Mutation to a cytochrome P450 -like gene alters the leaf color by affecting the heme and chlorophyll biosynthesis pathways in Brassica napus.

The regulated biosynthesis of chlorophyll is important because of its effects on plant photosynthesis and dry biomass production. In this study, a map-based cloning approach was used to isolate the cytochrome P450 -like gene BnaC08g34840D (BnCDE1) from a chlorophyll-deficient mutant (cde1) of Brassica napus obtained by ethyl methanesulfonate (EMS) mutagenization. Sequence analyses revealed that BnaC08g34840D in the cde1 mutant (BnCDE1I320T ) encodes a substitution at amino acid 320 (Ile320Thr) in the conserved region. The over-expression of BnCDE1I320T in ZS11 (i.e., gene-mapping parent with green leaves) recapitulated a yellow-green leaf phenotype. The CRISPR/Cas9 genome-editing system was used to design two single-guide RNAs (sgRNAs) targeting BnCDE1I320T in the cde1 mutant. The knockout of BnCDE1I320T in the cde1 mutant via a gene-editing method restored normal leaf coloration (i.e., green leaves). These results indicate that the substitution in BnaC08g34840D alters the leaf color. Physiological analyses showed that the over-expression of BnCDE1I320T leads to decreases in the number of chloroplasts per mesophyll cell and in the contents of the intermediates of the chlorophyll biosynthesis pathway in leaves, while it increases heme biosynthesis, thereby lowering the photosynthetic efficiency of the cde1 mutant. The Ile320Thr mutation in the highly conserved region of BnaC08g34840D inhibited chlorophyll biosynthesis and disrupted the balance between heme and chlorophyll biosynthesis. Our findings may further reveal how the proper balance between the chlorophyll and heme biosynthesis pathways is maintained.

Comparative proteomic and physiological analyses reveal tribenuron-methyl phytotoxicity and nontarget-site resistance mechanisms in Brassica napus.

The application of herbicides is the most effective strategy for weed control and the development of herbicide-resistant crops will facilitate the weed management. The acetolactate synthase-inhibiting herbicide, tribenuron-methyl (TBM), is broadly used for weed control. However, its application in rapeseed field is restricted since rapeseed is sensitive to TBM. Herein, an integrated study of cytological, physiological and proteomic analysis of the TBM-resistant rapeseed mutant M342 and its wild-type (WT) plants was conducted. After TBM spraying, M342 showed improved tolerance to TBM, and proteins implicated in non-target-site resistance (NTSR) to herbicides had a significantly higher level in M342 as compared with the WT. Differentially accumulated proteins (DAPs) between these two genotypes were enriched in glutathione metabolism and oxidoreduction coenzyme metabolic process, which protected the mutant from oxidative stress triggered by TBM. Important DAPs related to stress or defence response were up-accumulated in M342 regardless of the TBM treatment, which might serve as the constitutive part of NTSR to TBM. These results provide new clues for further exploration of the NTSR mechanism in plants and establish a theoretical basis for the development of herbicide-resistant crops.

Fine Mapping of a Pleiotropic Locus (BnUD1) Responsible for the Up-Curling Leaves and Downward-Pointing Siliques in Brassica napus.

Leaves and siliques are important organs associated with dry matter biosynthesis and vegetable oil accumulation in plants. We identified and characterized a novel locus controlling leaf and silique development using the Brassica napus mutant Bnud1, which has downward-pointing siliques and up-curling leaves. The inheritance analysis showed that the up-curling leaf and downward-pointing silique traits are controlled by one dominant locus (BnUD1) in populations derived from NJAU5773 and Zhongshuang 11. The BnUD1 locus was initially mapped to a 3.99 Mb interval on the A05 chromosome with a BC6F2 population by a bulked segregant analysis-sequencing approach. To more precisely map BnUD1, 103 InDel primer pairs uniformly covering the mapping interval and the BC5F3 and BC6F2 populations consisting of 1042 individuals were used to narrow the mapping interval to a 54.84 kb region. The mapping interval included 11 annotated genes. The bioinformatic analysis and gene sequencing data suggested that BnaA05G0157900ZS and BnaA05G0158100ZS may be responsible for the mutant traits. Protein sequence analyses showed that the mutations in the candidate gene BnaA05G0157900ZS altered the encoded PME in the trans-membrane region (G45A), the PMEI domain (G122S), and the pectinesterase domain (G394D). In addition, a 573 bp insertion was detected in the pectinesterase domain of the BnaA05G0157900ZS gene in the Bnud1 mutant. Other primary experiments indicated that the locus responsible for the downward-pointing siliques and up-curling leaves negatively affected the plant height and 1000-seed weight, but it significantly increased the seeds per silique and positively affected photosynthetic efficiency to some extent. Furthermore, plants carrying the BnUD1 locus were compact, implying they may be useful for increasing B. napus planting density. The findings of this study provide an important foundation for future research on the genetic mechanism regulating the dicotyledonous plant growth status, and the Bnud1 plants can be used directly in breeding.

Ammonia borane positively regulates cold tolerance in Brassica napus via hydrogen sulfide signaling.

BACKGROUND: Cold stress adversely influences rapeseeds (Brassica napus L.) growth and yield during winter and spring seasons. Hydrogen (H2) is a potential gasotransmitter that is used to enhance tolerance against abiotic stress, including cold stress. However, convenience and stability are two crucial limiting factors upon the application of H2 in field agriculture. To explore the application of H2 in field, here we evaluated the role of ammonia borane (AB), a new candidate for a H2 donor produced by industrial chemical production, in plant cold tolerance. RESULTS: The application with AB could obviously alleviate the inhibition of rapeseed seedling growth and reduce the oxidative damage caused by cold stress. The above physiological process was closely related to the increased antioxidant enzyme system and reestablished redox homeostasis. Importantly, cold stress-triggered endogenous H2S biosynthesis was further stimulated by AB addition. The removal or inhibition of H2S synthesis significantly abolished plant tolerance against cold stress elicited by AB. Further field experiments demonstrated that the phenotypic and physiological performances of rapeseed plants after challenged with cold stress in the winter and early spring seasons were significantly improved by administration with AB. Particularly, the most studied cold-stress response pathway, the ICE1-CBF-COR transcriptional cascade, was significantly up-regulated either. CONCLUSION: Overall, this study clearly observed the evidence that AB-increased tolerance against cold stress could be suitable for using in field agriculture by stimulation of H2S signaling.

A small chromosomal inversion mediated by MITE transposons confers cleistogamy in Brassica napus.

Cleistogamy, self-pollination within closed flowers, can help maintain seed purity, accelerate breeding speed, and aid in the development of ornamental flowers. However, the mechanism underlying petal closing/opening behavior remains elusive. Here, we found that a Brassica napus petal closing/opening behavior was inherited in a Mendelian manner. Fine mapping and positional cloning experiments revealed that the Mendelian factor originated from a short (29.8 kb) inversion mediated by BnDTH9 miniature inverted-repeat transposable elements (MITEs) on chromosome C03. This inversion led to tissue-specific gene promoter exchange between BnaC03.FBA (BnaC03G0156800ZS encoding an F-Box-associated domain-containing protein) and BnaC03.EFO1 (BnaC03G0157400ZS encoding an EARLY FLOWERING BY OVEREXPRESSION 1 protein) positioned near the respective inversion breakpoints. Our genetic transformation work demonstrated that the cleistogamy originated from high tissue-specific expression of the BnaC03.FBA gene caused by promoter changes due to the MITE-mediated inversion. BnaC03.FBA is involved in the formation of an SCF (Skp1-Cullin-F-box) complex, which participates in ubiquitin-mediated protein targeting for degradation through the ubiquitin 26S-proteasome system. Our results shed light on a molecular model of petal-closing behavior.

Identification and Fine Mapping of a Locus Related to Leaf Up-Curling Trait (Bnuc3) in Brassica napus.

Leaf trait is an important target trait in crop breeding programs. Moderate leaf curling may be a help for improving crop yield by minimizing the shadowing by leaves. Mining locus for leaf curling trait is of significance for plant genetics and breeding researches. The present study identified a novel rapeseed accession with up-curling leaf, analyzed the up-curling leaf trait inheritance, and fine mapped the locus for up-curling leaf property (Bnuc3) in Brassica napus. Genetic analysis revealed that the up-curling leaf trait is controlled by a single dominant locus, named BnUC3. We performed an association study of BnUC3 with single nucleotide polymorphism (SNP) markers using a backcross population derived from the homozygous up-curling leaf line NJAU-M1295 and the canola variety 'zhongshuang11' with typical flat leaves, and mapped the BnUC3 locus in a 1.92 Mb interval of chromosome A02 of B. napus. To further map BnUC3, 232 simple sequence repeat (SSR) primers and four pairs of Insertion/Deletion (InDel) primers were developed for the mapping interval. Among them, five SSR markers and two InDel markers were polymorphic. By these markers, the mapping interval was narrowed to 92.0 kb using another F2 population. This fine mapping interval has 11 annotated genes among which BnaA02T0157000ZS were inferred to be candidate casual genes for up-curling leaf based on the cloned sequence analysis, gene functionality, and gene expression analysis. The current study laid a foundational basis for further elucidating the mechanism of BnUC3 and breeding of variety with up-curling leaf.

Fine mapping of the BnaC04.BIL1 gene controlling plant height in Brassica napus L.

BACKGROUND: Plant height is an important architecture trait which is a fundamental yield-determining trait in crops. Variety with dwarf or semi-dwarf phenotype is a major objective in the breeding because dwarfing architecture can help to increase harvest index, increase planting density, enhance lodging resistance, and thus be suitable for mechanization harvest. Although some germplasm or genes associated with dwarfing plant type have been carried out. The molecular mechanisms underlying dwarfism in oilseed rape (Brassica napus L.) are poorly understood, restricting the progress of breeding dwarf varieties in this species. Here, we report a new dwarf mutant Bndwarf2 from our B. napus germplasm. We studied its inheritance and mapped the dwarf locus BnDWARF2. RESULTS: The inheritance analysis showed that the dwarfism phenotype was controlled by one semi-dominant gene, which was mapped in an interval of 787.88 kb on the C04 chromosome of B. napus by Illumina Brassica 60 K Bead Chip Array. To fine-map BnDWARF2, 318 simple sequence repeat (SSR) primers were designed to uniformly cover the mapping interval. Among them, 15 polymorphic primers that narrowed down the BnDWARF2 locus to 34.62 kb were detected using a F2:3 family population with 889 individuals. Protein sequence analysis showed that only BnaC04.BIL1 (BnaC04g41660D) had two amino acid residues substitutions (Thr187Ser and Gln399His) between ZS11 and Bndwarf2, which encoding a GLYCOGEN SYNTHASE KINASE 3 (GSK3-like). The quantitative real-time PCR (qRT-PCR) analysis showed that the BnaC04.BIL1 gene expressed in all tissues of oilseed rape. Subcellular localization experiment showed that BnaC04.BIL1 was localized in the nucleus in tobacco leaf cells. Genetic transformation experiments confirmed that the BnaC04.BIL1 is responsible for the plant dwarf phenotype in the Bndwarf2 mutants. Overexpression of BnaC04.BIL1 reduced plant height, but also resulted in compact plant architecture. CONCLUSIONS: A dominant dwarfing gene, BnaC04.BIL1, encodes an GSK3-like that negatively regulates plant height, was mapped and isolated. Our identification of a distinct gene locus may help to improve lodging resistance in oilseed rape.

Nitric Oxide Is Associated With Heterosis of Salinity Tolerance in Brassica napus L.

Heterosis is most frequently manifested as the superior performance of a hybrid than either of the parents, especially under stress conditions. Nitric oxide (NO) is a well-known gaseous signaling molecule that acts as a functional component during plant growth, development, and defense responses. In this study, the Brassica napus L. hybrid (F1, NJ4375 × MB1942) showed significant heterosis under salt stress, during both germination and post-germination periods. These phenotypes in the hybrid were in parallel with the better performance in redox homeostasis, including alleviation of reactive oxygen species accumulation and lipid peroxidation, and ion homeostasis, evaluated as a lower Na/K ratio in the leaves than parental lines. Meanwhile, stimulation of endogenous NO was more pronounced in hybrid plants, compared with parental lines, which might be mediated by nitrate reductase. Proteomic and biochemical analyses further revealed that protein abundance related to several metabolic processes, including chlorophyll biosynthesis, proline metabolism, and tricarboxylic acid cycle metabolism pathway, was greatly suppressed by salt stress in the two parental lines than in the hybrid. The above responses in hybrid plants were intensified by a NO-releasing compound, but abolished by a NO scavenger, both of which were matched with the changes in chlorophyll and proline contents. It was deduced that the above metabolic processes might play important roles in heterosis upon salt stress. Taken together, we proposed that heterosis derived from F1 hybridization in salt stress tolerance might be mediated by NO-dependent activation of defense responses and metabolic processes.

Multi-Walled Carbon Nanotubes Can Promote Brassica napus L. and Arabidopsis thaliana L. Root Hair Development through Nitric Oxide and Ethylene Pathways.

Here, we report that multi-walled carbon nanotubes (MWCNTs) can promote plant root hair growth in the species analyzed in this study; however, low and excessive concentrations of MWCNTs had no significant effect or even an inhibiting influence. Further results show that MWCNTs can enter rapeseed root cells. Meanwhile, nitrate reductase (NR)-dependent nitric oxide (NO) and ethylene syntheses, as well as root hair formation, were significantly stimulated by MWCNTs. Transcription of root hair growth-related genes were also modulated. The above responses were sensitive to the removal of endogenous NO or ethylene with a scavenger of NO or NO/ethylene synthesis inhibitors. Pharmacological and molecular evidence suggested that ethylene might act downstream of NR-dependent NO in MWCNTs-induced root hair morphogenesis. Genetic evidence in Arabidopsis further revealed that MWCNTs-triggered root hair growth was abolished in ethylene-insensitive mutants ein2-5 and ein3-1, and NR mutant nia1/2, but not in noa1 mutant. Further data placed NO synthesis linearly before ethylene production in root hair development triggered by MWCNTs. The above findings thus provide some insights into the molecular mechanism underlying MWCNTs control of root hair morphogenesis.

Fine mapping of the BnUC2 locus related to leaf up-curling and plant semi-dwarfing in Brassica napus.

BACKGROUND: Studies of leaf shape development and plant stature have made important contributions to the fields of plant breeding and developmental biology. The optimization of leaf morphology and plant height to improve lodging resistance and photosynthetic efficiency, increase planting density and yield, and facilitate mechanized harvesting is a desirable goal in Brassica napus. RESULTS: Here, we investigated a B. napus germplasm resource exhibiting up-curled leaves and a semi-dwarf stature. In progeny populations derived from NJAU5737 and Zhongshuang 11 (ZS11), we found that the up-curled leaf trait was controlled by a dominant locus, BnUC2. We then fine mapped the BnUC2 locus onto an 83.19-kb interval on chromosome A05 using single nucleotide polymorphism (SNP) and simple sequence repeat (SSR) markers. We further determined that BnUC2 was a major plant height QTL that explained approximately 70% of the phenotypic variation in two BC5F3 family populations derived from NJAU5737 and ZS11. This result implies that BnUC2 was also responsible for the observed semi-dwarf stature. The fine mapping interval of BnUC2 contained five genes, two of which, BnaA05g16700D (BnaA05.IAA2) and BnaA05g16720D, were revealed by comparative sequencing to be mutated in NJAU5737. This result suggests that the candidate gene mutation (BnaA05g16700D, encoding Aux/IAA2 proteins) in the conserved Degron motif GWPPV (P63S) was responsible for the BnUC2 locus. In addition, investigation of agronomic traits in a segregated population indicated that plant height, main inflorescence length, and branching height were significantly reduced by BnUC2, whereas yield was not significantly altered. The determination of the photosynthetic efficiency showed that the BnUC2 locus was beneficial to improve the photosynthetic efficiency. Our findings may provide an effective foundation for plant type breeding in B. napus. CONCLUSIONS: Using SNP and SSR markers, a dominant locus (BnUC2) related to up-curled leaves and semi-dwarf stature in B. napus has been fine mapped onto an 83.19-kb interval of chromosome A05 containing five genes. The BnaA05.IAA2 is inferred to be the candidate gene responsible for the BnUC2 locus.

Genome-Wide Analysis Reveals the Role of Mediator Complex in the Soybean-Phytophthora sojae Interaction.

The mediator complex is an essential link between transcription factors and RNA polymerase II, and mainly functions in the transduction of diverse signals to genes involved in different pathways. Limited information is available on the role of soybean mediator subunits in growth and development, and their participation in defense response regulation. Here, we performed genome-wide identification of the 95 soybean mediator subunits, which were unevenly localized on the 20 chromosomes and only segmental duplication events were detected. We focused on GmMED16-1, which is highly expressed in the roots, for further functional analysis. Transcription of GmMED16-1 was induced in response to Phytophthora sojae infection. Agrobacterium rhizogenes mediated soybean hairy root transformation was performed for the silencing of the GmMED16-1 gene. Silencing of GmMED16-1 led to an enhanced susceptibility phenotype and increased accumulation of P. sojae biomass in hairy roots of transformants. The transcript levels of NPR1, PR1a, and PR5 in the salicylic acid defense pathway in roots of GmMED16-1-silenced transformants were lower than those of empty-vector transformants. The results provide evidence that GmMED16-1 may participate in the soybean-P. sojae interaction via a salicylic acid-dependent process.

Fine mapping of an up-curling leaf locus (BnUC1) in Brassica napus.

BACKGROUND: Leaf shape development research is important because leaf shapes such as moderate curling can help to improve light energy utilization efficiency. Leaf growth and development includes initiation of the leaf primordia and polar differentiation of the proximal-distal, adaxial-abaxial, and centrolateral axes. Changes in leaf adaxial-abaxial polarity formation, auxin synthesis and signaling pathways, and development of sclerenchyma and cuticle can cause abnormal leaf shapes such as up-curling leaf. Although many genes related to leaf shape development have been reported, the detailed mechanism of leaf development is still unclear. Here, we report an up-curling leaf mutant plant from our Brassica napus germplasm. We studied its inheritance, mapped the up-curling leaf locus BnUC1, built near-isogenic lines for the Bnuc1 mutant, and evaluated the effect of the dominant leaf curl locus on leaf photosynthetic efficiency and agronomic traits. RESULTS: The up-curling trait was controlled by one dominant locus in a progeny population derived from NJAU5734 and Zhongshuang 11 (ZS11). This BnUC1 locus was mapped in an interval of 2732.549 kb on the A05 chromosome of B. napus using Illumina Brassica 60 K Bead Chip Array. To fine map BnUC1, we designed 201 simple sequence repeat (SSR) primers covering the mapping interval. Among them, 16 polymorphic primers that narrowed the mapping interval to 54.8 kb were detected using a BC6F2 family population with 654 individuals. We found six annotated genes in the mapping interval using the B. napus reference genome, including BnaA05g18250D and BnaA05g18290D, which bioinformatics and gene expression analyses predicted may be responsible for leaf up-curling. The up-curling leaf trait had negative effects on the agronomic traits of 30 randomly selected individuals from the BC6F2 population. The near-isogenic line of the up-curling leaf (ZS11-UC1) was constructed to evaluate the effect of BnUC1 on photosynthetic efficiency. The results indicated that the up-curling leaf trait locus was beneficial to improve the photosynthetic efficiency. CONCLUSIONS: An up-curling leaf mutant Bnuc1 was controlled by one dominant locus BnUC1. This locus had positive effects on photosynthetic efficiency, negative effects on some agronomic traits, and may help to increase planting density in B. napus.

Nitrate reductase-dependent nitric oxide is crucial for multi-walled carbon nanotube-induced plant tolerance against salinity.

Although there have been some studies on the plant-carbonaceous nanomaterials (CNMs) interactions, related conclusions were controversial. Here, we report that multi-walled carbon nanotubes (MWCNTs) can enter into rapeseed (Brassica napus L.) seedling root, and transport to stem. Further results showed that salinity-inhibited rapeseed seedling growth was obviously alleviated by MWCNTs. Meanwhile, NaCl-induced nitrate reductase (NR)-dependent NO production was significantly intensified by MWCNTs. The redox and ion imbalance was reestablished as well, confirmed by the reduction in reactive oxygen species (ROS) overproduction, the decrease in thiobarbituric acid reactive substance production, and the lower Na+/K+ ratio. These beneficial effects could be explained by the changes in related antioxidant defense genes, sodium hydrogen exchanger 1 (NHX1), salt overly sensitive 1 (SOS1), and K+transporter 1 (KT1) transcripts. The above responses were separately abolished after the removal of endogenous NO with its scavengers or the addition of the NR inhibitor. Genetic evidence revealed that the NaCl-triggered NO level in wild-type seedling roots was partly abolished in either the nitric reductase mutant (nia1/2) or noa1 mutant (exhibiting indirectly a reduced endogenous NO level). Treatment with MWCNTs could totally rescue the impaired NO production in the noa1 mutant rather than the nia1/2 mutant, suggesting that NR-dependent NO acts as a downstream signaling molecule in MWCNT signaling. This point was verified by phenotypic analyses, histochemical staining, and ion analysis. qPCR analysis further demonstrated that MWCNTs stimulated antioxidant genes and ion balance-related genes through NR-mediated NO. The above molecular and genetic evidence indicated that NR-dependent NO acts downstream of MWCNTs in salinity tolerance, which requires the reestablishment of redox and ion homeostasis.

L-Cysteine desulfhydrase-dependent hydrogen sulfide is required for methane-induced lateral root formation.

KEY MESSAGE: Methane-triggered lateral root formation is not only a universal event, but also dependent on L-cysteine desulfhydrase-dependent hydrogen sulfide signaling. Whether or how methane (CH4) triggers lateral root (LR) formation has not been elucidated. In this report, CH4 induction of lateral rooting and the role of hydrogen sulfide (H2S) were dissected in tomato and Arabidopsis by using physiological, anatomical, molecular, and genetic approaches. First, we discovered that CH4 induction of lateral rooting is a universal event. Exogenously applied CH4 not only triggered tomato lateral rooting, but also increased activities of L-cysteine desulfhydrase (DES; a major synthetic enzyme of H2S) and induced endogenous H2S production, and contrasting responses were observed in the presence of hypotaurine (HT; a scavenger of H2S) or DL-propargylglycine (PAG; an inhibitor of DES) alone. CH4-triggered lateral rooting were sensitive to the inhibition of endogenous H2S with HT or PAG. The changes in the transcripts of representative cell cycle regulatory genes, miRNA and its target genes were matched with above phenotypes. In the presence of CH4, Arabidopsis mutant Atdes1 exhibited defects in lateral rooting, compared with the wild-type. Molecular evidence showed that the transcriptional profiles of representative target genes modulated by CH4 in wild-type plants were impaired in Atdes1 mutant. Overall, our data demonstrate the main branch of the DES-dependent H2S signaling cascade in CH4-triggered LR formation.

Transcriptional and translational responses of rapeseed leaves to red and blue lights at the rosette stage.

Under different red (R):blue (B) photon flux ratios, the growth performance of rapeseed (Brassica napus L.) is significantly different. Rapeseed under high R ratios shows shade response, while under high B ratios it shows sun-type morphology. Rapeseed under monochromatic red or blue light is seriously stressed. Transcriptomic and proteomic methods were used to analyze the metabolic pathway change of rapeseed (cv. "Zhongshuang 11") leaves under different R:B photon flux ratios (including 100R:0B%, 75R:25B%, 25R:75B%, and 0R:100B%), based on digital gene expression (DGE) and two-dimensional gel electrophoresis (2-DE). For DGE analysis, 2054 differentially expressed transcripts (|log2(fold change)|≥1, q<0.005) were detected among the treatments. High R ratios (100R:0B% and 75R:25B%) enhanced the expression of cellular structural components, mainly the cell wall and cell membrane. These components participated in plant epidermis development and anatomical structure morphogenesis. This might be related to the shade response induced by red light. High B ratios (25R:75B% and 0R:100B%) promoted the expression of chloroplast-related components, which might be involved in the formation of sun-type chloroplast induced by blue light. For 2-DE analysis, 37 protein spots showed more than a 2-fold difference in expression among the treatments. Monochromatic light (ML; 100R:0B% and 0R:100B%) stimulated accumulation of proteins associated with antioxidation, photosystem II (PSII), DNA and ribosome repairs, while compound light (CL; 75R:25B% and 25R:75B%) accelerated accumulation of proteins associated with carbohydrate, nucleic acid, amino acid, vitamin, and xanthophyll metabolisms. These findings can be useful in understanding the response mechanisms of rapeseed leaves to different R:B photon flux ratios.

Nitric Oxide Is Required for Melatonin-Enhanced Tolerance against Salinity Stress in Rapeseed (Brassica napus L.) Seedlings.

Although melatonin (N-acetyl-5-methoxytryptamine) could alleviate salinity stress in plants, the downstream signaling pathway is still not fully characterized. Here, we report that endogenous melatonin and thereafter nitric oxide (NO) accumulation was successively increased in NaCl-stressed rapeseed (Brassica napus L.) seedling roots. Application of melatonin and NO-releasing compound not only counteracted NaCl-induced seedling growth inhibition, but also reestablished redox and ion homeostasis, the latter of which are confirmed by the alleviation of reactive oxygen species overproduction, the decreases in thiobarbituric acid reactive substances production, and Na⁺/K⁺ ratio. Consistently, the related antioxidant defense genes, sodium hydrogen exchanger (NHX1), and salt overly sensitive 2 (SOS2) transcripts are modulated. The involvement S-nitrosylation, a redox-based posttranslational modification triggered by NO, is suggested. Further results show that in response to NaCl stress, the increased NO levels are strengthened by the addition of melatonin in seedling roots. Above responses are abolished by the removal of NO by NO scavenger. We further discover that the removal of NO does not alter endogenous melatonin content in roots supplemented with NaCl alone or together with melatonin, thus excluding the possibility of NO-triggered melatonin production. Genetic evidence reveals that, compared with wild-type Arabidopsis, the hypersensitivity to NaCl in nia1/2 and noa1 mutants (exhibiting null nitrate reductase activity and indirectly reduced endogenous NO level, respectively) cannot be rescued by melatonin supplementation. The reestablishment of redox homeostasis and induction of SOS signaling are not observed. In summary, above pharmacological, molecular, and genetic data conclude that NO operates downstream of melatonin promoting salinity tolerance.

Histological, Physiological, and Comparative Proteomic Analyses Provide Insights into Leaf Rolling in Brassica napus.

Moderate leaf rolling is important in ideotype breeding, as it improves photosynthetic efficiency and therefore increases crop yields. To understand the regulatory network of leaf rolling in Brassica napus, a down-curved leaf mutant ( Bndcl1) has been investigated. Physiological analyses indicated that the chlorophyll contents and antioxidant enzyme activities were remarkably increased and the photosynthetic performance was significantly improved in Bndcl1. Consistent with these findings, 943 differentially accumulated proteins (DAPs) were identified in the Bndcl1 mutant and its wild-type plants using iTRAQ-based comparative proteomic analyses. Enrichment analysis of proteins with higher abundance in Bndcl1 revealed that the functional category "photosynthesis" was significantly overrepresented. Moreover, proteins associated with oxidative stress response and photosystem II repairing were also up-accumulated in Bndcl1, which might help the mutant to sustain the photosynthetic efficiency under unfavorable conditions. Histological observation showed that the mutant displayed defects in adaxial-abaxial patterning. Important DAPs associated with leaf polarity establishment were detected in Bndcl1, including ribosomal proteins, proteins involved in post-transcriptional gene silencing, and proteins related to brassinosteroid. Together, our findings may help clarify the mechanisms underlying leaf rolling and its physiological effects on plants and may facilitate ideotype breeding in Brassica napus.

Quantitative Trait Transcripts Mapping Coupled with Expression Quantitative Trait Loci Mapping Reveal the Molecular Network Regulating the Apetalous Characteristic in Brassica napus L.

The apetalous trait of rapeseed (Brassica napus, AACC, 2n = 38) is important for breeding an ideal high-yield rapeseed with superior klendusity to Sclerotinia sclerotiorum. Currently, the molecular mechanism underlying the apetalous trait of rapeseed is unclear. In this study, 14 petal regulators genes were chosen as target genes (TGs), and the expression patterns of the 14 TGs in the AH population, containing 189 recombinant inbred lines derived from a cross between apetalous "APL01" and normal "Holly," were analyzed in two environments using qRT-PCR. Phenotypic data of petalous degree (PDgr) in the AH population were obtained from the two environments. Both quantitative trait transcript (QTT)-association mapping and expression QTL (eQTL) analyses of TGs expression levels were performed to reveal regulatory relationships among TGs and PDgr. QTT mapping for PDgr determined that PLURIPETALA (PLP) was the major negative QTT associated with PDgr in both environments, suggesting that PLP negatively regulates the petal development of line "APL01." The QTT mapping of PLP expression levels showed that CHROMATIN-REMODELING PROTEIN 11 (CHR11) was positively associated with PLP expression, indicating that CHR11 acts as a positive regulator of PLP expression. Similarly, QTT mapping for the remaining TGs identified 38 QTTs, associated with 13 TGs, and 31 QTTs, associated with 10 TGs, respectively, in the first and second environments. Additionally, eQTL analyses of TG expression levels showed that 12 and 11 unconditional eQTLs were detected in the first and second environment, respectively. Based on the QTTs and unconditional eQTLs detected, we presented a hypothetical molecular regulatory network in which 14 petal regulators potentially regulated the apetalous trait in "APL01" through the CHR11-PLP pathway. PLP acts directly as the terminal signal integrator negatively regulating petal development in the CHR11-PLP pathway. These findings will aid in the understanding the molecular mechanism underlying the apetalous trait of rapeseed.

An innovative procedure of genome-wide association analysis fits studies on germplasm population and plant breeding.

KEY MESSAGE: The innovative RTM-GWAS procedure provides a relatively thorough detection of QTL and their multiple alleles for germplasm population characterization, gene network identification, and genomic selection strategy innovation in plant breeding. The previous genome-wide association studies (GWAS) have been concentrated on finding a handful of major quantitative trait loci (QTL), but plant breeders are interested in revealing the whole-genome QTL-allele constitution in breeding materials/germplasm (in which tremendous historical allelic variation has been accumulated) for genome-wide improvement. To match this requirement, two innovations were suggested for GWAS: first grouping tightly linked sequential SNPs into linkage disequilibrium blocks (SNPLDBs) to form markers with multi-allelic haplotypes, and second utilizing two-stage association analysis for QTL identification, where the markers were preselected by single-locus model followed by multi-locus multi-allele model stepwise regression. Our proposed GWAS procedure is characterized as a novel restricted two-stage multi-locus multi-allele GWAS (RTM-GWAS, https://github.com/njau-sri/rtm-gwas ). The Chinese soybean germplasm population (CSGP) composed of 1024 accessions with 36,952 SNPLDBs (generated from 145,558 SNPs, with reduced linkage disequilibrium decay distance) was used to demonstrate the power and efficiency of RTM-GWAS. Using the CSGP marker information, simulation studies demonstrated that RTM-GWAS achieved the highest QTL detection power and efficiency compared with the previous procedures, especially under large sample size and high trait heritability conditions. A relatively thorough detection of QTL with their multiple alleles was achieved by RTM-GWAS compared with the linear mixed model method on 100-seed weight in CSGP. A QTL-allele matrix (402 alleles of 139 QTL × 1024 accessions) was established as a compact form of the population genetic constitution. The 100-seed weight QTL-allele matrix was used for genetic characterization, candidate gene prediction, and genomic selection for optimal crosses in the germplasm population.

Mapping a major QTL responsible for dwarf architecture in Brassica napus using a single-nucleotide polymorphism marker approach.

BACKGROUND: Key genes related to plant type traits have played very important roles in the "green revolution" by increasing lodging resistance and elevating the harvest indices of crop cultivars. Although there have been numerous achievements in the development of dwarfism and plant type in Brassica napus breeding, exploring new materials conferring oilseed rape with efficient plant types that provide higher yields is still of significance in breeding, as well as in elucidating the mechanisms underlying plant development. Here, we report a new dwarf architecture with down-curved leaf mutant (Bndwf/dcl1) isolated from an ethyl methanesulphonate (EMS)-mutagenized B. napus line, together with its inheritance and gene mapping, and pleiotropic effects of the mapped locus on plant-type traits. RESULTS: We constructed a high-density single-nucleotide polymorphism (SNP) map using a backcross population derived from the Bndwf/dcl1 mutant and the canola cultivar 'zhongshuang11' ('ZS11') and mapped the dwarf architecture with the down-curved leaf dominant locus, BnDWF/DCL1, in a 6.58-cM interval between SNP marker bins M46180 and M49962 on the linkage group (LG) C05 of B. napus. Further mapping with other materials derived from Bndwf/dcl1 narrowed the interval harbouring BnDWF/DCL1 to 175 kb in length and this interval contained 16 annotated genes. Quantitative trait locus (QTL) mappings with the backcross population for plant type traits, including plant height, branching height, main raceme length and average branching interval, indicated that the mapped QTLs for plant type traits were located at the same position as the BnDWF/DCL1 locus. CONCLUSIONS: This study suggests that the BnDWF/DCL1 locus is a major pleiotropic locus/QTL in B. napus, which may reduce plant height, alter plant type traits and change leaf shape, and thus may lead to compact plant architecture. Accordingly, this locus may have substantial breeding potential for increasing planting density.