Haploid and Doubled Haploid Plant Production in Brassica rapa L. subsp. pekinensis Via Microspore Culture.

The production of haploid and doubled haploid plants is a biotechnological tool that shortens the breeding process of new cultivars in many species. Doubled haploid plants are homozygous at every locus and they can be utilized as parents to produce F1 hybrids. In this chapter, we describe a protocol for the production of doubled haploid plants in Brassica rapa L. subsp. pekinensis using androgenesis induced by isolated microspore cultures.

Mechanism of self/nonself-discrimination in Brassica self-incompatibility.

Self-incompatibility (SI) is a breeding system that promotes cross-fertilization. In Brassica, pollen rejection is induced by a haplotype-specific interaction between pistil determinant SRK (S receptor kinase) and pollen determinant SP11 (S-locus Protein 11, also named SCR) from the S-locus. Although the structure of the B. rapa S9-SRK ectodomain (eSRK) and S9-SP11 complex has been determined, it remains unclear how SRK discriminates self- and nonself-SP11. Here, we uncover the detailed mechanism of self/nonself-discrimination in Brassica SI by determining the S8-eSRK-S8-SP11 crystal structure and performing molecular dynamics (MD) simulations. Comprehensive binding analysis of eSRK and SP11 structures reveals that the binding free energies are most stable for cognate eSRK-SP11 combinations. Residue-based contribution analysis suggests that the modes of eSRK-SP11 interactions differ between intra- and inter-subgroup (a group of phylogenetically neighboring haplotypes) combinations. Our data establish a model of self/nonself-discrimination in Brassica SI.

HPLC and NMR quantification of bioactive compounds in flowers and leaves of Brassica rapa: the influence of aging.

Several members of the Brassicaeae family are known to possess beneficial properties which positively impact human diet, thanks to the presence of antioxidants, bioactive polyphenols and amino acids. B. rapa, one of the most widespread and economically relevant species, represents an outstanding example. The aim of this study is to investigate, at the molecular level, the effect of plant aging on the concentration of some biologically relevant compounds in different parts of the plant. Using HPLC and NMR techniques, the quantification of polyphenolic species (caffeic acid, quercetin and rutin), succinic acid and alanine was performed in flowers and leaves of young and mature B. rapa plants.

Systematic identification of long non-coding RNAs during pollen development and fertilization in Brassica rapa.

The importance of long non-coding RNAs (lncRNAs) in plant development has been established, but a systematic analysis of lncRNAs expressed during pollen development and fertilization has been elusive. We performed a time series of RNA-seq experiments at five developmental stages during pollen development and three different time points after pollination in Brassica rapa and identified 12 051 putative lncRNAs. A comprehensive view of dynamic lncRNA expression networks underpinning pollen development and fertilization was provided. B. rapa lncRNAs share many common characteristics of lncRNAs: relatively short length, low expression but specific in narrow time windows, and low evolutionary conservation. Gene modules and key lncRNAs regulating reproductive development such as exine formation were uncovered. Forty-seven cis-acting lncRNAs and 451 trans-acting lncRNAs were revealed to be highly coexpressed with their target protein-coding genes. Of particular importance are the discoveries of 14 lncRNAs that were highly coexpressed with 10 function-known pollen-associated coding genes. Fifteen lncRNAs were predicted as endogenous target mimics for 13 miRNAs, and two lncRNAs were proved to be functional target mimics for miR160 after experimental verification and shown to function in pollen development. Our study provides the systematic identification of lncRNAs during pollen development and fertilization in B. rapa and forms the foundation for future genetic, genomic, and evolutionary studies.

Improving tolerance against drought in canola by penconazole and calcium.

Drought stress is one of the most important environmental factors that limit plant growth. Canola is an important agricultural crop grown primarily for its edible oil. In this study, penconazole (PEN), a triazole growth regulator, and calcium (Ca2+), a secondary messenger, were used to analyses their role in decease sensitivity and induce tolerance to drought stress in canola. Plants were treated by various PEG concentrations (0, 5, 10, and 15%) without or with PEN (15 mg l-1) and Ca2+ (15 mM). According to the obtained results in two cultivars of canola (RGS003 and Sarigol), improvement of growth by PEN and Ca2+observed at low concentration of drought (5%). Betterment of growth by PEN can be explained by induction in DPPH (1, 1-diphenyl-2- picrylhydrazyl), chlorophyll and P content and reduction in Electrolyte leakage in cultivar RGS003. Growth recovery by PEN is concomitant by increase in DPPH, succinate dehydrogenase, chlorophyll, carotenoid and K+ content in cultivar Sarigol. Ca2+ treatment by induction in Relative water content, DPPH, chlorophyll, carotenoid, indole-3-acetic acid content and the content of Ca2+ and K+ causes increase in growth in cultivar Sarigol. Overall, application of PEN and Ca2+ improved the performance in canola under drought stress. These results provide novel insights about the physiological and molecular roles of PEN and Ca2+ in canola plant tolerance against drought stress.

Phytochrome B regulates resource allocation in Brassica rapa.

Crop biomass and yield are tightly linked to how the light signaling network translates information about the environment into allocation of resources, including photosynthates. Once activated, the phytochrome (phy) class of photoreceptors signal and re-deploy carbon resources to alter growth, plant architecture, and reproductive timing. Most of the previous characterization of the light-modulated growth program has been performed in the reference plant Arabidopsis thaliana. Here, we use Brassica rapa as a crop model to test for conservation of the phytochrome-carbon network. In response to elevated levels of CO2, B. rapa seedlings showed increases in hypocotyl length, shoot and root fresh weight, and the number of lateral roots. All of these responses were dependent on nitrogen and polar auxin transport. In addition, we identified putative B. rapa orthologs of PhyB and isolated two nonsense alleles. BrphyB mutants had significantly decreased or absent CO2-stimulated growth responses. Mutant seedlings also showed misregulation of auxin-dependent genes and genes involved in chloroplast development. Adult mutant plants had reduced chlorophyll levels, photosynthetic rate, stomatal index, and seed yield. These findings support a recently proposed holistic role for phytochromes in regulating resource allocation, biomass production, and metabolic state in the developing plant.

Allocation to male vs female floral function varies by currency and responds differentially to density and moisture stress.

Allocation of finite resources to separate reproductive functions is predicted to vary across environments and affect fitness. Biomass is the most commonly measured allocation currency; however, in comparison with nutrients it may be less limited and express different environmental and evolutionary responses. Here, we measured carbon, nitrogen, phosphorus, and biomass allocation among floral whorls in recombinant inbred lines of Brassica rapa in multiple environments to characterize the genetic architecture of floral allocation, including its sensitivity to environmental heterogeneity and to choice of currency. Mass, carbon, and nitrogen allocation to female whorls (pistils and sepals) decreased under high density, whereas nitrogen allocation to male organs (stamens) decreased under drought. Phosphorus allocation decreased by half in pistils under drought, while stamen phosphorus was unaffected by environment. While the contents of each currency were positively correlated among whorls, selection to improve fitness through female (or male) function typically favored increased allocation to pistils (or stamens) but decreased allocation to other whorls. Finally, genomic regions underlying correlations among allocation metrics were mapped, and loci related to nitrogen uptake and floral organ development were located within mapped quantitative trait loci. Our candidate gene identification suggests that nutrient uptake may be a limiting step in maintaining male allocation. Taken together, allocation to male vs female function is sensitive to distinct environmental stresses, and the choice of currency affects the interpretation of floral allocation responses to the environment. Further, genetic correlations may counter the evolution of allocation patterns that optimize fitness through female or male function.

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.

Concurrent elevation of CO2, O3 and temperature severely affects oil quality and quantity in rapeseed.

Plant oil is an essential dietary and bio-energy resource. Despite this, the effects of climate change on plant oil quality remain to be elucidated. The present study is the first to show changes in oil quality and quantity of four rapeseed cultivars in climate scenarios with elevated [CO2], [O3] and temperature (T) combined and as single factors. The combination of environmental factors resembled IPCC's 'business as usual' emission scenario predicted for late this century. Generally, the climate scenarios reduced the average amounts of the six fatty acids (FAs) analysed, though in some treatments single FAs remained unchanged or even increased. Most reduced was the FA essential for human nutrition, C18:3-ω3, which decreased by 39% and 45% in the combined scenarios with elevated [CO2]+T+[O3] and [CO2]+T, respectively. Average oil content decreased 3-17%. When [CO2] and T were elevated concurrently, the seed biomass was reduced by half, doubling the losses in FAs and oil content. This corresponded to a 58% reduction in the oil yield per hectare, and C18:3-ω3 decreased by 77%. Furthermore, the polyunsaturated FAs were significantly decreased. The results indicate undesirable consequences for production and health benefits of rapeseed oil with future climate change. The results also showed strong interactive effects of CO2, T and O3 on oil quality, demonstrating why prediction of climate effects requires experiments with combined factors and should not be based on extrapolation from single factor experiments.

[Effects of selenite addition on selenium absorption, root morphology and physiological characteristics of rape seedlings].

Abstract: The rape (Brassica napus L. cv. Xiangnongyou 571) was chosen as the experimental material to undergo solution cultivation at seedling stage to investigate the effects of selenite addition on the selenium (Se) absorption and distribution, root morphology and physiological characteristics of rape seedlings. The results showed that the bioaccumulation ability of Se decreased significantly with increasing the Se application rate, but the Se distribution coefficient remained around 0.9 with no significant influence. The application of 10 µmol . L-1 selenite stimulated the growth of rape seedlings through improving the root physiological characteristics and root morphology significantly, including significantly increasing the production of superoxide radical (O2∙-) rate and the activities of superoxide dismutase (SOD), peroxidase (POD) and fungal catalase (CAT) in the root system, which resulted in a reduction of the lipids peroxidation (MDA) content as much as 26.0%, consequently increasing the root activity as much as 17.4%. The promoting degrees of selenite on root morphological parameters were from strong to weak in such a tendency: root volume > total surface area > number of root forks > total root length > number of root tips > average diameter. However, such positive effects had no significant difference with those in treatment with 1 µmol . L-1 selenite, indicating that small amounts (≤ 10 Lmol . L-1) of selenite were able to increase the activity of antioxidant enzymes and reduce the content of MDA in root system, which could increase root activity and improve root morphology, hence increased the biomass of rape seedlings.

On the potential strength and consequences for nonrandom gene flow caused by local adaptation in flowering time.

Gene flow is generally considered a random process, that is the loci under consideration have no effect on dispersal success. Edelaar and Bolnick (Trends Ecol Evol, 27, 2012 659) recently argued that nonrandom gene flow could exert a significant evolutionary force. It can, for instance, ameliorate the maladaptive effects of immigration into locally adapted populations. I examined the potential strength for nonrandom gene flow for flowering time genes, a trait frequently found to be locally adapted. The idea is that plants that successfully export pollen into a locally adapted resident population will be a genetically biased subset of their natal population - they will have resident-like flowering times. Reciprocally, recipients will be more migrant-like than the resident population average. I quantified the potential for biased pollen exchange among three populations along a flowering time cline in Brassica rapa from southern California. A two-generation line cross experiment demonstrated genetic variance in flowering time, both within and among populations. Calculations based on the variation in individual flowering schedules showed that resident plants with the most migrant-like flowering times could expect to have up to 10 times more of the their flowers pollinated by immigrant pollen than the least migrant-like. Further, the mean flowering time of the pollen exporters that have access to resident mates differs by up to 4 weeks from the mean in the exporters' natal population. The data from these three populations suggest that the bias in gene flow for flowering time cuts the impact on the resident population by as much as half. This implies that when selection is divergent between populations, migrants with the highest mating success tend to be resident-like in their flowering times, and so, fewer maladaptive alleles will be introduced into the locally adapting gene pool.

Bees use honest floral signals as indicators of reward when visiting flowers.

Pollinators visit flowers for rewards and should therefore have a preference for floral signals that indicate reward status, so called 'honest signals'. We investigated honest signalling in Brassica rapa L. and its relevance for the attraction of a generalised pollinator, the bumble bee Bombus terrestris (L.). We found a positive association between reward amount (nectar sugar and pollen) and the floral scent compound phenylacetaldehyde. Bumble bees developed a preference for phenylacetaldehyde over other scent compounds after foraging on B. rapa. When foraging on artificial flowers scented with synthetic volatiles, bumble bees developed a preference for those specific compounds that honestly indicated reward status. These results show that the honesty of floral signals can play a key role in their attractiveness to pollinators. In plants, a genetic constraint, resource limitation in reward and signal production, and sanctions against cheaters may contribute to the evolution and maintenance of honest signalling.

Temporal variation in phenotypic gender and expected functional gender within and among individuals in an annual plant.

BACKGROUND AND AIMS: Adaptive explanations for variation in sex allocation centre on variation in resource status and variation in the mating environment. The latter can occur when dichogamy causes siring opportunity to vary across the flowering season. In this study, it is hypothesized that the widespread tendency towards declining fruit-set from first to last flowers on plants can similarly lead to a varying mating environment by causing a temporal shift in the quality (not quantity) of siring opportunities. METHODS: A numerical model was developed to examine the effects of declining fruit-set on the expected male versus female reproductive success (functional gender) of first and last flowers on plants, and of early- and late-flowering plants. Within- and among-plant temporal variation in pollen production, ovule production and fruit-set in 70 Brassica rapa plants was then characterized to determine if trends in male and female investment mirror expected trends in functional gender. KEY RESULTS: Under a wide range of model conditions, functional femaleness decreased sharply in the last flowers on plants, and increased from early- to late-flowering plants in the population. In B. rapa, pollen production decreased more rapidly than ovule production from first to last flowers, leading to a within-plant increase in phenotypic femaleness. Among plants, ovule production decreased from early- to late-flowering plants, causing a temporal decrease in phenotypic femaleness. CONCLUSIONS: The numerical model confirmed that declining fruit-set can drive temporal variation in functional gender, especially among plants. The discrepancy between observed trends in phenotypic gender in B. rapa and expected functional gender predicted by the numerical model does not rule out the possibility that male reproductive success decreases with later flowering onset. If so, plants may experience selection for early flowering through male fitness.

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.

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.

Ogura-CMS in Chinese cabbage (Brassica rapa ssp. pekinensis) causes delayed expression of many nuclear genes.

We investigated the mechanism regulating cytoplasmic male sterility (CMS) in Brassica rapa ssp. pekinensis using floral bud transcriptome analyses of Ogura-CMS Chinese cabbage and its maintainer line in B. rapa 300-K oligomeric probe (Br300K) microarrays. Ogura-CMS Chinese cabbage produced few and infertile pollen grains on indehiscent anthers. Compared to the maintainer line, CMS plants had shorter filaments and plant growth, and delayed flowering and pollen development. In microarray analysis, 4646 genes showed different expression, depending on floral bud size, between Ogura-CMS and its maintainer line. We found 108 and 62 genes specifically expressed in Ogura-CMS and its maintainer line, respectively. Ogura-CMS line-specific genes included stress-related, redox-related, and B. rapa novel genes. In the maintainer line, genes related to pollen coat and germination were specifically expressed in floral buds longer than 3mm, suggesting insufficient expression of these genes in Ogura-CMS is directly related to dysfunctional pollen. In addition, many nuclear genes associated with auxin response, ATP synthesis, pollen development and stress response had delayed expression in Ogura-CMS plants compared to the maintainer line, which is consistent with the delay in growth and development of Ogura-CMS plants. Delayed expression may reduce pollen grain production and/or cause sterility, implying that mitochondrial, retrograde signaling delays nuclear gene expression.

Development of self-compatible B. rapa by RNAi-mediated S locus gene silencing.

The self-incompatibility (SI) system is genetically controlled by a single polymorphic locus known as the S-locus in the Brassicaceae. Pollen rejection occurs when the stigma and pollen share the same S-haplotype. Recognition of S-haplotype specificity has recently been shown to involve at least two S-locus genes, S-receptor kinase (SRK) and S-locus protein 11 or S locus Cysteine-rich (SP11/SCR) protein. Here, we examined the function of S(60), one SP11/SCR allele of B. rapa cv. Osome, using a RNAi-mediated gene silencing approach. The transgenic RNAi lines were highly self-compatible, and this trait was stable in subsequent generations, even after crossing with other commercial lines. These findings also suggested that the resultant self-compatibility could be transferred to commercial cultivars with the desired performances in B. rapa.

Soil biota reduce allelopathic effects of the invasive Eupatorium adenophorum.

Allelopathy has been hypothesized to play a role in exotic plant invasions, and study of this process can improve our understanding of how direct and indirect plant interactions influence plant community organization and ecosystem functioning. However, allelopathic effects can be highly conditional. For example allelopathic effects demonstrated in vivo can be difficult to demonstrate in field soils. Here we tested phytotoxicity of Eupatorium adenophorum (croftonweed), one of the most destructive exotic species in China, to a native plant species Brassica rapa both in sand and in native soil. Our results suggested that natural soils from different invaded habitats alleviated or eliminated the efficacy of potential allelochemicals relative to sand cultures. When that soil is sterilized, the allelopathic effects returned; suggesting that soil biota were responsible for the reduced phytotoxicity in natural soils. Neither of the two allelopathic compounds (9-Oxo-10,11-dehydroageraphorone and 9b-Hydroxyageraphorone) of E. adenophorum could be found in natural soils infested by the invader, and when those compounds were added to the soils as leachates, they showed substantial degradation after 24 hours in natural soils but not in sand. Our findings emphasize that soil biota can reduce the allelopathic effects of invaders on other plants, and therefore can reduce community invasibility. These results also suggest that soil biota may have stronger or weaker effects on allelopathic interactions depending on how allelochemicals are delivered.

Targeted metabolite analysis and biological activity of Pieris brassicae fed with Brassica rapa var. rapa.

For the first time, an insect-plant system, Pieris brassicae fed with Brassica rapa var. rapa, was tested for its biological capacity, namely, antioxidant (DPPH*, *NO, and O(2)*- radicals) and antimicrobial (bacteria and fungi) activities. Samples from the insect's life cycle (larvae, excrements, exuviae, and butterfly) were always found to be more efficient than the host plant. Also, P. brassicae materials, as well as its host plant, were screened for phenolics and organic acids. The host plant revealed higher amounts of both compounds. Two phenolic acids, ferulic and sinapic, as well as kaempferol 3-Osophoroside, were common to insect (larvae and excrements) and plant materials, with excrements being considerably richer. Detection of sulfated compounds in excrements, absent in host plant, revealed that metabolic processes in this species involved sulfation. Additionally, deacylation and deglycosilation were observed. All matrices presented the same organic acids qualitative profile, with the exception of excrements.

Effects of genetically modified herbicide-tolerant cropping systems on weed seedbanks in two years of following crops.

The Farm Scale Evaluations (FSEs) showed that genetically modified herbicide-tolerant (GMHT) cropping systems could influence farmland biodiversity because of their effects on weed biomass and seed production. Recently published results for winter oilseed rape showed that a switch to GMHT crops significantly affected weed seedbanks for at least 2 years after the crops were sown, potentially causing longer-term effects on other taxa. Here, we seek evidence for similar medium-term effects on weed seedbanks following spring-sown GMHT crops, using newly available data from the FSEs. Weed seedbanks following GMHT maize were significantly higher than following conventional varieties for both the first and second years, while by contrast, seedbanks following GMHT spring oilseed rape were significantly lower over this period. Seedbanks following GMHT beet were smaller than following conventional crops in the first year after the crops had been sown, but this difference was much reduced by the second year for reasons that are not clear. These new data provide important empirical evidence for longer-term effects of GMHT cropping on farmland biodiversity.