG and SRK but also in the location of the S-locus genes. In order to compare the physical location of the S-locus genes in various S haplotypes, we used six class-I S haplotypes of B. rapa and seven class-I S haplotypes of B. oleracea in this study. DNA gel blot analysis using pulsed-field gel electrophoresis (PFGE) showed that the physical distances between SLG and SRK in B. rapa are significantly shorter than those in B. oleracea and that the sizes of MluI and BssHII fragments harboring SLG and SRK are less variable within B. rapa than within B. oleracea. We concluded that several large genomic fragments might have been inserted into the S-locus region of B. oleracea after allelic differentiation of S-locus genes. Received: 20 September 1999 / Accepted: 8 October 1999     

Comparative analysis of S haplotypes with very similar SLG alleles in Brassica rapa and Brassica oleracea

Self-incompatibility in Brassica is controlled by a single multi-allelic locus (the S locus) which harbors at least two highly polymorphic genes, SLG and SRK. SRK is a putative transmembrane receptor kinase and its amino acid sequence of the extracellular domain of SRK (the S domain) exhibits high homology to that of SLG. The amino acid sequences of the SLGs of S8 and S46 haplotypes of B. rapa are very similar and those of S23 and S29 haplotypes of B. oleracea were also found to be almost identical. In both cases, SLG and the S domain of SRK of the same haplotype were less similar. This seems to contradict the idea that SLG and SRK of the same haplotype have the same self-recognition specificity. In the transmembrane-kinase domain, the SRK alleles of the S8 and S46 haplotypes had almost identical nucleotide sequences in spite of their lower homology in the S domain. Such a cluster of nucleotide substitutions is probably due to recombination or related events, although recombination in the S locus is thought to be suppressed. Based on our observations, the recognition mechanism and the evolution of self-incompatibility in Brassica are discussed.     

Transformation and regeneration of Brassica rapa using Agrobacterium tumefaciens

Summary Transformation and regeneration procedures for obtaining transgenic Brassica rapa ssp. oleifera plants are described. Regeneration frequencies were increasedby using silver nitrate and by adjusting the duration of exposure to 2,4-D. For transformation, Agrobacterium tumefaciens strain EHA101 containing a binary plasmid with the neomycin phosphotransferase gene (NPT II) and the b-glucuronidase gene (GUS) was cocultivated with hypocotyl explants from the oilseed B. rapa cvs. Tobin and Emma. Transformed plants were obtained within three months of cocultivation. Transformation frequencies for the cultivars Tobin and Emma were 1–9%. Evidence for transformation was shown by NPT II dot blot assay, the GUS fluorometric assay, Southern analysis, and segregation of the kanamycin-resistance trait in the progeny. The transformation and regeneration procedure described here has been used routinely to transform two cultivars of B. rapa and 18 cultivars of B. napus.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Atrazine-resistant cytoplasmic male-sterile-nigra broccoli obtained by protoplast fusion between cytoplasmic male-sterile Brassica oleracea and atrazine-resistant Brassica campestris

Summary By using restriction endonuclease digestion patterns, the degree of intraspecific polymorphism of mitochondrial DNA in four diploid species of wheat and Aegilops, Ae. speltoides, Ae. longissima, Ae. squarrosa, and Triticum monococcum, was assessed. The outbreeding Ae. speltoides was found to possess the highest degree of variability, the mean number of nucleotide substitutions among conspecific individuals being 0.027 substitutions per nucleotide site. A very low degree of mtDNA variation was detected among Ae. longissima accessions, with most of the enzyme-probe combinations exhibiting uniform hybridization patterns. The mean number of substitutions among Ae. longissima individuals was 0.001 substitutions per nucleotide site. The domesticated diploid wheat T. monococcum var. monococcum and its conspecific variant T. monococcum var. boeoticum seem to lack mitochondrial DNA variability altogether. Thus, the restriction fragment pattern can be used as a characteristic identifier of the T. monococcum cytoplasmic genome. Similarly, Ae. squarrosa accessions were found to be genetically uniform. A higher degree of variation among accessions is observed when noncoding sequences are used as probes then when adjacent coding regions are used. Thus, while noncoding regions may contain regulatory functions, they are subject to less stringent functional constraints than protein-coding regions. Intraspecific variation in mitochondrial DNA correlates perfectly with the nuclear variability detected by using protein electrophoretic characters. This correlation indicates that both types of variation are selectively neutral and are affected only by the effective population size.     

Time-lapse imaging of self- and cross-pollinations in Brassica rapa

Background and Aims

Pollination is an important process in the life cycle of plants and is the first step in bringing together the male and female gametophytes for plant reproduction. While pollination has been studied for many years, accurate knowledge of the morphological aspects of this process is still far from complete. This study therefore focuses on a morphological characterization of pollination, using time-series image analysis of self- and cross-pollinations in Brassica rapa.

Methods

Time-lapse imaging of pollen behaviour during self- and cross-pollinations was recorded for 90 min, at 1 min intervals, using a stereoscopic microscope. Using time-series digital images of pollination, characteristic features of pollen behaviours during self- and cross-pollinations were studied.

Key Results

Pollen exhibited various behaviours in both self- and cross-pollinations, and these were classified into six representative patterns: germination, expansion, contraction, sudden contraction, pulsation and no change. It is noteworthy that in ‘contraction’ pollen grains shrunk within a short period of 30–50 min, and in ‘pulsation’ repeated expansion and contraction occurred with an interval of 10 min, suggesting that a dehydration system is operating in pollination. All of the six patterns were observed on an individual stigma with both self- and cross-pollinations, and the difference between self- and cross-pollinations was in the ratios of the different behaviours. With regard to water transport to and from pollen grains, this occurred in multiple steps, before, during and after hydration. Thus, pollination is regulated by a combination of multiple components of hydration, rehydration and dehydration systems.

Conclusions

Regulated hydration of pollen is a key process for both pollination and self-incompatibility, and this is achieved by a balanced complex of hydration, dehydration and nutrient supply to pollen grains from stigmatic papilla cells.     

Biosynthesis of glycoproteins involved in the pollen-stigma interaction of incompatibility in developing flowers of Brassica oleracea L.

De-novo synthesis of the S-allele-specific glycoproteins of Brassica oleracea is demonstrated in stigmas at different developmental stages. Excised stigmas incorporate ¹⁴C-labeled amino acids into their S-glycoproteins early in development and before the self-incompatibility response is acquired, but the rate of synthesis accelerates prior to anthesis, resulting in the accumulation of high levels of the S-glycoproteins in the stigma and coinciding with the acquisition of the pollen-stigma incompatibility response. Since the self-compatible and self-incompatible zones of developing inflorescences are very sharply delineated, a threshold quantity of S-glycoproteins appears to be critical for the onset of self-incompatibility. Incorporation experiments in which [^35Smethionine was applied to intact stigma surfaces indicate that the papillae are the main sites of synthesis of the S-specific glycoproteins.Abbreviations IEF isoelectric focusing - SC self-compatibility - SDS sodium dodecyl sulfate - SI self-incompatibility     

Gene conversion from SLG to SRK resulting in self-compatibility in Brassica rapa

Self-compatible S-54 homozygotic plants were found in progenies of an F(1) hybrid cultivar in Chinese cabbage. Pollination tests revealed that this self-compatibility is controlled by the S locus and caused by the loss of the recognition function of the stigma. SRK, the gene for the recognition molecule in the stigma, was normally transcribed and translated in the self-compatible plants. The 1034-bp region in the receptor domain of SRK in the self-compatible plants was 100% identical to SLG in S-54, while that in self-incompatible S-54 homozygotic plants was 95.1% identical. These results suggest that the self-compatibility of the S-54 homozygotes is due to amino-acid changes caused by gene conversion from SLG to SRK.     

Selection of Brassica rapa genotypes for tolerance to boron toxicity

Although boron (B) is a micronutrient essential for the growth of vascular plants, it reduces growth and seed yield when present in excessive amounts. A hydroponic assay of nineteen Brassica rapa genotypes resulted in the identification of two tolerant genotypes, WWY Sarson and Local at a range of boron concentrations (15–165 μM). The most tolerant and sensitive genotypes were assessed for shoot boron concentrations in a soil assay with 4, 29 and 54 mg B kg⁻¹ soil. The soil assay confirmed the results of the hydroponic screening. Shoot boron uptake was at least three times lower and shoot boron concentrations about 10 times lower in the tolerant than sensitive genotypes, indicating that boron tolerance involved boron exclusion from the shoot.     

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.     

Synthesis and phosphorylation of pollen proteins during the pollen-stigma interaction in self-compatible Brassica napus L. and self-incompatible Brassica oleracea L.

A technique is described which permits the in vivo study of protein synthesis and phosphorylation in the pollen of Brassica spp. during the early stages of the pollen-stigma interaction. In Brassica napus and B. oleracea, compatible pollination is followed by a dramatic activation of protein synthesis in the pollen involving the synthesis of approximately 40 proteins. After incompatible pollinations in B. oleracea, virtually no newly synthesised polypeptides were detected in the pollen except for a small group of high molecular weight proteins which were not normally synthesised during compatible pollinations. Both compatible and incompatible pollinations were followed by the appearance of newly phosphorylated proteins in the pollen; these fell into four distinct groups. In B. oleracea, the number of phosphorylated proteins and the degree of phosphorylation of individual proteins within the four groups differed between compatible and incompatible pollinations. One group of phosphorylated proteins appeared to correspond with the small group of high molecular weight polypeptides which were synthesised in pollen after incompatible pollinations. These findings are discussed in the perspective of cell signalling during the pollen-stigma interaction in Brassica and also in terms of their possible implication in sporophytic self-incompatibility.     

Artificial synthesis of interspecific chimeras between tuber mustard (Brassica juncea) and cabbage (Brassica oleracea) and cytological analysis

Interspecific chimeras between tuber mustard and red cabbage were obtained by in vitro graft-culture method. Before grafting, 6-day-old seedlings of tuber mustard and red cabbage were vertically half-cut and treated with different concentrations of 6-BA and NAA for 1 min, then, they were symmetrically fit together. As a result, sectorial chimeras were initially produced from the united shoot tips. The maximum frequency of chimeral bud formation reached 6.33% when the vertical sections of tuber mustard and cabbage were treated with 2 mg/l 6-BA and 1 mg/l NAA. When sectorial chimeras were propagated on MS medium containing 1 mg/l 6-BA, periclinal and mericlinal chimeras gradually developed. Chimeral shoots were rooted on half-strength MS medium containing 0.1 mg/l NAA. The rooted chimeras were acclimatized and transferred to the field for cytological and morphological analysis. The results showed that stomata density in the chimeras was significantly higher than that of their parents, while chloroplast size, starch grain size and number were intermediate between the two parents. The chimeras were further analyzed by flow cytometry, and the results indicated that they contained both sets of parental chromosomes. Moreover, chimeral plants possessed valuable characters from the two parents.     

Seed predation,pathogen infection and life-history traits in Brassica rapa

Herbivory and disease can shape the evolution of plant populations, but their joint effects are rarely investigated. Families of plants of Brassica rapa (Brassicaceae) were grown from seeds collected in two naturalized populations in an experimental garden. We examined leaf infection by the fungus Alternaria, seed predation by a gall midge (Cecidomyiidae) and plant life-history traits. Plants from one population had heavier seeds, were more likely to flower, had less fungal infection, had more seed predation and were more fecund. Fungal infection score and seed predation rate increased with plant size, but large plants still had the greatest number of undamaged fruits. Spatial heterogeneity in the experimental garden was significant; seed predation rate and fecundity varied among blocks. An apparent tradeoff existed between susceptibility to disease and seed predation: plants with the highest fungal infection score had the lowest seed predation rate. Alternaria infection varied between populations, but the disease had no effect on fecundity. Seed predation did reduce fecundity. Damaged fruits had 31.4% fewer intact seeds. However, evidence for additive genetic variation in resistance to seed predation was weak. Therefore, neither disease nor seed predation was likely to be a strong agent of genetically based fecundity selection.     

Atrazine-resistant cauliflower obtained by somatic hybridization between Brassica oleracea and ATR-B. napus

Summary Somatic hybridization between Brassica oleracea ssp. botrytis (cauliflower, 2n=18), carrying the Ogura (R1) male-sterile cytoplasm and B. napus (2n= 38), carrying a male-fertile, atrazine-resistant (ATR) cytoplasm, yielded three hybrids (2n=56) and six cauliflower cybrids (2n=18), which were selected for resistance to the herbicide in vitro. The hybrids and cybrids were male fertile and self-compatible. They contained both chloroplasts and mitochondria from the ATR cytoplasm. We found no evidence for mtDNA recombination in any of the regenerated plants. Selfed progeny of the B. oleracea atrazine-resistant cybrids were evaluated for tolerance to the herbicide in the field. Resistant plants exposed to 0.56–4.48 kg/ha (0.5–4.0 pounds/acre) atrazine in the soil showed no damage at any herbicide level, whereas plants of a susceptible alloplasmic line were severely damaged at the lowest level of herbicide application and killed at all higher levels. These atrazine-resistant cauliflower may have potential horticultural use, especially in fields where atrazine carry over is a serious problem.