Molecular mechanism of flowering time regulation in Brassica rapa:similarities and differences with Arabidopsis

Properly regulated flowering time is pivotal for successful plant reproduction.The floral transition from vegetative growth to reproductive growth is regulated by a complex gene regulatory network that integrates environmental signals and internal conditions to ensure that flowering takes place under favorable conditions.Brassica rapa is a diploid Cruciferae species that includes several varieties that are cultivated as vegetable or oil crops.Flowering time is one of the most important agricultural traits of B.rapa crops because of its influence on yield and quality.The transition to flowering in B.rapa is regulated by several environmental and developmental cues,which are perceived by several signaling pathways,including the vernalization pathway,the autonomous pathway,the circadian clock,the thermosensory pathway,and gibberellin(GA)signaling.These signals are integrated to control the expression of floral integrators BrFTs and BrSOC1s to regulate flowering.In this review,we summarized current research advances on the molecular mechanisms that govern flowering time regulation in B.rapa and compare this to what is known in Arabidopsis.

Editing of eIF(iso)4E.c confers resistance against Turnip mosaic virus in Brassica rapa

Turnip mosaic virus(TuMV)constitutes one of the primary diseases affecting Brassica rapa,severely impacting its production and resulting in crop failures in various regions worldwide.Recent research has demonstrated the significance of plant translation initiation factors,specifically the eIF4E and eIF4G family genes,as essential recessive disease resistance genes.In our study,we conducted evolutionary and gene expression studies,leading us to identify e IF(iso)4E.c as a potential TuMV-resistant gene.Leveraging CRISPR/Cas9 technology,we obtained mutant B.rapa plants with edited eIF(iso)4E.c gene.We confirmed eIF(iso)4E.c confers resistance against TuMV through phenotypic observations and virus content evaluations.Furthermore,we employed ribosome profiling assays on eif(iso)4e.c mutant seedlings to unravel the translation landscape in response to TuMV.Interestingly,we observed a moderate correlation between the fold changes in gene expression at the transcriptional and translational levels(R^(2)=0.729).Comparative analysis of ribosome profiling and RNA-seq data revealed that plant-pathogen interaction,and MAPK signaling pathway-plant pathways were involved in eIF(iso)4E.c-mediated TuMV resistance.Further analysis revealed that sequence features,coding sequence length,and normalized minimal free energy,influenced the translation efficiency of genes.Our study highlights that the loss of e IF(iso)4E.c can result in a highly intricate translation mechanism,acting synergistically with transcription to confer resistance against TuMV.

Comparison Between a Tetraploid Turnip and Its Diploid Progenitor(Brassica rapa L.):The Adaptation to Salinity Stress

Polyploidy is pursued in plant breeding programs due mainly to its ability to yield larger vegetative or reproductive organs. In controlled growth chamber experiments, a tetraploid turnip （cv. Aijiaohuang, 4n） and its diploid progenitor （cv. Aijiaohuang, 2n） were evaluated for their tolerance to salinity stress via investigations on a group of physiological parameters. The results indicate that the tetraploid turnip exhibit better adaptation to a high concentration salt medium （200 mmol L-1）, as evidenced by a less-affected germination rate and a healthier morphological appearance at the seedling stage. Furthermore, an extension of salinity stress up to a certain period of time at the 5-7-leaf stage shows differences between the tetraploid turnip and its diploid progenitor. The former had a higher K＋/Na＋ ratio in the roots, higher glutathione concentration and antioxidant activities in the leaves, and smaller reductions in photosynthetic capacity in terms of leaf chlorophyll content. Studies on the differences between an autopolyploid and its respective relative, from which the autopolyploid originated, in terms of their tolerance to salinity and/or other abiotic stresses, have remained rather limited. The comparison is interesting due to a homogenous genetic background.

Development and Characterization of Microsatellite Markers in Brassica rapa ssp.chinensis and Transferability Among Related Species

Simple sequence repeat （SSR） or microsatellite marker is a valuable tool for several purposes, such as mapping, fingerprinting, and breeding. In the present study, an intersimple sequence repeat （ISSR）-PCR technique was applied for developing SSR markers in non-heading Chinese cabbage （Brassica rapa）. A total of 190 SSRs were obtained. Among these, AG or CT （54.7%） was the most frequent repeat, followed by AC or GT （31.6%） of the microsatellites. The average number of the SSRs length array was 16 and 10 times, respectively. Based on the determined SSR sequences, 143 SSR primer pairs were designed to evaluate their transferabilities among the related species of Brassica. The number of alleles produced per marker averaged 2.91, and the polymorphism information content （PIC） value ranged from 0 to 0.863 with an average of 0.540. Monomorphism was observed in 16 primer pairs. The transferability percentage in CC genome was higher than in BB genome. More loci occurred in the BBCC genome. This result supported the hypothesis that BB genome was divergent from A and C genomes, and AA and CC genomes were relatively close. The polymorphic primers can be exploited for further evolution, fingerprinting, and variety identification.

Transformation of Chinese Cabbage(Brassica rapa L. ssp. pekinensis)by Agrobacterium Micro-Injection into Flower Bud

We obtained two lines of Chinese head cabbage(Brassica rapa L. ssp. pekinensis)selfed progenies containing both an anti-sense gene of BcpLH and a gene for resistance to kanamycin by micro-injecting buds of their primary transformants(T0)with Agrobacterium tumefaciens strain LBA4404. 31 positive plants resistant to kanamycien were recovered. Southern blot analysis confirmed the presence of T-DNA in two transgenic plants. One(DHZ-13-1)exhibits the characteristics of out-toward rosette and cauline leaves, and nested flower model in which secondary complete flower developed from the base of the primary ovary and the third flower from the ovary in the secondary flower, and so on, while another(DHZ-6-1)has no phenotype change. ABA and IAA affected the root growth of progeny of DHZ-13-1, but 6-BA was insensitive to hypocotyl growth during its seedling development.

Development of InDel Markers for Brassica rapa Based on a High-resolution Melting Curve

Brassica rapa is one of the most important leafy vegetable crops with large cultivated area in China.To increase the availability of DNA markers in B.rapa,we developed insertion-deletion(InDel)markers utilizing high-resolution melting(HRM)curve analysis.We designed primers for 252 InDels(≥3 bp)evenly distributed in the genome and tested gene polymorphisms with eight accessions.In total,208 markers were specifically amplified,and 148 InDels with polymorphism were genotyped successfully using HRM.We further analyzed the correlation with InDel size,GC number,and predicted the difference in Tm values(Tm)using 208 markers with specific amplification.We found that the success rate of InDel markers was correlated with the GC number of InDel and the predicted-Tm,but not clearly correlated with the length of InDel.When the GC number within InDel was≥8,the successful rate exceeded 90.0%.When the predicted-Tm reached 0.5°C,the success rate was greater than 90.0%,and when it was≥0.6°C,the rate climbed to 100.0%,indicating their role as the optimal parameter for successful development of an applicable InDel marker.The polymorphic InDel markers can be easily genotyped using HRM.They are of great value in genetic analysis,construction of linkage map,and molecular marker-assisted selection in B.rapa.

Genetic Diversity of European and Chinese Oilseed Brassica rapa Cultivars from Different Breeding Periods

The Brassica oilseed crops went through two major breeding bottlenecks during the introgression of genes for zero erucic acid and low glucosinolate content, respectively, which may lead to reduced genetic biodiversity of the crop. This study investigates the impact of these bottlenecks on the genetic diversity within and across European and Chinese winter B. rapa cultivars. We compared eight cultivars from Europe and China, representing three different seed qualities from three different breeding periods： （1） high erucic acid, high glucosinolates （＋＋）; （2） zero erucic acid, high glucosinolates（0＋）; （3） zero erucic acid, low glucosonolates （00, canola quality）. Diversity was estimated on 32 plants per cultivar, with 16 simple sequence repeat （SSR） markers covering each of the B. rapa linkage groups. The analysis of molecular variance （AMOVA） showed that genetic variations within cultivars, across cultivars and across regions （Europe and China） were significant, with about 60% of the total variation within cultivars. There was a slight, but non-significant loss in genetic diversity within cultivars when comparing the three breeding periods as indicated by effective number of alleles （2.39, 2.23, and 1.99 for breeding periods 1, 2, and 3, respectively）, Shannon information index （0.93, 0.90, 0.75）, and expected heterozygosity （0.51, 0.49, 0.42）. By cluster analysis （UPGMA dendrogram） and principal coordinate analysis, Chinese and European cultivars were clearly divided into two distinct groups. In conclusion, quality improvement did not significantly reduce the genetic diversity of European and Chinese B. rapa cultivars.

Molecular cloning and expression analysis of turnip (Brassica rapa var. rapa) sucrose transporter gene family

In higher plants, sugars （mainly sucrose） are produced by photosynthetically assimilated carbon in mesophyll cells of leaves and translocated to heterotrophic organs to ensure plant growth and devel- opment. Sucrose transporters, or sucrose carriers （SUCs）, play an important role in the long-distance transportation of sucrose from source organs to sink organs, thereby affecting crop yield and quality. The identification, characterization, and molecular function analysis of sucrose transporter genes have been reported for monocot and dicot plants. However, no relevant study has been reported on sucrose transporter genes in Brassica rapa vat. rapa, a cruciferous root crop used mainly as vegetables and fodder. We identified and cloned 12 sucrose transporter genes from turnips, named BrrSUC1.1 to BrrSUCB.2 according to the SUC gene sequences of B. rapa pekinensis. We constructed a phylogenetic tree and analyzed conserved motifs for all 12 sucrose transporter genes identified. Real-time quantitative poly- merase chain reaction was conducted to understand the expression levels of SUC genes in different tissues and developmental phases of the turnip. These findings add to our understanding of the genetics and physiology of sugar transport during taproot formation in turnips.

A Genetic Linkage Map of Brassica rapa Based on AFLP Markers

A F2 mapping population was developed by crossing a Chinese cabbage-pe-tsai variety CC156 and an oil type Rapid cycling RC144 which were different from each other in morphology, maturity, self-compatibility, plant height, etc. Using 244 AFLP markers a map was constructed containing 10 main linkage groups covering a total distance of 857 cM, corresponding to 3.5 cM per marker. Length of linkage groups varied from 43 to 125 cM and the number of AFLP markers linkage to each group ranged from 7 to 41.

Genome-wide identification,evolutionary selection,and genetic variation of DNA methylation-related genes in Brassica rapa and Brassica oleracea

DNA methylation plays an important role in plant growth and development,and in regulating the activity of transposable elements(TEs).Research on DNA methylation-related(DMR)genes has been reported in Arabidopsis,but little research on DMR genes has been reported in Brassica rapa and Brassica oleracea,the genomes of which exhibit significant differences in TE content.In this study,we identified 78 and 77 DMR genes in Brassica rapa and Brassica oleracea,respectively.Detailed analysis revealed that the numbers of DMR genes in different DMR pathways varied in B.rapa and B.oleracea.The evolutionary selection pressure of DMR genes in B.rapa and B.oleracea was compared,and the DMR genes showed differential evolution between these two species.The nucleotide diversity(π)and selective sweep(Tajima’s D)revealed footprints of selection in the B.rapa and B.oleracea populations.Transcriptome analysis showed that most DMR genes exhibited similar expression characteristics in B.rapa and B.oleracea.This study dissects the evolutionary differences and genetic variations of the DMR genes in B.rapa and B.oleracea,and will provide valuable resources for future research on the divergent evolution of DNA methylation between B.rapa and B.oleracea.

An optimized protocol for detecting guard cell specific gene expression by in situ RT-PCR in Brassica rapa

It is important to detect specific genes expressed in the guard cells,which control gas exchange and play key roles in response to drought and salt stresses.Due to the genetic transformation of Chinese cabbage(Brassica rapa)has not been well developed,in situ RT-PCR is a valuable option for detecting guard cell specific genes.We reported an optimized protocol of in situ RT-PCR by using an FAMA homologous gene Bra001929 in Brassica rapa.FAMA in Arabidopsis has been verified to be specially expressed in guard cells.We designed specific RT-PCR primers and optimized the protocol in terms of the(a)reverse transcription time,(b)blocking time,(c)antigen-antibody incubation time,and(d)washing temperature.Our approach provides a sensitive and effective in situ RT-PCR method for locating expression in the guard cells in Brassica rapa.Moreover,we proved the guard cell specific expression of Bra001929 in the epidermis indicating its’applicability as a marker gene for guard cells of Brassica rapa.

Inheritance Analysis of Bolting Associated Traits Using Mixed Major Gene Plus Polygene Model in Brassica rapa

[ Objective ] This study aimed to analyze the inheritance of bolting associated traits in Brassica rapa, which will provide useful information in a breeding program for late-bolting or bolting-resistant cultivars of Chinese cabbage. [ Method] Three phenotypic measurements, bolting index, flowering time, days to 5 cm elongated stalk, respectively were used for inheritance analysis of six generations, P, （bolting resistant inbreed line ）, P2 （vernalization independent type） and their filial generations F1 , B1, B2 and F2, using the mixed major-gene plus polygene inheritance model. [ Result] The two traits, bolting index and days to 5 cm elongated stalk, both were controlled by two major genes with additive-dominant-epistatic effects （ B-1 model） in hybrid. The flowering time was controlled by one major gene with addltive-dominant effects plus additive-dominant-epistatic effects （D model）. The heritability of the major genes in B1, B2 and F2 were 96.22%, 93.33%, 93.55% for bolting index, 70.68%, 70.68%, 70.64% for flowering time, 79.44%, 79.55%, 79.38% for days to 5-cm elongated stalk, respectively, but no polygene heritability was detected in BI, B2 and F2 generation. It indicated that the bolting trait in Brassica rapa was controlled by one or tow major genes. [ Conclusion] This implied that in the genetic improvement for bolting resistant trait major gene was a main factor. It is fit for early selection and environment factor should be mentioned.

Identiffcation of soft rot resistance loci in Brassica rapa with SNP markers

Soft rot caused by Pectobacterium carotovorum(Pc)is a devastating disease of Brassica rapa,causing substantial reductions in crop yield and quality.Identifying genes related to soft rot resistance is the key to solving this problem.To characterize soft rot resistance,we screened a soft rot-susceptible Chinese cabbage(A03),a resistant pakchoi(‘Huaguan’),and a resistant mutant(sr).An F_(2) population was generated by crossing susceptible Chinese cabbage A03and resistant pakchoi‘Huaguan’to identify quantitative trait loci(QTLs)that confer soft rot resistance.A high-density genetic map was constructed and the three QTLs identified contain 166 genes.Based on available transcriptome data,we analyzed the expression of the 166 genes during an important defense regulatory period in Pc infection in both A03and the resistant mutant sr.Among the 166 genes,six candidate genes were related to the soft rot defense response in B.rapa.TIFY10B(JAZ2,BraA07g038660.3C)was located in the major soft rot resistance QTL,DRQTL-3 on A07,and we speculate that this gene may play an important role in the defense mechanism against soft rot in B.rapa.This study lays the foundation for further investigations on the mechanism of soft rot resistance in B.rapa crops.

Variation and Correlation of Erucic Acid,Oleic Acid and Glucosinolate Contents in Brassica rapa Seeds

In order to screen and identify excellent breeding resources and provide basic materials for Brassica rapa breeding,the contents of erucic acid,oleic acid and glucosinolate in 84 kinds of B. rapa were determined by near-infrared spectroscopy,and the correlations among them were also analyzed. The results showed that the content of erucic acid and oleic acid were significantly negatively correlated,the contents of erucic acid and glucosinolate were significantly positively correlated,while the contents of oleic acid and glucosinolate were significantly negatively correlated; principal component analysis( PCA) were performed on the population materials,factors 1 and 2 were extracted for plotting,factor 1 and factor 2 could explain 73. 7% and 23. 2% of the phenotypic variation,respectively; through cluster analysis,79 materials aggregated to form group I,and five special variants deviated from the population. The variation of erucic acid,oleic acid and glucosinolates in B. rapa populations was rich and there was significant correlation. Through cluster analysis,five excellent B. rapa breeding materials( No. 32,No. 45,No. 46,No. 50,and No. 59) were screened.

QTL Mapping of Vernalization Requirement in Brassica rapa

An F2 population, derived from a cross between the vernalization independent genotype ＇ RcBr＇ and the vernalization dependent genotype＇08A061 ＇ , was developed to eonstruet a linkage map with 165 SSR and InDel markers. QTL analysis was performed by two phenotypie evaluation （ days to 5 em elongate stalk and flowering time） based on the difference in F2：3 families under vernalization and no vernalization. The results showed that the vernalization requirement was reeessive in Brassica rapa. Seven QTLs that controlled vernalization requirement were detected on A02 and A06 linkage groups, which explained phenotypic variation ranging from 0.08% to 22.52%. Two QTLs （ VR-DE01, VR-b391 ） were detected on the top of A02, which explained 22.52% and 14. 54% of the phenotypic variation, respectively, and closely linked with BrFLC2 gene. Two QTLs （VR-DE03, VR-DE04） were deteeted on A06 with 13.30% and 13.64% of phenotypie variation. These detected QTLs will provide useful information on understanding the genetic basis between vernalization requirement and bolting, and that will be useful for marker-assisted selection （MAS） in a breeding program for bolting resistant eultivars.

Antioxidant metabolic system and comparative proteomics analysis in winter turnip rape(Brassica rapa L.)under cold stress

Winter turnip rape(Brassica rapa L.)is widely cultivated in winter in Northwest China,however,its cold-tolerant mechanism remains insufficiently understood.In this study,winter turnip rape cultivar Longyou 7,a cold-tolerant variety,was used as material,whose accumulation of H_(2)O_(2) and O_(2)^(·-),antioxidant enzyme activity as well as differences in protein expression based on two-dimensional gel electrophoresis(2-DE)technique under -4℃ stress were analyzed.Results showed that,production of H_(2)O_(2) and O_(2)^(·-) were increased in Longyou 7 leaves,simultaneously,SOD and POD activities were also obviously rosed up,but the activities of CAT and APX were gradually reduced with the temperature.Thirty-six differential protein spots were successfully identified between control and treatments group by using mass spectrometry analysis.Among them,4 differential protein spots were induced under cold stress,and 2 were inhibited at-4℃.Functional analysis found that these identified proteins mainly participated in photosynthesis,carbohydrate metabolism,defense,protein synthesis,enzyme activity,redox and membrane metabolism,respectively.Additionally,13 proteins'function were still unknown.In conclusion,strong antioxidant capacity and cell defense ability might play important roles in Longyou 7 response to cold stress.

Developing Stable Cultivar through Microspore Mutagenesis in ×<i>Brassicoraphanus koranhort</i>, Inter-Generic Allopolyploid between <i>Brassica rapa</i>and <i>Raphanus sativus</i>

A stable progeny was developed through induced mutation, using microspore culture, of the hybrid (F1F1) produced by crossing a newly synthesized, unstable allopolyploid (F1) and a stable cultivar, BB#1(F1) in xBrassicoraphanus. An F1F1 plant was subjected to the induced mutation system established during production of BB#1. Morphological characteristics of the progeny such as color, and leaf number and length, differed from those of BB#1. The bolting time of the progeny in spring cropping was very late compared to BB#1, allowing it to be grown to an adult plant in spring. Genomic in situ hybridization analysis of pollen mother cells at prophase I identified 19 bivalents, 10 from Brassica rapa and 9 from Raphanus sativus. The glucoraphenin content was almost identical to that of BB#1. Two cultivars are available in the baemoochae crop now. These results indicate that induced mutation using microspore culture is a viable method of stabilizing intergeneric allopolyploids between B. rapa and R. sativus.

Effects of Fermented Nori (<i>Pyropia yezoensis</i>) Liquid Fertilizer on Plant Growth Characteristics and Nutrient Content of Komatsuna (<i>Brassica rapa</i>L. var. Wakana Komatsuna) Cultivated in Vermiculite

We conducted plant growth experiments in microbe-free vermiculite to study the effects of four types of fermented seaweed liquid fertilizer (SLF) made from nori (Pyropia yezoensis) seaweed on the germination, plant growth characteristics, SPAD value, and nutrient content and uptake of komatsuna (Brassica rapa L. var. wakana komatsuna). The four types of fermented nori SLF used in this study were prepared by anaerobic fermentation of unwashed nori (SLF1), aerobic fermentation of unwashed nori (SLF2), anaerobic fermentation of washed nori (SLF3), and aerobic fermentation of washed nori (SLF4). Komatsuna seeds treated with 200-, 300-, and 400-fold dilutions of SLFs exhibited improved relative germination ratios (RGRs) at 3 and 4 days after sowing (DAS). At 4 DAS, the RGRs of seeds treated with 10-, 100-, 200-, 300-, and 400-fold SLF dilutions showed no differential effect. Seeds treated with undiluted SLFs did not germinate by 4 DAS. SLF1 may promote komatsuna seed germination. The nitrogen (N), calcium, magnesium, sodium (Na), and iodine (I) contents of plants treated with SLF1 were significantly increased relative to plants treated with the other SLFs. Moreover, the I and Na contents of plants were significantly increased by foliar spray application of different dilutions of SLF1. However, SLF treatment markedly reduced the shoot dry weight compared with 1/2-strength modified Hoagland nutrient (MHN) solution, although the same amounts of N and K were applied. SPAD values of the plants treated with SLFs were significantly higher than those of plants treated with MHN. Foliar treatment with SLFs had no significant effect on plant growth, SPAD value, or uptake of nutrients (except Na) relative to the control, but the I content was increased. Plants treated with SLF1 and SLF2 exhibited the highest Na uptake. Foliar spray treatments with SLF1 resulted in the highest I contents in plants. Based on our results, SLF1 is suitable for use as a liquid fertilizer to promote germination and increase nutrient content in komatsuna. These results need to be followed up in soil experiments in the presence of microbes in the rhizosphere.

Costs of glucosinolates in <i>Brassica rapa</i>: Are they context dependent?

Models predicting optimal levels of plant defense against herbivores typically include two assumptions: 1) defense is both beneficial and costly;and 2) the relationship between costs and benefits of a defense is consistent across environments. However, the expression of costs and benefits of defense may be environmentally dependent. We examined lines of Brassica rapa, previously divergently selected for the defensive trait foliar glucosinolate content. In one set of experiments (Experiment #1), plants were grown in herbivore-free and herbivore-present environments to investigate the costs and benefits of this defense. In a second set of experiments (Experiment #2), plants were grown at two nutrient levels and two temperatures to examine the environmental context of costs of defense. In Experiment #1, increased levels of damage resulted in decreased flower production and plants from high glucosinolate lines received less damage than those from low glucosinolate lines, suggesting a benefit of this defense. In this experiment no cost of defense was detected. In Experiment #2, nutrients had a significant positive effect on flower production at 23°C, but not at 32°C. No significant effects of glucosinolate line nor interaction between nutrient environment and glucosinolate line were detected at 23°C, suggesting that no cost of defense occurred at this lower temperature. Similarly, no significant nutrient environment by glucosenolate line interaction was detected at 32°C. However, a significant effect of glucosinolate line was observed suggesting that at 32°C costs were incurred, but nutrient environment had no mitigating effect. While results from Experiment #1 suggested that defense was beneficial, but not costly, results from Experiment #2 suggested that costs of defense were temperature dependent. For species occupying broad geographic ranges, these findings of temperature-dependent costs are especially insightful with regard to the evolution of defense because differing geographic populations are likely to experience differing temperature environments.

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 Asand 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 T3F4strain(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 10μmol/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.