Heterogeneous expression of stearoyl-acyl carrier protein desaturase genes SAD1 and SAD2 from Linum usitatissimum enhances seed oleic acid accumulation and seedling cold and drought tolerance in Brassica napus

Flax(Linum usitatissimum L.)is a versatile crop and its seeds are a major source of unsaturated fatty acids.Stearoyl-acyl carrier protein desaturase(SAD)is a dehydrogenase enzyme that plays a key role in oleic acid biosynthesis as well as responses to biotic and abiotic stresses.However,the function of SAD orthologs from L.usitatissimum has not been assessed.Here,we found that two LuSAD genes,LuSAD1 and LuSAD2,are present in the genome of L.usitatissimum cultivar‘Longya 10’.Heterogeneous expression of either LuSAD1 or LuSAD2 in Arabidopsis thaliana resulted in higher contents of total fatty acids and oleic acid in the seeds.Interestingly,ectopic expression of LuSAD2 in A.thaliana caused altered plant architecture.Similarly,the overexpression of either LuSAD1 or LuSAD2 in Brassica napus also resulted in increased contents of total fatty acids and oleic acid in the seeds.Furthermore,we demonstrated that either LuSAD1 or LuSAD2 enhances seedling resistance to cold and drought stresses by improving antioxidant enzyme activity and nonenzymatic antioxidant levels,as well as reducing membrane damage.These findings not only broaden our knowledge of the LuSAD functions in plants,but also offer promising targets for improving the quantity and quality of oil,and the abiotic stress tolerance of oil-producing crops,through molecular manipulation.

Knock-out of BnHva22c reduces the susceptibility of Brassica napus to infection with the fungal pathogen Verticillium longisporum

Verticillium longisporum(Vl43)is a soilborne hemibiotrophic fungal pathogen causing stem striping on oilseed rape(OSR)and severe yield losses.Breeding for resistant varieties is the most promising approach to control this disease.Here,we report the identification of Hva22c as a novel susceptibility factor and its potential for improving OSR resistance.Hva22c is a member of the Hva22 gene family,originally described for barley(Hordeum vulgare).Several Hva22 members have been located at the endoplasmic reticulum.Hva22c is up-regulated in response to Vl43 in both Arabidopsis and OSR.We demonstrate that knock-out of Hva22c in OSR by CRISPR/Cas9 and its homolog in Arabidopsis by T-DNA insertion reduced plants’susceptibility to Vl43 infection and impaired the development of disease symptoms.To understand the underlying mechanism,we analysed transcriptomic data from infected and non-infected roots of hva22c knock-out and wild type plants.We identified a homozygous mutant with frame-shifts in all four BnHva22c loci displaying a vastly altered transcriptional landscape at 6 dpi.Significantly,a large set of genes was suppressed under mock conditions including genes related to the endomembrane systems.Among the up-regulated genes we found several defense-related and phytohormone-responsive genes when comparing mutant to the wild type.These results demonstrate that Hva22c is functionally required for a fully compatible plant-fungus interaction.Its loss of function reduces plant susceptibility,most likely due to endoplasmatic reticulum and Golgi dysfunction accompanied by additionally activated defense responses.These findings can help improve OSR resistance to V.longisporum infection.

BnaC03.BIN2 regulates plant height by affecting the main inflorescence length and first effective branch height in Brassica napus L.

Rapeseed(Brassica napus L.)is one of the main oil crops in the world,and increasing its yield is of great significance for ensuring the safety of edible oil.Presently,improving rapeseed plant architecture is an effective way to increase rapeseed yield with higher planting density.However,the regulatory mechanism of rapeseed plant architecture is poorly understood.In this study,a dwarf rapeseed mutant dwarf08(df08)is obtained by ethyl methane sulfonate(EMS)-mutagenesis.The decrease in plant height of df08 is mainly caused by the reduction in main inflorescence length and first effective branch height and controlled by a single semi-dominant gene.The hybrid plants(F1)show a semi-dwarf phenotype.Through map-based cloning and transgenic assay,we confirm that the nonsynonymous single nucleotide variant(SNV)(C to T)in BnaC03.BIN2,which is homologous with Arabidopsis(Arabidopsis thaliana)BIN2,is responsible for the dwarfism of df08.BnaC03.BIN2 interacts with BnaBZR1/BES1 and involves in brassinosteroids(BRs)signal transduction.Proline to Leucine substitution in 284(P284L)enhances the protein stability of BnaC03.bin2-D,disrupts BRs signal transduction and affects the expression of genes regulating cell division,leading to dwarfism of df08.This study provides a new insight for the mechanism of rapeseed plant height regulation and creates an elite germplasm that can be used for genetic improvement of rapeseed architecture.

BnaWRKY75 positively regulates the resistance against Sclerotinia sclerotiorum in ornamental Brassica napus

With the development of tourism at home and abroad,Rapeseed(Brassica napus)has become an important ornamental plant.However,its ornamental value at the inflorescence stage is greatly reduced by Sclerotinia sclerotiorum.Identification of important genes in the defense responses is critical for molecular breeding,which is an important strategy for controlling the disease.In this study,we isolated a B.napus WRKY transcription factor gene,BnaWRKY75.BnaWRKY75 was found to encode a nucleus-localized protein and exhibited relatively high expression in the stems.Arabidopsis thaliana transgenic plants expressing BnaWRKY75 showed enhanced resistance to S.sclerotiorum,and both ProBnaWRKY75:GUS and gene expression analyses showed that BnaWRKY75 was highly responsive to S.sclerotiorum infection,indicating the involvement of BnaWRKY75 in response to this infection.Furthermore,overexpression(OE)of BnaWRKY75 in B.napus significantly enhanced the resistance to S.sclerotiorum,whereas the resistance was reduced in RNAi transgenic B.napus plants.Moreover,the BnaWRKY75-OE B.napus plants exhibited constitutive activation of salicylic acid-,jasmonic acid-,and ethylene-mediated defense responses and the inhibition of both H_(2)O_(2)and O_(2)·^(-)accumulation in response to this pathogen.By contrast,BnaWRKY75-RNAi plants showed a reverse pattern,suggesting that BnaWRKY75 is involved in hormonal signaling pathways and in the control of reactive oxygen species accumulation.In conclusion,these data indicate that BnaWRKY75,a regulator of multiple defense responses,positively regulates resistance against S.sclerotiorum,which may guide the improvement of resistance in rapeseed.

Landscape of Sequence Variations in Homologous Copies of FAD2 and FAD3 in Rapeseed(Brassica napus L.)Germplasm with High/Low Linolenic Acid Trait

Genetic manipulation(either restraint or enhancement)of the biosynthesis pathway ofα-linolenic acid(ALA)in seed oil is an important goal in Brassica napus breeding.B.napus is a tetraploid plant whose genome often har-bors four and six homologous copies,respectively,of the two fatty acid desaturases FAD2 and FAD3,which con-trol the last two steps of ALA biosynthesis during seed oil accumulation.In this study,we compared their promoters,coding sequences,and expression levels in three high-ALA inbred lines 2006L,R8Q10,and YH25005,a low-ALA line A28,a low-ALA/high-oleic-acid accession SW,and the wildtype ZS11.The expression levels of most FAD2 and FAD3 homologs in the three high-ALA accessions were higher than those in ZS11 and much higher than those in A28 and SW.The three high-ALA accessions shared similar sequences with the pro-moters and CDSs of BnFAD3.C4 and BnFAD3.A3.In A28 and SW,substitution of three amino acid residues in BnFAD2.A5 and BnFAD2.C5,an absence of BnFAD2.C1 locus,and a 549 bp long deletion on the BnFAD3.A3 promoter were detected.The profile of BnFAD2 mutation in the two low-ALA accessions A28 and SW is different from that reported in previous studies.The mutations in BnFAD3 in the high-ALA accessions are reported for thefirst time.In identifying the sites of these mutations,we provide detailed information to aid the design of mole-cular markers for accelerated breeding schemes.

The influence of Lactobacillus plantarum fermentation in selenium-enriched Brassica napus L.:changes in the nutritional constituents,bioactivities and bioaccessibility

Selenium(Se)-enriched Brassica napus L.is a valuable organic Se supplement.In this study,the fermentation broth enriched with organic Se(FFS)was prepared using Lactobacillus plantarum to ferment the substrate of Se-enriched Brassica napus L.Significant increases were observed after fermentation in total sugars,reducing sugars,soluble proteins,total phenolic content(TPC),and total flavonoid content(TFC).The organic Se was retained at a concentration of 54.75 mg/g in the freeze-dried sample.Principal component analysis and cluster analysis showed good separation between the FFS and unfermented(FS)groups.Fragrant 2-ethyloxetane had the highest content among all volatiles,while sinapine had the highest content among all phenolic compounds.The fermentation process showed remarkable improvement in the abundance and concentration of volatile compounds and phenolic contents,making FFS exhibit strong antioxidant activity and inhibitory capacity againstα-glucosidase activity.The bioaccessibility of phenolic compounds was significantly greater in FFS compared to FS.ADMET analysis revealed that the majority of phenolic compounds contained in FFS did not exhibit mutagenicity toxicity,hepatotoxicity,skin sensitization,or blood-brain barrier penetration,indicating a favorable level of biosafety.Overall,our study provides a new insight into the further utilization of Se-enriched Brassica napus L.in foods.

Dissecting the genetic architecture of glucosinolate compounds for quality improvement in flowering stalk tissues of Brassica napus

Glucosinolates(GSLs) and their hydrolytic products contribute to the quality traits of rapeseed flowering stalk tissues, such as taste, flavor and anticarcinogenic properties(Glucoraphanin). However, little is known about the genetic mechanisms of GSL accumulation in rapeseed flowering stalks. In this study, the variation and genetic architecture of GSL metabolites in flowering stalk tissues were investigated for the first time among a panel of 107 accessions. All GSL compounds exhibited continuous and wide variations in the present population. Progoitrin,glucobrassicanapin and gluconapin were the most abundant GSL compounds. Five quantitative trait loci(QTL) significantly associated with three GSL compounds were identified by genome-wide association study. GRA_C04 was under selected during modern breeding, in which the ratio of lower GSL haplotype(HAP2) in the accessions bred before 1990(52.56%) was significantly lower than that after 1990(78.95%). Four candidate genes, BnaA01. SOT16, BnaA06. SOT17, Bna A06. MYB51a, and Bna A06. MYB51b, were identified in the GTL_A01 and 4OH_A06 regions.These findings provide new insights into GSL biosynthesis in flowering stalk tissues and facilitate quality improvement in rapeseed flowering stalks.

BnaUBP15s positively regulates seed size and seed weight in Brassica napus

Brassica napus(B.napus)is a globally significant oilseed crop,making a substantial contribution to both human oil and livestock feed production.Enhancing seed weight is crucial for improving rapeseed yield;however,only a limited number of seed weight-related genes have been functionally validated in B.napus thus far.UBIQUITINSPECIFIC PROTEASE 15(UBP15)belongs to the ubiquitin protease pathway and plays a maternal role in prolonging seed development in Arabidopsis.The potential utilization of UBP15 for enhancing seed yield in B.napus has remained unexplored until now.In this study,we identified the orthologs of UBP15 in B.napus and investigated its functionality using the CRISPR-Cas9 system.We generated mutant plants with multiple editing types targeting Bnaubp15s and successfully isolated T-DNA-free homozygous mutant lines that exhibited edits across four homologs of BnaUBP15 in T2 generation plants.Our preliminary data demonstrated that mutation of BnaUBP15s significantly reduced seed size,seed weight,and plant height while noticeably increasing the number of primary branches.These findings not only provide crucial evidence for further elucidating the molecular mechanism underlying the regulation of seed weight and size by BnaUBP15s but also offer promising novel germplasm for enhancing plant architecture.

First report of Leptosphaeria sclerotioides on Brassica napus in Northwestern China

In 2020,diseased seedlings of winter oilseed rape(Brassica napus)with cankered taproots as well as abundant sclerotium-like structures in the soil surrounding the roots were found in Longxi County of Gansu province of northwestern China.A fungus with production of pycnidia was isolated from the diseased roots,and it was identified based on morphological characteristics,molecular phylogeny(ITS,LSU)and PCR detection with the specific primers.The fungus was identified as Leptosphaeria sclerotioides Gruyter,Aveskamp&Verkley[anamorph:Phoma sclerotioides(Preuss)ex Sacc.].Re-inoculation of isolates of P9 and P10 of L.sclerotioides on winter oilseed rape(B.napus cultivar‘Zhongshuang No.9’)in Wuhan caused formation of abundant sclerotium-like structures in soil surrounding the roots,but failed to produce root cankers as those observed in Gansu possibly due to lack of long peroid of low-temperature conditioning in Wuhan.In spite of this,plant height,pod number and seed yield of oilseed rape were significantly reduced in the treatment with L.sclerotioides P9 and P10,compared to the control treatment.To the best of our knowledge,this is the first report of L.sclerotioides on B.napus in China and the finding broadened our understanding about the natural distribution of this psychrophilic fungus.

BnbHLH92a negatively regulates anthocyanin and proanthocyanidin biosynthesis in Brassica napus

Yellow seed trait is a desirable characteristic with potential for increasing seed quality and commercial value in rapeseed,and anthocyanin and proanthocyanidins(PAs)are major seed-coat pigments.Few transcription factors involved in the regulation of anthocyanin and PAs biosynthesis have been characterized in rapeseed.In this study,we identified a transcription factor gene BnbHLH92a(BnaA06T0441000ZS)in rapeseed.Overexpressing BnbHLH92a both in Arabidopsis and in rapeseed reduced levels of anthocyanin and PAs.Correspondingly,the expression profiles of anthocyanin and PA biosynthesis genes(TT3,BAN,TT8,TT18,and TTG1)were shown by quantitative real-time PCR to be inhibited in BnbHLH92a-overexpressing Arabidopsis seeds,indicating that BnbHLH92a represses the anthocyanin and PA biosynthesis pathway in Arabidopsis.BnbHLH92a physically interacts with the BnTTG1 protein and represses the biosynthesis of anthocyanins and PAs in rapeseed.BnbHLH92a also binds directly to the BnTT18 promoter and represses its expression.These results suggest that BnbHLH92a is a novel upstream regulator of flavonoid biosynthesis in B.napus.

Genome-wide recombination variation in biparental segregating and reciprocal backcross populations provides information for introgression breeding in Brassica napus

Variation in patterns of recombination in plant genomes provides information about species evolution,genetic diversity and crop improvement. We investigated meiotic crossovers generated in biparental segregating and reciprocal backcross populations of the allopolyploid genome of rapeseed(Brassica napus)(AACC, 2n = 38). A structured set of 1445 intercrossed lines was derived from two homozygous de novo genome-assembled parents that represented the major genetic clusters of semi-winter Chinese and winter European rapeseeds, and was used to increase QTL resolution and achieve genomic reciprocal introgression. A high-density genetic map constructed with 6161 genetic bins and anchored centromere regions was used to establish the pattern of recombination variation in each chromosome. Around 93%of the genome contained crossovers at a mean rate of 3.8 c M Mb^(-1), with the remaining 7% attributed to centromeres or low marker density. Recombination hotspots predominated in the A genome, including two-thirds of those associated with breeding introgression from B. rapa. Genetic background might affect recombination variation. Introgression of genetic diversity from European winter to Chinese semi-winter rapeseed showed an increase in crossover rate under the semi-winter environment. Evidence for an elevated recombination rate having historically contributed to selective trait improvement includes accumulation of favorable alleles for seed oil content on hotspots of chromosome A10. Conversely, strong artificial selection may affect recombination rate variation, as appears to be the case with a coldspot resulting from strong selection for glucosinolate alleles on A09. But the cold region would be promptly reactivated by crossing design indicated by the pedigree analysis. Knowledge of recombination hotspots and coldspots associated with QTL for 22 traits can guide selection strategies for introgression breeding between the two gene pools. These results and rich genomic resources broaden our understanding of recombination behavior in allopolyploids and may advance rapeseed genetic improvement.

BnaSD.C3 is a novel major quantitative trait locus affecting semi-dwarf architecture in Brassica napus L.

Plant height is a key plant architectural trait that affects the seed yield,harvest index and lodging resistance in Brassica napus L.,although the genetic mechanisms affecting plant height remain unclear.Here,a semi-dwarf mutant,df34,was obtained by ethyl methanesulphonate-induced mutagenesis.Genetic analysis showed that the semi-dwarf phenotype is controlled by one semi-dominant gene,which was located on chromosome C03 using a bulked segregant analysis coupled with whole-genome sequencing,and this gene was named BnaSD.C3.Then BnaSD.C3 was fine-mapped to a 297.35-kb segment of the“Darmor-bzh”genome,but there was no potential candidate gene for the semi-dwarf trait underlying this interval.Furthermore,the interval was aligned to the Zhongshuang 11 reference genome.Finally,combining structural variation analysis,transcriptome sequencing,phytohormone analyses and gene annotation information,BnaC03G0466900ZS and BnaC03G0478900ZS were determined to be the most likely candidate genes affecting the plant height of df34.This study provides a novel major locus for breeding and new insights into the genetic architecture of plant height in B.napus.

Identification of a candidate QTG for seed number per silique by integrating QTL mapping and RNA-seq in Brassica napus L.

Seed number per silique(SNPS)is one of seed yield components in rapeseed,but its genetic mechanism remains elusive.Here a double haploid(DH)population derived from a hybrid between female 6Q006with 35–40 SNPS and male 6W26 with 10–15 SNPS was investigated for SNPS in the year 2017,2018,2019 and 2021,and genotyped with Brassica 60K Illumina Infinium SNP array.An overlapping major QTL(qSNPS.C09)explaining 51.50%of phenotypic variance on average was narrowed to a 0.90 Mb region from 44.87 Mb to 45.77 Mb on chromosome C09 by BSA-seq.Subsequently,two DEGs in this interval were detected between extreme individuals in DH and F_2populations by transcriptome sequencing at7 and 14 days after pollination siliques.Of which,BnaC09g45400D encoded an adenine phosphoribosyltransferase 5(APT5)has a 48-bp InDel variation in the promoter of two parents.Candidate gene association analysis showed that this InDel variation was associated with SNPS in a nature population of rapeseed,where 54 accessions carrying the same haplotype as parent 6Q006 had higher SNPS than103 accessions carrying the same haplotype as parent 6W26.Collectively,the findings are helpful for rapeseed molecular breeding of SNPS,and provide new insight into the genetic and molecular mechanism of SNPS in rapeseed.

Detection of new candidate genes controlling seed weight by integrating gene coexpression analysis and QTL mapping in Brassica napus L.

Seed weight is a component of seed yield in rapeseed(Brassica napus L.).Although quantitative trait loci(QTL)for seed weight have been reported in rapeseed,only a few causal quantitative trait genes(QTGs)have been identified,resulting in a limitation in understanding of seed weight regulation.We constructed a gene coexpression network at the early seed developmental stage using transcripts of 20,408 genes in QTL intervals and 1017 rapeseed homologs of known genes from other species.Among the 10 modules in this gene coexpression network,modules 1 and 2 were core modules and contained genes involved in source–flow–sink processes such as synthesis and transportation of fatty acid and protein,and photosynthesis.A hub gene SERINE CARBOXYPEPTIDASE-LIKE 19(SCPL19)was identified by candidate gene association analysis in rapeseed and functionally investigated using Arabidopsis T-DNA mutant and overexpression lines.Our study demonstrates the power of gene coexpression analysis to prioritize candidate genes from large candidate QTG sets and enhances the understanding of molecular mechanism for seed weight at the early developmental stage in rapeseed.

Biotechnology ofα-linolenic acid in oilseed rape(Brassica napus)using FAD2 and FAD3 from chia(Salvia hispanica)

α-Linolenic acid(ALA,18:3Δ9,12,15)is an essential fatty acid for humans since it is the precursor for the biosynthesis of omega-3 long-chain polyunsaturated fatty acids(LC-PUFA).Modern people generally suffer from deficiency of ALA because most staple food oils are low or lack ALA content.Biotechnological enrichment of ALA in staple oil crops is a promising strategy.Chia(Salvia hispanica)has the highest ALA content in its seed oil among known oil crops.In this study,the FAD2 and FAD3 genes from chia were engineered into a staple oil crop,oilseed rape(Brassica napus),via Agrobaterium tumefaciens-mediated transformation of their LP4-2A fusion gene construct driven by the seed-specific promoter P_(NapA).In seeds of T0,T1,and T2 lines,the average ALA contents were 20.86,23.54,and 24.92%,respectively,which were 2.21,2.68,and 3.03 folds of the non-transformed controls(9.42,8.78,and 8.22%),respectively.The highest seed ALA levels of T0,T1,and T2 plants were 38.41,35.98,and 39.19%respectively,which were 4.10-4.77 folds of the respective controls.FA-pathway enzyme genes(BnACCD,BnFATA,BnSAD,BnSCD,BnDGAT1,BnDGAT2,and BnDGAT3)and positive regulatory genes(BnWRI1,BnLEC1,BnL1L,BnLEC2,BnABI3,BnbZIP67,and BnMYB96)were all significantly up-regulated.In contrast,BnTT1,BnTT2,BnTT8,BnTT16,BnTTG1,and BnTTG2,encoding negative oil accumulation regulators but positive secondary metabolism regulators,were all significantly down-regulated.This means the foreign ShFAD2-ShFAD3 fusion gene,directly and indirectly,remodeled both positive and negative loci of the whole FA-related network in transgenic B.napus seeds.

Identification and characterization of a curly-leaf locus CL1 encoding an IAA2 protein in Brassica napus

The leaf is the main organ for rapeseed photosynthesis,and its morphology influences photosynthetic efficiency and supports increased planting density and yield.However,the molecular regulatory mechanism of leaf morphology in Brassica napus is poorly understood,restricting progress in breeding for the trait.We describe a novel dominant mutation,curly leaf 1(cl1),which confers uneven dorsal–ventral axis development,irregular cellular structure and influenced gravitropic response in the seedling stage.The CL1 locus was mapped to a 1.573-Mb interval on chromosome A05 using simple sequence repeat(SSR)markers,and co-segregated with the phenotype of plants in the curly F2 population.A substitution(P62S)was identified in the highly conserved degron motif(GWSPV)of the IAA2 protein in the cl1mutant,and the P62S substitution impaired the interaction between IAA2 and TIR1 in the presence of auxin,influencing auxin signaling.The P62S substitution-induced curly leaf phenotype was verified by ectopic expression of Bna A05.iaa2 in Arabidopsis and B.napus.Our findings explain the function of IAA2 in rapeseed,providing a foundation for future investigation of auxin signaling and the mechanisms underlying leaf development in B.napus.

Global and Comparative Proteome Analysis of Nitrogen-Stress Responsive Proteins in the Root, Stem and Leaf of Brassica napus

Nitrogen(N)is one of the basic nutrients and signals for plant development and deficiency of it would always limit the productions of crops in the field.Quantitative research on expression of N-stress responsive proteins on a proteome level remains elusive.In order to gain a deep insight into the proteins responding to nitrogen stress in rapeseed(Brassica napus L.),comparative proteomic analysis was performed to investigate changes of protein expression profiles from the root,stem and leaf under different N concentrations,respectively.More than 200 differential abundance proteins(DAPs)were detected and categorized into groups according to annotations,including“binding and catalytic activity”,“involved in primary metabolism and cellular processes”,“stress-response”and so on.Variation in chlorophyll(Chl)content and antioxidant activities further revealed that oxidative stress raised with the increase of N concentration.Bioinformatics analysis based on the expression level of total proteins suggested these DAPs might play important roles in adaptation to N-stress conditions.Generally,these results provides a new aspect into N-stress responding proteins in Brassica plants.

Proteome-wide identification of S-sulfenylated cysteines response to salt stress in Brassica napus root

Reactive oxygen species(ROS)play a key role in a variety of biological processes,such as the perception of abiotic stress,the integration of different environmental signals,and the activation of stress response networks.Salt stress could induce an increased ROS accumulation in plants,disrupting intracellular redox homeostasis,leading to posttranslational modifications(PTMs)of specific proteins,and eventually causing adaptive changes in metabolism.Here,we performed an iodoTMT-based proteomic approach to identify the sulfenylated proteins in B.napus root responsing to salt stress.Totally,1348 sulfenylated sites in 751 proteins were identified and these proteins were widely existed in different cell compartments and processes.Our study revealed that proteins with changed abundance and sulfenylation level in B.napus root under salt stress were mainly enriched in the biological processes of ion binding,glycolysis,ATP binding,and oxidative stress response.This study displays a landscape of sulfenylated proteins response to salt stress in B.napus root and provides some theoretical support for further understanding of the molecular mechanisms of redox regulation under salt stress in plants.

Effects of Heat Stress during Seed Filling Stage on Brassica napus Seed Oil Accumulation and Chlorophyll Fluorescence Characteristics

As global temperature rise,the threat of heat stress to rapeseed production is becoming more obvious.Exploring the response characteristics of two important biological pathways,oil accumulation and photosynthesis,to heat stress during B.napus seed filling is helpful in the genetic improvement of heat-tolerant rapeseed.The effects of heat stress on seed oil accumulation and chlorophyll fluorescence characteristics of 29 B.napus germplasms with different oil content and environmental sensitivity,including 6 rapeseed varieties which exhibited environmentsensitive/insensitive and with high,medium or low oil content,were tested by whole plant heat stress or the in vitro silique culture system.Both assay exhibited similar trend on oil content of the rapeseed germplasms.The heat effect on the chlorophyll fluorescence kinetic parameters F_(v)/F_(m),ETR and Y(Ⅱ)were also consistent.Heat stress significantly decreased oil content,although there was abundant genetic variation on heat tolerance among the genotypes.Correlation analysis showed that the decrease rate of F_(v)/F_(m) of silique heat-stressed B.napus developing seed was positive correlative to the decrease rate of mature seed oil content of the whole plant heat-stressed rapeseed(R=0.9214,P-value<0.01).Overall,the results indicated that heat stress inhibited oil accumulation and photosynthesis in B.napus developing seed.The decrease rate of chlorophyll fluorescence parameter F_(v)/F_(m) of heat-stressed developing seed could be used as the index of heat tolerant rapeseed identification.Further,two heat insensitive rapeseed varieties with high oil content were identified.

Genome-Wide Identification, Evolution and Expression Analyses of GA2ox Gene Family in Brassica napus L.

Gibberellin 2-oxidases(GA2ox)are important enzymes that maintain the balance of bioactive GAs in plants.GA2ox genes have been identified and characterized in many plants,but these genes were not investigated in Brassica napus.Here,we identified 31 GA2ox genes in B.napus and 15 of these BnaGA2ox genes were distributed in the A and C subgenomes.Subcellular localization predictions suggested that all BnaGA2ox proteins were localized in the cytoplasm,and gene structure analysis showed that the BnaGA2ox genes contained 2–4 exons.Phylogenetic analysis indicated that BnGA2ox family proteins in monocotyledons and dicotyledons can be divided into four groups,including two C_(19)-GA2ox and two C_(20)-GA2ox clades.Group 4 is a C_(20)-GA2ox Class discovered recently.Most BnaGA2ox genes had a syntenic relationship with AtGA2ox genes.BnaGA2ox genes in the C subgenome had experienced stronger selection pressure than genes in the A subgenome.BnaGA2ox genes were highly expressed in specific tissues such as those involved in growth and development,and most of them were mainly involved in abiotic responses,regulation of phytohormones and growth and development.Our study provided a valuable evolutionary analysis of GA2ox genes in monocotyledons and dicotyledons,as well as an insight into the biological functions of GA2ox family genes in B.napus.