A cell wall invertase controls nectar volume and sugar composition.

Nectar volume and sugar composition are key determinants of the strength of plant-pollinator mutualisms. The main nectar sugars are sucrose, glucose and fructose, which can vary widely in ratio and concentration across species. Brassica spp. produce a hexose-dominant nectar (high in the monosaccharides glucose and fructose) with very low levels of the disaccharide sucrose. Cell wall invertases (CWINVs) catalyze the irreversible hydrolysis of sucrose into glucose and fructose in the apoplast. We found that BrCWINV4A is highly expressed in the nectaries of Brassica rapa. Moreover, a brcwinv4a null mutant: (i) has greatly reduced CWINV activity in the nectaries; (ii) produces a sucrose-rich nectar; but (iii) with significantly less volume. These results definitively demonstrate that CWINV activity is not only essential for the production of a hexose-rich nectar, but also support a hypothetical model of nectar secretion in which its hydrolase activity is required for maintaining a high intracellular-to-extracellular sucrose ratio that facilitates the continuous export of sucrose into the nectary apoplast. The extracellular hydrolysis of each sucrose into two hexoses by BrCWINV4A also likely creates the osmotic potential required for nectar droplet formation. These results cumulatively indicate that modulation of CWINV activity can at least partially account for naturally occurring differences in nectar volume and sugar composition. Finally, honeybees prefer nectars with some sucrose, but wild-type B. rapa flowers were much more heavily visited than flowers of brcwinv4a, suggesting that the potentially attractive sucrose-rich nectar of brcwinv4a could not compensate for its low volume.

Reduced fertility caused by meiotic defects and micronuclei formation during microsporogenesis in xBrassicoraphanus.

BACKGROUND: Hybridization and polyploidization events are important driving forces in plant evolution. Allopolyploids formed between different species can be naturally or artificially created but often suffer from genetic instability and infertility in successive generations. xBrassicoraphanus is an intergeneric allopolyploid obtained from a cross between Brassica rapa and Raphanus sativus, providing a useful resource for genetic and genomic study in hybrid species. OBJECTIVE: The current study aims to understand the cause of hybrid sterility and pollen abnormality in different lines of synthetic xBrassicoraphanus from the cytogenetic perspective. METHODS: Alexander staining was used to assess the pollen viability. Cytogenetic analysis was employed to monitor meiotic chromosome behaviors in pollen mother cells (PMCs). Origins of parental chromosomes in xBrassicoraphanus meiocytes were determined by genome in situ hybridization analysis. RESULTS: The xBrassicoraphanus lines BB#4 and BB#6 showed high rates of seed abortion and pollen deformation. Abnormal chromosome behaviors were observed in their PMCs, frequently forming univalents and inter-chromosomal bridges during meiosis. A positive correlation also exists between meiotic defects and the formation of micronuclei, which is conceivably responsible for unbalanced gamete production and pollen sterility. CONCLUSION: These results suggest that unequal segregation of meiotic chromosomes, due in part to non-homologous interactions, is responsible for micronuclei and unbalanced gamete formation, eventually leading to pollen degeneration and inferior fertility in unstable xBrassicoraphanus lines.

Duplicated pollen-pistil recognition loci control intraspecific unilateral incompatibility in Brassica rapa.

In plants, cell-cell recognition is a crucial step in the selection of optimal pairs of gametes to achieve successful propagation of progeny. Flowering plants have evolved various genetic mechanisms, mediated by cell-cell recognition, to enable their pistils to reject self-pollen, thus preventing inbreeding and the consequent reduced fitness of progeny (self-incompatibility, SI), and to reject foreign pollen from other species, thus maintaining species identity (interspecific incompatibility)1. In the genus Brassica, the SI system is regulated by an S-haplotype-specific interaction between a stigma-expressed female receptor (S receptor kinase, SRK) and a tapetum cell-expressed male ligand (S locus protein 11, SP11), encoded by their respective polymorphic genes at the S locus2-6. However, the molecular mechanism for recognition of foreign pollen, leading to reproductive incompatibility, has not yet been identified. Here, we show that recognition between a novel pair of proteins, a pistil receptor SUI1 (STIGMATIC UNILATERAL INCOMPATIBILITY 1) and a pollen ligand PUI1 (POLLEN UNILATERAL INCOMPATIBILITY 1), triggers unilateral reproductive incompatibility between plants of two geographically distant self-incompatible Brassica rapa lines, even though crosses would be predicted to be compatible based on the S haplotypes of pollen and stigma. Interestingly, SUI1 and PUI1 are similar to the SI genes, SRK and SP11, respectively, and are maintained as cryptic incompatibility genes in these two populations. The duplication of the SRK and SP11 followed by reciprocal loss in different populations would provide a molecular mechanism of the emergence of a reproductive barrier in allopatry.

[Effect of Genotype and Medium Culture Content on Microspore-Derived Embryo Formation in Chinese Cabbage (Brassica rapa ssp. chinensis) Cv. Lastochka].

The influence of different factors on microspore embryogenesis in Chinese cabbage (B. rapa ssp. chinensis) was studied. A genotype dependence for embryo formation was observed. The majority of embryos and plants were obtained from microspores isolated from flower buds (2-2.9 mm in length) and cultured in the NLN liquid medium with 13% sucrose (w/v) supplemented with 24-epibrassinolide and 1% activated charcoal. Embryos cultured on the 1/2 Murashige-Skoog culture medium with 2% sucrose (w/v), 0.1 mg/L benzylaminopurine, and 3g/L Phytagel stimulated the formation of secondary embryos that resulted in development of large number of doubled haploid plants.

Characterization and classification of one new cytoplasmic male sterility (CMS) line based on morphological, cytological and molecular markers in non-heading Chinese cabbage (Brassica rapa L.).

KEY MESSAGE: A new non-heading Chinese cabbage CMS line M119A was characterized and specific molecular markers were developed to classify different CMS types. One new non-heading Chinese cabbage (Brassica rapa L.) cytoplasmic male sterile (CMS) line M119A was obtained by interspecific crosses between the recently discovered hau CMS line of Brassica juncea and B. rapa. Furthermore, the line was characterized and compared with other five isonuclear-alloplasmic CMS lines. The M119A line produced six stamens without pollen and only two stamen fused together in fewer flowers. Tissue section indicated that anther abortion in M119A may have occurred during differentiation of the archesporial cells without pollen sac. All the six CMS lines were grouped into three types based on the presence of three PCR fragments of 825, 465 and 772 bp amplified with different mitochondrial genes specific primers. The 825-bp fragment was amplified both in 09-10A and H201A using the specific primer pair P-orf224-atp6, and showed 100 % identity with the mitochondrial gene of pol CMS. The 465-bp fragment was amplified in 30A and 105A using the primer pair P-orf138 and shared 100 % identity with the mitochondrial gene of ogu CMS. The 772-bp fragment was amplified in M119A and H203A using the primer pair P-orf288 and showed 100 % identity with the mitochondrial gene of hau CMS. Therefore, these markers could efficiently distinguish different types of isonuclear-alloplasmic CMS lines of non-heading Chinese cabbage, which were useful for improving the efficiency of cross-breeding and heterosis utilization in cruciferous vegetables.

Effect of enzyme-aided cell wall disintegration on protein extractability from intact and dehulled rapeseed (Brassica rapa L. and Brassica napus L.) press cakes.

Cell-wall- and pectin-degrading enzyme preparations were used to enhance extractability of proteins from rapeseed press cake. Rapeseed press cakes from cold pressing of intact Brassica rapa and partially dehulled Brassica napus seeds, containing 36-40% protein and 35% carbohydrates, were treated with pectinolytic (Pectinex Ultra SP-L), xylanolytic (Depol 740L), and cellulolytic (Celluclast 1.5L) enzyme preparations. Pectinex caused effective disintegration of embryonic cell walls through hydrolysis of pectic polysaccharides and glucans and increased protein extraction by up to 1.7-fold in comparison to treatment without enzyme addition. Accordingly, 56% and 74% of the total protein in the intact and dehulled press cakes was extracted. Light microscopy of the press cakes suggested the presence of pectins colocalized with proteins inside the embryo cells. Hydrolysis of these intracellular pectins and deconstruction of embryonic cell walls during Pectinex treatment were concluded to relate with enhanced protein release.

A pollen coat-inducible autoinhibited Ca2+-ATPase expressed in stigmatic papilla cells is required for compatible pollination in the Brassicaceae.

In the Brassicaceae, intraspecific non-self pollen (compatible pollen) can germinate and grow into stigmatic papilla cells, while self-pollen or interspecific pollen is rejected at this stage. However, the mechanisms underlying this selective acceptance of compatible pollen remain unclear. Here, using a cell-impermeant calcium indicator, we showed that the compatible pollen coat contains signaling molecules that stimulate Ca(2+) export from the papilla cells. Transcriptome analyses of stigmas suggested that autoinhibited Ca(2+)-ATPase13 (ACA13) was induced after both compatible pollination and compatible pollen coat treatment. A complementation test using a yeast Saccharomyces cerevisiae strain lacking major Ca(2+) transport systems suggested that ACA13 indeed functions as an autoinhibited Ca(2+) transporter. ACA13 transcription increased in papilla cells and in transmitting tracts after pollination. ACA13 protein localized to the plasma membrane and to vesicles near the Golgi body and accumulated at the pollen tube penetration site after pollination. The stigma of a T-DNA insertion line of ACA13 exhibited reduced Ca(2+) export, as well as defects in compatible pollen germination and seed production. These findings suggest that stigmatic ACA13 functions in the export of Ca(2+) to the compatible pollen tube, which promotes successful fertilization.

Physiological and genetic analysis of CO2-induced breakdown of self-incompatibility in Brassica rapa.

Self-incompatibility (SI) of the Brassicaceae family can be overcome by CO2 gas treatment. This method has been used for decades as an effective means to obtain a large amount of inbred seeds which can then be used for F1 hybrid seed production; however, the molecular mechanism by which CO2 alters the SI pathway has not been elucidated. In this study, to obtain new insights into the mechanism of CO2-induced SI breakdown, the focus was on two inbred lines of Brassica rapa (syn. campestris) with different CO2 sensitivity. Physiological examination using X-ray microanalysis suggested that SI breakdown in the CO2-sensitive line was accompanied by a significant accumulation of calcium at the pollen-stigma interface. Pre-treatment of pollen or pistil with CO2 gas before pollination showed no effect on the SI reaction, suggesting that some physiological process after pollination is necessary for SI to be overcome. Genetic analyses using F1 progeny of a CO2-sensitive × CO2-insensitive cross suggested that CO2 sensitivity is a semi-dominant trait in these lines. Analysis of F2 progeny suggested that CO2 sensitivity could be a quantitative trait, which is controlled by more than one gene. Quantitative trait locus (QTL) analyses identified two major loci, BrSIO1 and BrSIO2, which work additively in overcoming SI during CO2 treatment. No QTL was detected at the loci previously shown to affect SI stability, suggesting that CO2 sensitivity is determined by novel genes. The QTL data presented here should be useful for determining the responsible genes, and for the marker-assisted selection of desirable parental lines with stable but CO2-sensitive SI in F1 hybrid breeding.

Cell type-specific transcriptome of Brassicaceae stigmatic papilla cells from a combination of laser microdissection and RNA sequencing.

Pollination is an early and critical step in plant reproduction, leading to successful fertilization. It consists of many sequential processes, including adhesion of pollen grains onto the surface of stigmatic papilla cells, foot formation to strengthen pollen-stigma interaction, pollen hydration and germination, and pollen tube elongation and penetration. We have focused on an examination of the expressed genes in papilla cells, to increase understanding of the molecular systems of pollination. From three representative species of Brassicaceae (Arabidopsis thaliana, A. halleri and Brassica rapa), stigmatic papilla cells were isolated precisely by laser microdissection, and cell type-specific gene expression in papilla cells was determined by RNA sequencing. As a result, 17,240, 19,260 and 21,026 unigenes were defined in papilla cells of A. thaliana, A. halleri and B. rapa, respectively, and, among these, 12,311 genes were common to all three species. Among the17,240 genes predicted in A. thaliana, one-third were papilla specific while approximately half of the genes were detected in all tissues examined. Bioinformatics analysis revealed that genes related to a wide range of reproduction and development functions are expressed in papilla cells, particularly metabolism, transcription and membrane-mediated information exchange. These results reflect the conserved features of general cellular function and also the specific reproductive role of papilla cells, highlighting a complex cellular system regulated by a diverse range of molecules in these cells. This study provides fundamental biological knowledge to dissect the molecular mechanisms of pollination in papilla cells and will shed light on our understanding of plant reproduction mechanisms.

Seed structure characteristics to form ultrahigh oil content in rapeseed.

BACKGROUND: Rapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding. METHODOLOGY/PRINCIPAL FINDINGS: Here we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition. CONCLUSIONS/SIGNIFICANCE: Our results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.

Effect of temperature on cortical infection by Plasmodiophora brassicae and clubroot severity.

A study was conducted to assess the effect of temperature on infection and development of Plasmodiophora brassicae in the root cortex of Shanghai pak choy (Brassica rapa subsp. chinensis) and on subsequent clubroot severity. Ten-day-old seedlings were grown individually, inoculated with resting spores, and maintained in growth cabinets at 10, 15, 20, 25, and 30?C. Seedlings were harvested at 2-day intervals, starting 8 days after inoculation (DAI) and continuing until 42 DAI. Roots were assessed at 4-day intervals for the incidence of cortical infection and stage of infection (young plasmodia, mature plasmodia, and resting spores), at 2-day intervals for symptom development and clubroot severity, and at 8-day intervals for the number of spores per gram of gall. Temperature affected every stage of clubroot development. Cortical infection was highest and symptoms were observed earliest at 25?C, intermediate at 20 and 30?C, and lowest and latest at 15?C. No cortical infection or symptoms were observed at 42 DAI in plants grown at 10?C. A substantial delay in the development of the pathogen was observed at 15?C. Resting spores were first observed at 38 DAI in plants at 15?C, 26 DAI at 20 and 30?C, and 22 DAI at 25?C. The yield of resting spores from galls was higher in galls that developed at 20 to 30?C than those that developed at 15?C over 42 days of assessment. These results support the observation in companion studies that cool temperatures result in slower development of clubroot symptoms in brassica crops, and demonstrate that the temperature has a consistent pattern of effect throughout the life cycle of the pathogen.

[Structural and cytochemical aspects of Brassica rapa embryogenesis under conditions of clinorotation].

The results of study of embryo development in B. rapa plants, as well as the rate and the character of nutrient substances accumulation in their cells under slow horizontal clinorotation and laboratory control are presented. Significant similarity of the peculiarities of embryo differentiation and character of nutrient substance accumulation in both variants was shown. The cases of different deviations during embryo differentiation, and rate and quantity of reserve nutrient substances in their cells are revealed under clinorotation compared to the laboratory control.

Formation of green-blue compounds in Brassica rapa root by high pressure processing and subsequent storage.

The effect of high pressure treatment on biochemical changes during storage was investigated using Brassica rapa root. High pressure treated samples with 400 and 600 MPa formed unique green-blue color during 7-d storage at 4 degrees C. The mechanism of green-blue compound formation would be based on biochemical pathway for a unique green-blue pigment synthesis, containing O2-dependent steps and possibly enzymatic reactions.

Chromosome 'speed dating' during meiosis of polyploid Brassica hybrids and haploids.

Given their tremendous importance for correct chromosome segregation, the number and distribution of crossovers are tightly controlled during meiosis. In this review, we give an overview of crossover formation in polyploid Brassica hybrids and haploids that illustrates or underscores several aspects of crossover control. We first demonstrate that multiple targets for crossover formation (i.e. different but related chromosomes or duplicated regions) are sorted out during meiosis based on their level of relatedness. In euploid Brassica napus (AACC; 2n = 38), crossovers essentially occur between homologous chromosomes and only a few of them form between homeologues. The situation is different in B. napus haploids in which crossovers preferentially occur between homeologous chromosomes and a few can then form between more divergent duplicated regions. We then provide evidence that the frequency of crossovers between a given pair of chromosomes is influenced by the karyotypic and genetic composition of the plants that undergo meiosis. For instance, genetic evidence indicates that the number of crossovers between exactly the same pairs of homologous A chromosomes gets a boost in Brassica digenomic tetraploid (AACC) and triploid (AAC) hybrids. Increased autosyndesis within B. napus haploids as compared to monoploid B. rapa and B. oleracea is another illustration of this process. All these observations may suggest that polyploidization overall boosts up crossover machinery and/or that the number of crossovers is modulated through inter-bivalents or univalent-bivalent cross-talk effects. The last part of this review gives an up-to-date account of what we know about the genetic control of homologous and homeologous crossover formation among Brassica species.

Microtubules spatial alterations in root cells of Brassica rapa under clinorotation.

Organization of tubulin cytoskeleton in epidermis and cortex cells in different root growth zones in Brassica rapa L. 6-day-old seedlings under clinorotation has been investigated. It was shown that changes in cortical microtubules orientation occur only in the distal elongation zone. In control, cortical microtubule arrays oriented transversely to the root long axis. Whereas under clinorotation an appearance of shorter randomly organized cortical microtubules was observed. Simultaneously, a significant decrease in a cell length in the central elongation zone under clinorotation was revealed. It is suggested that the decline of anisotropic growth, typical for central elongation zone cells, is connected with cortical microtubules disorientation under clinorotation.

Production and genetic analysis of partial hybrids in intertribal crosses between Brassica species (B. rapa, B. napus) and Capsella bursa-pastoris.

Capsella bursa-pastoris (L.) Medic (2n = 4x = 32) is a natural double-low (erucic acid < 1%, glucosinolates < 30 micromol/g) germplasm and shows high degree of resistance to Sclerotinia sclerotiorum. Hybridizations were carried out between two Brassica species viz. B. rapa (2n = 20) and B. napus (2n = 38) as female and C. bursa-pastoris as male parent to introduce these desirable traits into cultivated Brassica species. Majority of F(1) plants resembled female parents in morphology and only a few expressed some characters of male parent, including the white petals. Based on cytological observation of somatic cells, the F(1) plants were classified into five types: two types from the cross with B. rapa, type I had 2n = 27-29; type II had 2n = 20; three types from the crosses with B. napus, type III was haploids with 2n = 19; type IV had 2n = 29; type V had 2n = 38. One to two chromosomes of C. bursa-pastoris were detected in pollen mother cells (PMCs) of type I plant by genomic in situ hybridization (GISH), together with chromosomal segments in ovary cells and PMCs of some F1 plants. Amplified fragment length polymorphism (AFLP) bands specific for the male parent, novel for two parents and absent bands in Brassica parents were generated in majority of F1 plants, even in Brassica-types and haploids, indicating the introgressions at various levels from C. bursa-pastoris and genomic alterations following hybridization. Some Brassica-type progeny plants had reduced contents of erucic acid and glucosinolates associated with improved resistance to S. sclerotiorum. The cytological and molecular mechanisms behind these results are discussed.

Differences in cell death induction by Phytophthora Elicitins are determined by signal components downstream of MAP kinase kinase in different species of Nicotiana and cultivars of Brassica rapa and Raphanus sativus.

Elicitins are small, secreted proteins produced by species of the plant-pathogenic oomycete Phytophthora. They induce hypersensitive cell death in most Nicotiana species and in some cultivars of Brassica rapa and Raphanus sativus. In this study, two true-breeding Fast Cycling B. rapa lines were established that showed severe necrosis (line 7-R) or no visible response (line 18-NR) after treatment with elicitin. Unexpectedly, microscopic examination revealed localized cell death in line 18-NR plants, and expression levels of various defense-marker genes were comparable in both lines. These results suggested that both "responsive" and "nonresponsive" plants responded to elicitin but differed in the extent of the cell death response. Expression of a constitutively active form of Arabidopsis (Arabidopsis thaliana) MAP kinase kinase 4 (AtMEK4(DD)) also induced rapid development of confluent cell death in line 7-R, whereas line 18-NR showed no visible cell death. Similarly, elicitin-responsive Nicotiana species and R. sativus cultivars showed significantly stronger cell death responses following expression of AtMEK4(DD) compared with nonresponsive species/cultivars. Line 7-R also showed higher sensitivity to toxin-containing culture filtrates produced by Alternaria brassicicola, and toxin sensitivity cosegregated with elicitin responsiveness, suggesting that the downstream responses induced by elicitin and Alternaria toxin share factors that control the extent of cell death. Interestingly, elicitin responsiveness was shown to correlate with greater susceptibility to A. brassicicola (a necrotroph) in B. rapa but less susceptibility to Phytophthora nicotianae (a hemibiotroph) in Nicotiana, suggesting a more extensive cell death response could cause opposite effects on the outcomes of biotrophic versus necrotrophic plant-pathogen interactions.

Cytology, RAPD, and seed colour of progeny plants from Brassica rapa-alboglabra aneuploids and development of monosomic addition lines.

Progeny plants from Brassica rapa-alboglabra aneuploids were characterized genetically by scoring random amplified polymorphic DNA (RAPD) markers and seed colour and cytologically as to chromosome number and pairing. Sets of RAPD markers specific for each of the encountered eight alien Brassica alboglabra chromosomes were defined. The finding of subsets of markers associated with the presence or absence of alien chromosomes inferred the frequent occurrence of intergenomic genetic recombination and introgression. The chromosome numbers were in the range 2n = 20-28, with a maximum of seven alien B. alboglabra chromosomes and one trisomic B. rapa chromosome. Five types of monosomic addition lines were obtained, two of which have not been developed before. Differences in chromatin condensation patterns made it possible to differentiate between the B. rapa and B. alboglabra chromosomes at diakinesis, and to detect intergenomic homoeological pairing. In addition to the frequent formation of trivalents by homoeologous pairing of an alien B. alboglabra chromosome and a background B. rapa pair, occasional heteromorphic intergenomic bivalents and B. rapa univalents were encountered. Homoeological intergenomic pairing occurred between chromosomes with similar centromeric and karyotypic positions. Plants with structurally changed alien chromosomes were found. The RAPD and cytological data substantiated each other. Observations of the colour of sown and harvested seeds indicated that B. alboglabra chromosome 4 carries a gene for brown seed colour. It exerts its control embryonically, and thus it differs from chromosome 1 which controls seed colour maternally.