Assembly and comparative analysis of the complete mitochondrial genome of Brassica rapa var. Purpuraria.

BACKGROUND: Purple flowering stalk (Brassica rapa var. purpuraria) is a widely cultivated plant with high nutritional and medicinal value and exhibiting strong adaptability during growing. Mitochondrial (mt) play important role in plant cells for energy production, developing with an independent genetic system. Therefore, it is meaningful to assemble and annotate the functions for the mt genome of plants independently. Though there have been several reports referring the mt genome of in Brassica species, the genome of mt in B. rapa var. purpuraria and its functional gene variations when compared to its closely related species has not yet been addressed. RESULTS: The mt genome of B. rapa var. purpuraria was assembled through the Illumina and Nanopore sequencing platforms, which revealed a length of 219,775 bp with a typical circular structure. The base composition of the whole B. rapa var. purpuraria mt genome revealed A (27.45%), T (27.31%), C (22.91%), and G (22.32%). 59 functional genes, composing of 33 protein-coding genes (PCGs), 23 tRNA genes, and 3 rRNA genes, were annotated. The sequence repeats, codon usage, RNA editing, nucleotide diversity and gene transfer between the cp genome and mt genome were examined in the B. rapa var. purpuraria mt genome. Phylogenetic analysis show that B. rapa var. Purpuraria was closely related to B. rapa subsp. Oleifera and B. juncea. Ka/Ks analysis reflected that most of the PCGs in the B. rapa var. Purpuraria were negatively selected, illustrating that those mt genes were conserved during evolution. CONCLUSIONS: The results of our findings provide valuable information on the B.rapa var. Purpuraria genome, which might facilitate molecular breeding, genetic variation and evolutionary researches for Brassica species in the future.

Brassica rapa CURLY LEAF is a major H3K27 methyltransferase regulating flowering time.

MAIN CONCLUSION: In Brassica rapa, the epigenetic modifier BraA.CLF orchestrates flowering by modulating H3K27me3 levels at the floral integrator genes FT, SOC1, and SEP3, thereby influencing their expression. CURLY LEAF (CLF) is the catalytic subunit of the plant Polycomb Repressive Complex 2 that mediates the trimethylation of histone H3 lysine 27 (H3K27me3), an epigenetic modification that leads to gene silencing. While the function of CURLY LEAF (CLF) has been extensively studied in Arabidopsis thaliana, its role in Brassica crops is barely known. In this study, we focused on the Brassica rapa homolog of CLF and found that the loss-of-function mutant braA.clf-1 exhibits an accelerated flowering together with pleiotropic phenotypic alterations compared to wild-type plants. In addition, we carried out transcriptomic and H3K27me3 genome-wide analyses to identify the genes regulated by BraA.CLF. Interestingly, we observed that several floral regulatory genes, including the B. rapa homologs of FT, SOC1 and SEP3, show reduced H3K27me3 levels and increased transcript levels compared to wild-type plants, suggesting that they are direct targets of BraA.CLF and key players in regulating flowering time in this crop. In addition, the results obtained will enhance our understanding of the epigenetic mechanisms regulating key developmental traits and will aid to increase crop yield by engineering new Brassica varieties with different flowering time requirements.

Too hot to handle: temperature-induced plasticity influences pollinator behaviour and plant fitness.

Increased temperature can induce plastic changes in many plant traits. However, little is known about how these changes affect plant interactions with insect pollinators and herbivores, and what the consequences for plant fitness and selection are. We grew fast-cycling Brassica rapa plants at two temperatures (ambient and increased temperature) and phenotyped them (floral traits, scent, colour and glucosinolates). We then exposed plants to both pollinators (Bombus terrestris) and pollinating herbivores (Pieris rapae). We measured flower visitation, oviposition of P. rapae, herbivore development and seed output. Plants in the hot environment produced more but smaller flowers, with lower UV reflectance and emitted a different volatile blend with overall lower volatile emission. Moreover, these plants received fewer first-choice visits by bumblebees and butterflies, and fewer flower visits by butterflies. Seed production was lower in hot environment plants, both because of a reduction in flower fertility due to temperature and because of the reduced visitation of pollinators. The selection on plant traits changed in strength and direction between temperatures. Our study highlights an important mechanism by which global warming can change plant-pollinator interactions and negatively impact plant fitness, as well as potentially alter plant evolution through changes in phenotypic selection.

Uncovering genes involved in pollinator-driven mating system shifts and selfing syndrome evolution in Brassica rapa.

Shifts in pollinator occurrence and their pollen transport effectiveness drive the evolution of mating systems in flowering plants. Understanding the genomic basis of these changes is essential for predicting the persistence of a species under environmental changes. We investigated the genomic changes in Brassica rapa over nine generations of pollination by hoverflies associated with rapid morphological evolution toward the selfing syndrome. We combined a genotyping-by-sequencing (GBS) approach with a genome-wide association study (GWAS) to identify candidate genes, and assessed their functional role in the observed morphological changes by studying mutations of orthologous genes in the model plant Arabidopsis thaliana. We found 31 candidate genes involved in a wide range of functions from DNA/RNA binding to transport. Our functional assessment of orthologous genes in A. thaliana revealed that two of the identified genes in B. rapa are involved in regulating the size of floral organs. We found a protein kinase superfamily protein involved in petal width, an important trait in plant attractiveness to pollinators. Moreover, we found a histone lysine methyltransferase (HKMT) associated with stamen length. Altogether, our study shows that hoverfly pollination leads to rapid evolution toward the selfing syndrome mediated by polygenic changes.

Combining PSII photochemistry and hydraulics improves predictions of photosynthesis and water use from mild to lethal drought.

Rising temperatures and increases in drought negatively impact the efficiency and sustainability of both agricultural and forest ecosystems. Although hydraulic limitations on photosynthesis have been extensively studied, a solid understanding of the links between whole plant hydraulics and photosynthetic processes at the cellular level under changing environmental conditions is still missing, hampering our predictive power for plant mortality. Here, we examined plant hydraulic traits and CO2 assimilation rate under progressive water limitation by implementing Photosystem II (PSII) dynamics with a whole plant process model (TREES). The photosynthetic responses to plant water status were parameterized based on measurements of chlorophyll a fluorescence, gas exchange and water potential for Brassica rapa (R500) grown in a greenhouse under fully watered to lethal drought conditions. The updated model significantly improved predictions of photosynthesis, stomatal conductance and leaf water potential. TREES with PSII knowledge predicted a larger hydraulic safety margin and a decrease in percent loss of conductivity. TREES predicted a slower decrease in leaf water potential, which agreed with measurements. Our results highlight the pressing need for incorporating PSII drought photochemistry into current process models to capture cross-scale plant water dynamics from cell to whole plant level.

Characterization and anti-tumor activities of polysaccharide isolated from Brassica rapa L. via activation of macrophages through TLR2-and TLR4-Dependent pathways.

We have previously shown the immunostimulatory effects by Nozawana (Brassica rapa L.). In this report, we determined the characteristics of Nozawana polysaccharide (NPS) and evaluated the immunomodulatory effects and anti-tumor activity of NPS mediated by macrophage activation. The molecular weight of NPS was determined by gel filtration chromatography with an average molecular weight of approximately 100.6 kDa. HPLC analysis showed that NPS contained glucose, galacturonic acid, galactose, and arabinose. NPS increased cytokine and nitric oxide (NO) production by macrophages in a Toll-like receptor (TLR)2 and TLR4-dependent manner. Furthermore, NPS induced apoptosis significantly against 4T1 murine breast cancer cells cultured in conditioned medium from NPS-treated macrophages through tumor necrosis factor-α. In tumor-bearing mouse model, tumor growth was significantly reduced in NPS-treated mice compared with control mice. These results support the potential use of NPS as an immunotherapeutic material found in health food products.

The evolution and functional divergence of FT-related genes in controlling flowering time in Brassica rapa ssp. rapa.

KEY MESSAGE: The BrrFT paralogues exhibit distinct expression patterns and play different roles in regulating flowering time, and BrrFT4 competes with BrrFT1 and BrrFT2 to interact with BrrFD proteins. Flowering time is an important agricultural trait for Brassica crops, and early bolting strongly affects the yield and quality of Brassica rapa ssp. rapa. Flowering Locus T paralogues play an important role in regulating flowering time. In this study, we identified FT-related genes in turnip by phylogenetic classification, and four BrrFT homoeologs that shared with high identities with BraFT genes were isolated. The different gene structures, promoter binding sites, and expression patterns observed indicated that these genes may play different roles in flowering time regulation. Further genetic and biochemical experiments showed that as for FT-like paralogues, BrrFT2 acted as the key floral inducer, and BrrFT1 seems to act as a mild 'florigen' protein. However, BrrFT4 acts as a floral repressor and antagonistically regulates flowering time by competing with BrrFT1 and BrrFT2 to bind BrrFD proteins. BrrFT3 may have experienced loss of function via base shift mutation. Our results revealed the potential roles of FT-related genes in flowering time regulation in turnip.

Contributions of selenium-oxidizing bacteria to selenium biofortification and cadmium bioremediation in a native seleniferous Cd-polluted sandy loam soil.

Selenium (Se) is a trace element that is essential for human health. Daily dietary Se intake is governed by the food chain through soil-plant systems. However, the cadmium (Cd) content tends to be excessive in seleniferous soil, in which Se and Cd have complex interactions. Therefore, it is a great challenge to grow crops containing appreciable amounts of Se but low amounts of Cd. We compared the effects of five Se-transforming bacteria on Se and Cd uptake by Brassica rapa L. in a native seleniferous Cd-polluted soil. The results showed that three Se-oxidizing bacteria (LX-1, LX-100, and T3F4) increased the Se content of the aboveground part of the plant by 330.8%, 309.5%, and 724.3%, respectively, compared to the control (p < 0.05). The three bacteria also reduced the aboveground Cd content by 15.1%, 40.4%, and 16.4%, respectively (p < 0.05). In contrast, the Se(IV)-reducing bacterium ES2-45 and weakly Se-transforming bacterium LX-4 had no effect on plant Se uptake, although they did decrease the aboveground Cd content. In addition, the three Se-oxidizing bacteria increased the Se available in the soil by 38.4%, 20.4%, and 24.0%, respectively, compared to the control (p < 0.05). The study results confirm the feasibility of using Se-oxidizing bacteria to simultaneously enhance plant Se content and reduce plant Cd content in seleniferous Cd-polluted soil.

Integrating Dynamic 3D Chromatin Architecture and Gene Expression Alterations Reveal Heterosis in Brassica rapa.

Heterosis plays a significant role in enhancing variety, boosting yield, and raising economic value in crops, but the molecular mechanism is still unclear. We analyzed the transcriptomes and 3D genomes of a hybrid (F1) and its parents (w30 and 082). The analysis of the expression revealed a total of 485 specially expressed genes (SEGs), 173 differentially expressed genes (DEGs) above the parental expression level, more actively expressed genes, and up-regulated DEGs in the F1. Further study revealed that the DEGs detected in the F1 and its parents were mainly involved in the response to auxin, plant hormone signal transduction, DNA metabolic process, purine metabolism, starch, and sucrose metabolism, which suggested that these biological processes may play a crucial role in the heterosis of Brassica rapa. The analysis of 3D genome data revealed that hybrid F1 plants tend to contain more transcriptionally active A chromatin compartments after hybridization. Supplementaryly, the F1 had a smaller TAD (topologically associated domain) genome length, but the number was the highest, and the expression change in activated TAD was higher than that of repressed TAD. More specific TAD boundaries were detected between the parents and F1. Subsequently, 140 DEGs with genomic structural variants were selected as potential candidate genes. We found two DEGs with consistent expression changes in A/B compartments and TADs. Our findings suggested that genomic structural variants, such as TADs and A/B chromatin compartments, may affect gene expression and contribute to heterosis in Brassica rapa. This study provides further insight into the molecular mechanism of heterosis in Brassica rapa.

Selenium-oxidizing Agrobacterium sp. T3F4 decreases arsenic uptake by Brassica rapa L. under a native polluted soil.

Toxic arsenic (As) and trace element selenium (Se) are transformed by microorganisms but their complex interactions in soil-plant systems have not been fully understood. An As- and Se- oxidizing bacterium, Agrobacterium sp. T3F4, was applied to a native seleniferous As-polluted soil to investigate As/Se uptake by the vegetable Brassica rapa L. and As-Se interaction as mediated by strain T3F4. The Se content in the aboveground plants was significantly enhanced by 34.1%, but the As content was significantly decreased by 20.5% in the T3F4-inoculated pot culture compared to the control (P < 0.05). Similar result was shown in treatment with additional 5 mg/kg of Se(IV) in soil. In addition, the As contents in roots were significantly decreased by more than 35% under T3F4 or Se(IV) treatments (P<0.05). Analysis of As-Se-bacterium interaction in a soil simulation experiment showed that the bioavailability of Se significantly increased and As was immobilized with the addition of the T3F4 strain (P < 0.05). Furthermore, an As/Se co-exposure hydroponic experiment demonstrated that As uptake and accumulation in plants was reduced by increasing Se(IV) concentrations. The 50% growth inhibition concentration (IC50) values for As in plants were increased about one-fold and two-fold under co-exposure with 5 and 10 µmol/L Se(IV), respectively. In conclusion, strain T3F4 improves Se uptake but decreases As uptake by plants via oxidation of As and Se, resulting in decrease of soil As bioavailability and As/Se competitive absorption by plants. This provides a potential bioremediation strategy for Se biofortification and As immobilization in As-polluted soil.

Genome-wide identification and expression analysis of the AUX/IAA gene family in turnip (Brassica rapa ssp. rapa).

BACKGROUND: Auxin/indoleacetic acid (AUX/IAA) genes encoding short-lived proteins participate in AUX signaling transduction and play crucial roles in plant growth and development. Although the AUX/IAA gene family has been identified in many plants, a systematic analysis of AUX/IAA genes in Brassica rapa ssp. rapa has not yet been reported. RESULTS: We performed a comprehensive genome-wide analysis and found 89 AUX/IAA genes in turnip based on the conserved AUX/IAA domain (pfam02309). Phylogenetic analysis of AUX/IAA genes from turnip, Arabidopsis, and cabbage revealed that these genes cluster into six subgroups (A1, A2, A3, A4, B1, and B2). The motif distribution was also conservative among the internal members of the clade. Enhanced yellow fluorescent protein (EYFP) signals of BrrIAA-EYFPs showed that BrrIAA members functioned as nucleoproteins. Moreover, transcriptional analysis revealed that the expression patterns of AUX/IAA genes in turnip were tissue-dependent. Because orthologs have similar biological functions and interaction networks in plant growth and development, BrrIAA66 in turnip possibly played a role in embryo axis formation, vascular development, lateral root formation, and floral organ development by interacting with BrrARF19 and BrrTIR1. CONCLUSION: These results provide a theoretical basis for further investigation of BrrAUX/IAA genes and lay the foundation for functional analysis of BrrIAA66 in turnip.

Genome-wide identification of RNA recognition motif (RRM1) in Brassica rapa and functional analysis of RNA-binding protein (BrRBP) under low-temperature stress.

BACKGROUND: The RNA recognition motif (RRM) is primarily engaged in the processing of mRNA and rRNA following gene transcription as well as the regulation of RNA transport; it is critical in preserving RNA stability. RESULTS: In this study, we identified 102 members of the RRM1 gene family in Brassica rapa, which were dispersed across 10 chromosomes with the ninth chromosome being the most extensively distributed. The RRM1 gene family members of Brassica rapa and Arabidopsis thaliana were grouped into 14 subclades (I-XIV) using phylogenetic analysis. Moreover, the results of transcriptome analysis and RT-qPCR indicated that the expression of Brapa05T000840 was upregulated in the cultivars 'Longyou 7' and 'Longyou 99' following exposure to cold stress at a temperature of 4 °C for 24 h. The levels of expression in the leaves and growth cones of the 'Longyou 7' variety were found to be significantly higher than those observed in the 'Longyou 99' variety under conditions of low temperature and NaCl stress. It illustrates the involvement of the RRM1 gene in the physiological response to both low temperature and salt stress. In addition, it was observed that the survival rate of transgenic BrRBP (Brapa05T000840) Arabidopsis thaliana plants was notably higher compared to that of wild-type plants when subjected to varying durations of low temperature treatment. Furthermore, the expression of the BrRBP gene in transgenic plants exhibited an upward trend as the duration of low temperature treatment increased, reaching its peak at 24 h. The in-vivo enzymatic activity of reactive oxygen species-scavenging enzymes were found to be significantly elevated in comparison to wild-type plants, suggesting that the BrRBP gene may enhance the cold tolerance of Arabidopsis thaliana. CONCLUSIONS: This study offers a significant foundation for comprehending the regulation mechanism of the RRM1 gene family in winter Brassica rapa subjected to cold stress, as well as for finding key genes associated with cold resistance.

Identification and characterization of the gene BraANS.A03 associated with purple leaf color in pak choi (Brassica rapa L. ssp. chinensis).

MAIN CONCLUSION: BraANS.A3 was the key gene controlling purple leaf color in pak choi, and two short fragments of promoter region in green pak choi might be interfering its normal expression. Pak choi (B. rapa L. ssp. chinensis) is an influential and important vegetable with green, yellow, or purple leaves that is cultivated worldwide. The purple leaves are rich in anthocyanins, but the underlying genetics and evolution have yet to be extensively studied. Free-hand sections of the purple leaves indicated that anthocyanins mainly accumulate throughout the adaxial and abaxial epidermal leaf cells. Segregation analyses of an F2 population of a B. rapa ssp. chinensis L. purple leaf mutant ZBC indicated that the purple trait is controlled by an incompletely dominant nuclear gene. Bulked segregant analysis (BSA) showed that the key genes controlling the trait were between 24.25 and 38.10 Mb on chromosome A03 of B. rapa. From the annotated genes, only BraA03g050560.3C, homologous to Arabidopsis AtANS, was related to the anthocyanin synthesis pathway. Genome annotation results and transcriptional sequencing analyses revealed that the BraANS.A3 gene was involved in the purple leaf trait. qRT-PCR analyses showed that BraANS.A3 was highly upregulated in ZBC but hardly expressed in the leaves of an inbred homozygous line of B. campestris ssp. chinensis L. green leaf mutant WTC, indicating that BraANS.A3 played a key role catalyzing anthocyanin synthesis in ZBC. Full-length sequence alignment of BraANS.A3 in WTC and ZBC showed that it was highly conserved in the gene region, with significant variation in the promoter region. In particular, the insertion of two short fragments of the promoter region in WTC may interfere with its normal expression. The promoter regions of ANS in six Brassica species all had multiple cis-elements involved in responses to abscisic acid, light, and stress, suggesting that ANS may be involved in multiple metabolic pathways or biological processes. Protein-protein interactions predicted that BraANS.A3 interacts with virtually all catalytic proteins in the anthocyanin synthesis pathway and has a strong relationship with Transparent Testa 8 (TT8). These results suggest that BraANS.A3 promotes anthocyanin accumulation in purple pak choi and provide new insights into the functional analysis of anthocyanin-related genes in Chinese cabbage and transcriptional regulatory networks.

Database of space life investigations and information on spaceflight plant biology.

Extensive spaceflight life investigations (SLIs) have revealed observable space effects on plants, particularly their growth, nutrition yield, and secondary metabolite production. Knowledge of these effects not only facilitates space agricultural and biopharmaceutical technology development but also provides unique perspectives to ground-based investigations. SLIs are specialized experimental protocols and notable biological phenomena. These require specialized databases, leading to the development of the NASA Science Data Archive, Erasmus Experiment Archive, and NASA GeneLab. The increasing interests of SLIs across diverse fields demand resources with comprehensive content, convenient search facilities, and friendly information presentation. A new database SpaceLID (Space Life Investigation Database http://bidd.group/spacelid/ ) was developed with detailed menu search tools and categorized contents about the phenomena, protocols, and outcomes of 459 SLIs (including 106 plant investigations) of 92 species, where 236 SLIs and 57 plant investigations are uncovered by the existing databases. The usefulness of SpaceLID as an SLI information source is illustrated by the literature-reported analysis of metabolite, nutrition, and symbiosis variations of spaceflight plants. In conclusion, this study extensively investigated the impact of the space environment on plant biology, utilizing SpaceLID as an information source and examining various plant species, including Arabidopsis thaliana, Brassica rapa L., and Glycyrrhiza uralensis Fisch. The findings provide valuable insights into the effects of space conditions on plant physiology and metabolism.

Wall-associated kinase BrWAK1 confers resistance to downy mildew in Brassica rapa.

The plant cell wall is the first line of defence against physical damage and pathogen attack. Wall-associated kinase (WAK) has the ability to perceive the changes in the cell wall matrix and transform signals into the cytoplasm, being involved in plant development and the defence response. Downy mildew, caused by Hyaloperonospora brassicae, can result in a massive loss in Chinese cabbage (Brassica rapa L. ssp. pekinensis) production. Herein, we identified a candidate resistant WAK gene, BrWAK1, in a major resistant quantitative trait locus, using a double haploid population derived from resistant inbred line T12-19 and the susceptible line 91-112. The expression of BrWAK1 could be induced by salicylic acid and pathogen inoculation. Expression of BrWAK1 in 91-112 could significantly enhance resistance to the pathogen, while truncating BrWAK1 in T12-19 increased disease susceptibility. Variation in the extracellular galacturonan binding (GUB) domain of BrWAK1 was found to mainly confer resistance to downy mildew in T12-19. Moreover, BrWAK1 was proved to interact with BrBAK1 (brassinosteroid insensitive 1 associated kinase), resulting in the activation of the downstream mitogen-activated protein kinase (MAPK) cascade to trigger the defence response. BrWAK1 is the first identified and thoroughly characterized WAK gene conferring disease resistance in Chinese cabbage, and the plant biomass is not significantly influenced by BrWAK1, which will greatly accelerate Chinese cabbage breeding for downy mildew resistance.

A near-complete genome assembly of Brassica rapa provides new insights into the evolution of centromeres.

Brassica rapa comprises many important cultivated vegetables and oil crops. However, Chiifu v3.0, the current B. rapa reference genome, still contains hundreds of gaps. Here, we presented a near-complete genome assembly of B. rapa Chiifu v4.0, which was 424.59 Mb with only two gaps, using Oxford Nanopore Technology (ONT) ultralong-read sequencing and Hi-C technologies. The new assembly contains 12 contigs, with a contig N50 of 38.26 Mb. Eight of the ten chromosomes were entirely reconstructed in a single contig from telomere to telomere. We found that the centromeres were mainly invaded by ALE and CRM long terminal repeats (LTRs). Moreover, there is a high divergence of centromere length and sequence among B. rapa genomes. We further found that centromeres are enriched for Copia invaded at 0.14 MYA on average, while pericentromeres are enriched for Gypsy LTRs invaded at 0.51 MYA on average. These results indicated the different invasion mechanisms of LTRs between the two structures. In addition, a novel repetitive sequence PCR630 was identified in the pericentromeres of B. rapa. Overall, the near-complete genome assembly, B. rapa Chiifu v4.0, offers valuable tools for genomic and genetic studies of Brassica species and provides new insights into the evolution of centromeres.

Battle for control of anthocyanin biosynthesis in two Brassicaceae species infected with turnip mosaic virus.

It has previously been found that turnip mosaic virus (TuMV) greatly suppresses anthocyanin accumulation (AA) in Brassica rapa leaves, and that such leaves become infected whilst anthocyanin-enriched leaves on the same plants are rarely infected. To clarify whether AA is a defense against TuMV, in this study we examined tissue-level patterns of spontaneous AA in relation to the cellular localization of a TuMV strain that expresses a yellow fluorescent protein. We found that TuMV infection was significantly blocked by AA, suggesting that it functions as a chemical barrier against TuMV. We next analysed changes in expression of genes related to anthocyanin biosynthesis in TuMV-infected leaves of Arabidopsis. TuMV also suppressed AA that is induced by high light in Arabidopsis, and this this suppression was mainly due to inhibited expression of anthocyanin late-biosynthesis genes (LBGs). Most positive transcription factors of LBGs were also down-regulated, while the negative regulator SPL15 was highly up-regulated. Cucumber mosaic virus (CMV) also moderately suppressed AA in Arabidopsis, but in a different manner. Since it appeared that anthocyanin-enriched leaves of Arabidopsis were resistant to TuMV but not CMV, our results suggested that the anthocyanin-associated resistance that we observed was specific to TuMV.

Rapid, nonparallel genomic evolution of Brassica rapa (field mustard) under experimental drought.

While we know that climate change can potentially cause rapid phenotypic evolution, our understanding of the genetic basis and degree of genetic parallelism of rapid evolutionary responses to climate change is limited. In this study, we combined the resurrection approach with an evolve-and-resequence design to examine genome-wide evolutionary changes following drought. We exposed genetically similar replicate populations of the annual plant Brassica rapa derived from a field population in southern California to four generations of experimental drought or watered conditions in a greenhouse. Genome-wide sequencing of ancestral and descendant population pools identified hundreds of SNPs that showed evidence of rapidly evolving in response to drought. Several of these were in stress response genes, and two were identified in a prior study of drought response in this species. However, almost all genetic changes were unique among experimental populations, indicating that the evolutionary changes were largely nonparallel, despite the fact that genetically similar replicates of the same founder population had experienced controlled and consistent selection regimes. This nonparallelism of evolution at the genetic level is potentially because of polygenetic adaptation allowing for multiple different genetic routes to similar phenotypic outcomes. Our findings help to elucidate the relationship between rapid phenotypic and genomic evolution and shed light on the degree of parallelism and predictability of genomic evolution to environmental change.

Evaluation of a Series of Turnip Mosaic Virus Chimeric Clones Reveals Two Amino Acid Sites Critical for Systemic Infection in Chinese Cabbage.

Two infectious clones of turnip mosaic virus (TuMV), pKBC-1 and pKBC-8, with differential infectivity in Chinese cabbage (Brassica rapa subsp. pekinensis), were obtained. Both infected Nicotiana benthamiana systemically, inducing similar symptoms, whereas only virus KBC-8 infected Chinese cabbage systemically. To identify the determinants affecting infectivity on Chinese cabbage, chimeric clones were constructed by restriction fragment exchange between the parental clones and tested on several Chinese cabbage cultivars. Chimeric clones p1N8C and p8N1C demonstrated that the C-terminal portion of the polyprotein determines systemic infection of Chinese cabbage despite only three amino acid differences in this region, in the cylindrical inclusion (CI), viral protein genome-linked (VPg), and coat protein (CP). A second pair of hybrid constructs, pHindIII-1N8C and pHindIII-8N1C, failed to infect cultivars CR Victory and Jinseonnorang systemically, yet pHindIII-1N8C caused hypersensitive response-like lesions on inoculated leaves of these cultivars, and could systemically infect cultivars CR Chusarang and Jeongsang; this suggests that R genes effective against TuMV may exist in the first two cultivars but not the latter two. Constructs with single amino acid changes in both VPg (K2045E) and CP (Y3095H) failed to infect Chinese cabbage, implying that at least one of these two amino acid substitutions is essential for successful infection on Chinese cabbage. Successful infection by mutant KBC-8-CP-H and delayed infection with mutant HJY1-VPg-E following mutation or reversion suggested that VPg (2045K) is the residue required for infection of Chinese cabbage and involved in the interaction between VPg and eukaryotic initiation factor eIF(iso)4E, confirmed by yeast two-hybrid assay.

Genome-wide analysis reveals the crucial role of lncRNAs in regulating the expression of genes controlling pollen development.

KEY MESSAGE: The genomic location and stage-specific expression pattern of many long non-coding RNAs reveal their critical role in regulating protein-coding genes crucial in pollen developmental progression and male germ line specification. Long non-coding RNAs (lncRNAs) are transcripts longer than 200 bp with no apparent protein-coding potential. Multiple investigations have revealed high expression of lncRNAs in plant reproductive organs in a cell and tissue-specific manner. However, their potential role as essential regulators of molecular processes involved in sexual reproduction remains largely unexplored. We have used developing field mustard (Brassica rapa) pollen as a model system for investigating the potential role of lncRNAs in reproductive development. Reference-based transcriptome assembly performed to update the existing genome annotation identified novel expressed protein-coding genes and long non-coding RNAs (lncRNAs), including 4347 long intergenic non-coding RNAs (lincRNAs, 1058 expressed) and 2,045 lncRNAs overlapping protein-coding genes on the opposite strand (lncNATs, 780 expressed). The analysis of expression profiles reveals that lncRNAs are significant and stage-specific contributors to the gene expression profile of developing pollen. Gene co-expression networks accompanied by genome location analysis identified 38 cis-acting lincRNA, 31 cis-acting lncNAT, 7 trans-acting lincRNA and 14 trans-acting lncNAT to be substantially co-expressed with target protein-coding genes involved in biological processes regulating pollen development and male lineage specification. These findings provide a foundation for future research aiming at developing strategies to employ lncRNAs as regulatory tools for gene expression control during reproductive development.