The complete chloroplast genome sequence of yellow mustard (Sinapis alba L.) and its phylogenetic relationship to other Brassicaceae species.

As a member of the large Brassicaceae family, yellow mustard (Sinapis alba L.) has been used as an important gene pool for the genetic improvement of cash crops in Brassicaceae. Understanding the phylogenetic relationship between Sinapis alba (S. alba) and other Brassicaceae crops can provide guidance on the introgression of its favorable alleles into related species. The chloroplast (cp) genome is an ideal model for assessing genome evolution and the phylogenetic relationships of complex angiosperm families. Herein, we de novo assembled the complete cp genome of S. alba by integrating the PacBio and Illumina sequencing platforms. A 153,760 bp quadripartite cycle without any gap was obtained, including a pair of inverted repeats (IRa and IRb) of 26,221 bp, separated by a large single copy (LSC) region of 83,506 bp and a small single copy (SSC) region of 17,821 bp. A total of 78 protein-coding genes, 30 tRNA genes, and four rRNA genes were identified in this cp genome, as were 89 simple sequence repeat (SSR) loci of 18 types. The codon usage analysis revealed a preferential use of the Leu codon with the A/U ending. The phylogenetic analysis using 82 Brassicaceae species demonstrated that S. alba had a close relationship with important Brassica and Raphanus species; moreover, it likely originated from a separate evolutionary pathway compared with the congeneric Sinapis arvensis. The synonymous (Ks) and non-synonymous (Ks) substitution rate analysis showed that genes encoding "Subunits of cytochrome b/f complex" were under the lowest purifying selection pressure, whereas those associated with "Maturase", "Subunit of acetyl-CoA", and "Subunits of NADH-dehydrogenase" underwent relatively higher purifying selection pressures. Our results provide valuable information for fully utilizing the S. alba cp genome as a potential genetic resource for the genetic improvement of Brassica and Raphanus species.

A part of the upstream promoter region of SHN2 gene directs trichome specific expression in Arabidopsis thaliana and heterologous plants.

A promoter trap mutant line of Arabidopsis carrying a promoterless ß-glucuronidase (uidA) gene exhibited GUS expression predominantly in all the trichomes. In this mutant, the T-DNA insertion was localized at 147bp upstream of the putative start codon, ATG, of the At5g11190 (SHN2) gene. Transcript profiling of the SHN2 suggested a constitutive expression of the gene in all the tissues. Deletion analysis of the upstream sequences established that a 565bp (-594/-30) region confers trichome-specific gene expression. The trichomes isolated from young, mature and senesced leaf tissues also showed the presence of SHN2 transcript. The occurrence of multiple TSSs on the SHN2 gene sequence, presence of the SHN2 transcript in the homozygous trip mutant, despite an insertional mutation event, and diverse reporter gene expression pattern driven by 5' and 3' promoter deletion fragments, suggest a complex transcriptional regulation of SHN2 gene in Arabidopsis. The promoter sequence -594/-30 showed a conserved functional role in conferring non-glandular trichome-specific expression in other heterologous systems like Brassica juncea and Solanum lycopersicon. Thus, in the present study T-DNA tagging has led to the identification of a trichome-specific regulatory sequence in the upstream region of a constitutively expressed SHN2 gene. The study also suggests a complex regulation of SHN2 gene. Isolated trichome specific region retains its functions in other systems like Brassica and tomato, hence could be effectively exploited in engineering trichome cells in heterologous crop plants to manipulate traits like biopharming and insect herbivory.

Expression of Arabidopsis acyl-CoA-binding proteins AtACBP1 and AtACBP4 confers Pb(II) accumulation in Brassica juncea roots.

In Arabidopsis thaliana, the expression of two genes encoding acyl-CoA-binding proteins (ACBPs) AtACBP1 and AtACBP4, were observed to be induced by lead [Pb(II)] in shoots and roots in qRT-PCR analyses. Quantitative GUS (ß-glucuronidase) activity assays confirmed induction of AtACBP1pro::GUS by Pb(II). Electrophoretic mobility shift assays (EMSAs) revealed that Pas elements in the 5'-flanking region of AtACBP1 were responsive to Pb(II) treatment. AtACBP1 and AtACBP4 were further compared in Pb(II) uptake using Brassica juncea, a potential candidate for phytoremediation given its rapid growth, large roots, high biomass and good capacity to accumulate heavy metals. Results from atomic absorption analyses on transgenic B. juncea expressing AtACBP1 or AtACBP4 indicated Pb(II) accumulation in roots. Subsequent Pb(II)-tracing assays demonstrated Pb(II) accumulation in the cytosol of root tips and vascular tissues of transgenic B. juncea AtACBP1-overexpressors (OXs) and AtACBP4-OXs and transgenic Arabidopsis AtACBP1-OXs. Transgenic Arabidopsis AtACBP1-OXs sequestered Pb(II) in the trichomes and displayed tolerance to hydrogen peroxide (H2 O2 ) treatment. In addition, AtACBP1 and AtACBP4 were H2 O2 -induced in the roots of wild-type Arabidopsis, while lipid hydroperoxide (LOOH) measurements of B. juncea AtACBP1-OX and AtACBP4-OX roots suggested that AtACBP1 and AtACBP4 can protect lipids against Pb(II)-induced lipid peroxidation.

Attenuation of hydrogen peroxide-mediated oxidative stress by Brassica juncea annexin-3 counteracts thiol-specific antioxidant (TSA1) deficiency in Saccharomyces cerevisiae.

Brassica juncea annexin-3 (BjAnn3) was functionally characterized for its ability to modulate H2O2-mediated oxidative stress in Saccharomyces cerevisiae. BjAnn3 showed a significant protective role in cellular-defense against oxidative stress and partially alleviated inhibition of mitochondrial respiration in presence of exogenously applied H2O2. Heterologous expression of BjAnn3 protected membranes from oxidative stress-mediated damage and positively regulated antioxidant gene expression for ROS detoxification. We conclude that, BjAnn3 partially counteracts the effects of thioredoxin peroxidase 1 (TSA1) deficiency and aids in cellular-protection across kingdoms. Despite partial compensation of TSA1 by BjAnn3 in cell-viability tests, the over-complementation in ROS-related features suggests the existence of both redundant (e.g. ROS detoxification) and distinct features (e.g. membrane protection versus proximity-based redox regulator) of both proteins.

HsfA1d, a protein identified via FOX hunting using Thellungiella salsuginea cDNAs improves heat tolerance by regulating heat-stress-responsive gene expression.

Thellungiella salsuginea (formerly T. halophila), a species closely related to Arabidopsis (Arabidopsis thaliana), is tolerant not only to high salt levels, but also to chilling, freezing, and ozone. Here, we report that T. salsuginea also shows greater heat tolerance than Arabidopsis. We identified T. salsuginea HsfA1d (TsHsfA1d) as a gene that can confer marked heat tolerance on Arabidopsis. TsHsfA1d was identified via Full-length cDNA Over-eXpressing gene (FOX) hunting from among a collection of heat-stress-related T. salsuginea cDNAs. Transgenic Arabidopsis overexpressing TsHsfA1d showed constitutive up-regulation of many genes in the Arabidopsis AtHsfA1 regulon under normal growth temperature. In Arabidopsis mesophyll protoplasts, TsHsfA1d was localized in both the nucleus and the cytoplasm. TsHsfA1d also interacted with AtHSP90, which negatively regulates AtHsfA1s by forming HsfA1-HSP90 complexes in the cytoplasm. It is likely that the partial nuclear localization of TsHsfA1d induced the expression of the AtHsfA1d regulon in the transgenic plants at normal temperature. We also discovered that transgenic Arabidopsis plants overexpressing AtHsfA1d were more heat-tolerant than wild-type plants and up-regulated the expression of the HsfA1d regulon, as was observed in TsHsfA1d-overexpressing plants. We propose that the products of both TsHsfA1d and AtHsfA1d function as positive regulators of Arabidopsis heat-stress response and would be useful for the improvement of heat-stress tolerance in other plants.

Heterosis as investigated in terms of polyploidy and genetic diversity using designed Brassica juncea amphiploid and its progenitor diploid species.

Fixed heterosis resulting from favorable interactions between the genes on their homoeologous genomes in an allopolyploid is considered analogous to classical heterosis accruing from interactions between homologous chromosomes in heterozygous plants of a diploid species. It has been hypothesized that fixed heterosis may be one of the causes of low classical heterosis in allopolyploids. We used Indian mustard (Brassica juncea, 2n = 36; AABB) as a model system to analyze this hypothesis due to ease of its resynthesis from its diploid progenitors, B. rapa (2n = 20; AA) and B. nigra (2n = 16; BB). Both forms of heterosis were investigated in terms of ploidy level, gene action and genetic diversity. To facilitate this, eleven B. juncea genotypes were resynthesized by hybridizing ten near inbred lines of B. rapa and nine of B. nigra. Three half diallel combinations involving resynthesized B. juncea (11×11) and the corresponding progenitor genotypes of B. rapa (10×10) and B. nigra (9×9) were evaluated. Genetic diversity was estimated based on DNA polymorphism generated by SSR primers. Heterosis and genetic diversity in parental diploid species appeared not to predict heterosis and genetic diversity at alloploid level. There was also no association between combining ability, genetic diversity and heterosis across ploidy. Though a large proportion (0.47) of combinations showed positive values, the average fixed heterosis was low for seed yield but high for biomass yield. The genetic diversity was a significant contributor to fixed heterosis for biomass yield, due possibly to adaptive advantage it may confer on de novo alloploids during evolution. Good general/specific combiners at diploid level did not necessarily produce good general/specific combiners at amphiploid level. It was also concluded that polyploidy impacts classical heterosis indirectly due to the negative association between fixed heterosis and classical heterosis.

Calcium supplementation modulates arsenic-induced alterations and augments arsenic accumulation in callus cultures of Indian mustard (Brassica juncea (L.) Czern.).

In the present study, the effect of arsenate (AsV) exposure either alone or in combination with calcium (Ca) was investigated in callus cultures of Brassica juncea (L.) Czern. cv. Pusa Bold grown for a period up to 24 h. The AsV (250 µM) + Ca (10 mM) treatment resulted in a significantly higher level of As (464 µg g(-1) dry weight (DW)) than AsV without Ca (167 µg g(-1) DW) treatment at 24 h. Furthermore, AsV + Ca-treated calli had a higher percent of AsIII (24-47%) than calli subjected to AsV treatment (12-14%). Despite this, AsV + Ca-treated calli did not show any signs of hydrogen peroxide (H(2)O(2)) accumulation or cell death upon in vivo staining, while AsV-exposed calli had increased H(2)O(2), shrinkage of cytoplasmic contents, and cell death. Thus, AsV treatment induced oxidative stress, which in turn elicited a response of antioxidant enzymes and metabolites as compared with control and AsV + Ca treatment. The positive effects of Ca supplementation were also correlated to an increase in thiolic constituents', viz., cysteine, reduced glutathione, and glutathione reductase in AsV + Ca than in AsV treatment. An analysis of selected signaling related genes, e.g., mitogen-activated protein kinases (MAPK3 and MAPK6) and jasmonate ZIM-domain (JAZ3) suggested that AsV and AsV + Ca followed variable pathways to sense and signal the As stress. In AsV-alone treatment, jasmonate signaling was seemingly activated, while MAPK3 was not involved. In contrast, AsV + Ca treatment appeared to specifically inhibit jasmonate signaling and activate MAPK3. In conclusion, Ca supplementation may hold promise for achieving increased As accumulation in plants without compromising their tolerance.

Brassica juncea plant cadmium resistance 1 protein (BjPCR1) facilitates the radial transport of calcium in the root.

Calcium (Ca) is an important structural component of plant cell walls and an intracellular messenger in plants and animals. Therefore, plants tightly control the balance of Ca by regulating Ca uptake and its transfer from cell to cell and organ to organ. Here, we propose that Brassica juncea PCR1 (PCR1), a member of the plant cadmium resistance (PCR) protein family in Indian mustard, is a Ca(2+) efflux transporter that is required for the efficient radial transfer of Ca(2+) in the root and is implicated in the translocation of Ca to the shoot. Knock-down lines of BjPCR1 were greatly stunted and translocated less Ca to the shoot than did the corresponding WT. The localization of BjPCR1 to the plasma membrane and the preferential expression of BjPCR1 in the root epidermal cells of WT plants suggest that BjPCR1 antisense plants could not efficiently transfer Ca(2+) from the root epidermis to the cells located inside the root. Protoplasts isolated from BjPCR1 antisense lines had lower Ca(2+) efflux activity than did those of the WT, and membrane vesicles isolated from BjPCR1-expressing yeast exhibited increased Ca(2+) transport activity. Inhibitor studies, together with theoretical considerations, indicate that BjPCR1 exports one Ca(2+) in exchange for three protons. Root hair-specific expression of BjPCR1 in Arabidopsis results in plants that exhibit increased Ca(2+) resistance and translocation. In conclusion, our data support the hypothesis that BjPCR1 is an exporter required for the translocation of Ca(2+) from the root epidermis to the inner cells, and ultimately to the shoot.

Generation of B. nigra-B. rapa chromosome addition stocks: cytology and microsatellite markers (SSRs) based characterization.

To improve Brassica nigra, the B-genome donor for Brassica juncea through selective introgression of useful variation from A-genome chromosomes, B. nigra-B. rapa chromosome addition stocks were successfully synthesized for the first time. Resynthesized B. juncea was used as B-genome donor species and A-genome addition stocks were developed by hybridizing sesquidiploid plant (ABB) as female and using B. nigra as the male parent. Various cycles of backcrossing and/or selfing were utilized to isolate plants carrying addition of three A-genome chromosomes in the background of B. nigra. These chromosome addition stocks were characterized by chromosome counts, pollen and seed fertility and chromosome specific microsatellite (SSRs) markers. The chromosome number in different backcross/self generations ranged between 2n=26 and 2n=19 with relatively high frequency of univalents (8-10I) at in meiotic configurations observed, suggesting the role of preferential transmission of A-genome chromosomes. SSRs analysis revealed that B. rapa chromosomes 3 and 4 were the first to get eliminated followed by chromosome 10. Remaining chromosomes were maintained till BC(1)F(4). However, second cycle of backcrossing (BC(2)) led to the elimination of chromosome numbers 1 and 2. BC(2)F(2) plants carried the chromosome numbers 6, 7, 8 and 9. Generation BC(3) having plants with 2n=19 carried chromosome numbers 6, 7 and 8. It is possible that chromosomes 6, 7 and 8 had higher transmission frequency and these were better tolerated by the B. nigra genome.

Assessment of genetic variation within Indian mustard (Brassica juncea) germplasm using random amplified polymorphic DNA markers.

Genetic diversity among 45 Indian mustard (Brassica juncea L.) genotypes comprising 37 germplasm collections, five advance breeding lines and three improved cultivars was investigated at the DNA level using the random amplified polymorphic DNA (RAPD) technique. Fifteen primers used generated a total of 92 RAPD fragments, of which 81 (88%) were polymorphic. Of these, 13 were unique to accession 'Pak85559'. Each primer produced four to nine amplified products with an average of 6.13 bands per primer. Based on pairwise comparisons of RAPD amplification products, Nei and Li's similarity coefficients were calculated to evaluate the relationships among the accessions. Pairwise similarity indices were higher among the oilseed accessions and cultivars showing narrow ranges of 0.77-0.99. An unweighted pair-group method with arithmetic averages cluster analysis based on these genetic similarities placed most of the collections and oilseed cultivars close to each other, showing a low level of polymorphism between the accessions used. However, the clusters formed by oilseed collections and cultivars were comparatively distinct from that of advanced breeding lines. Genetically, all of the accessions were classified into a few major groups and a number of individual accessions. Advanced breeding lines were relatively divergent from the rest of the accessions and formed independent clusters. Clustering of the accessions did not show any pattern of association between the RAPD markers and the collection sites. A low level of genetic variability of oilseed mustard was attributed to the selection for similar traits and horticultural uses. Perhaps close parentage of these accessions further contributed towards their little diversity. The study demonstrated that RAPD is a simple and fast technique to compare the genetic relationship and pattern of variation among the gene pool of this crop.

Indian mustard [Brassica juncea (L.) Czern.].

All economically important Brassica species have been successfully transformed using Agrobacterium tumefaciens. Although different tissues have been used as explants, hypocotyls remain the most desirable explants for Brassica tissue culture owing to their amenability to regeneration. Young explants excised from 3- to 4-d-old seedlings have exhibited optimal regeneration potential; the addition of adjuvants such as silver nitrate to the selection medium is necessary to achieve high efficiency of transformation. This chapter describes an Agrobacterium-mediated transformation protocol for Indian mustard based on inoculation of hypocotyls. The selectable marker gene used encodes for neomycin phosphotransferase II (nptII), and the selection agent is kanamycin.

Cadmium-induced plant stress investigated by scanning electrochemical microscopy.

In vivo oxygen evolution above single stomata in Brassica juncea has been used to investigate, for the first time, the effect of Cd-induced stress as imaged by scanning electrochemical microscopy (SECM). SECM images showed a clear stomatal structure-a pore, whose aperture is modulated by two guard cells, serving as the conduit for the oxygen produced. Lower stomatal density and larger stoma size were found in plants treated with 0.2 mM CdCl2 compared with control plants. Either the introduction of Cd caused a slower cell replication in the plane of the epidermis, hence fewer stomata, and/or the number of open stomata was reduced when plants were under Cd-stress. Oxygen evolution above individual stomatal complexes in Cd-treated plants was lower than that from control plants, as determined from the electrochemical current above the middle of each stoma. All guard cells under illumination were swollen, indicating that the stomata were open in both control and treated plants. Thus, decreased oxygen evolution in response to Cd cannot be attributed to simple closing of the stomata, but to a lower photosynthetic yield. SECM provides an excellent tool for monitoring the effects of Cd on photosynthetic activity at the scale of individual stomata.

The shoot apical meristem of Sinapis alba L. expands its central symplasmic field during the floral transition.

The shoot apical meristem (SAM) is functionally subdivided into zones with distinct tasks. During vegetative growth the peripheral zone of the meristem gives rise to leaf primordia that develop into dorsiventral leaves under the influence of signals from the central zone. During the floral transition the function of the SAM is altered and its peripheral zone starts to form floral structures in a specific pattern. This requires alterations in the signal networks that coordinate the activities of the peripheral and central zone of the SAM. These signal networks are partly housed in the symplasmic space of the SAM. Dye-coupling experiments demonstrate that in the superficial layer of the Sinapis alba meristem this space is radially subdivided. The cells of the central zone are coupled into a symplasmic field, which is shielded from the peripheral zone by the positional closing of plasmodesmata. In the vegetative meristems, most of these central symplasmic fields have a triangular geometry and are relatively small in size. Plants that are induced to flower by exposure to a single long day alter the geometry as well as the size of their central symplasmic field. After two subsequent days under short photoperiod the central symplasmic fields exhibit a circular form. Simultaneously, their size strongly increases both in an absolute sense and relative to the enlarging meristem. The geometric change in the fields is hypothesized to be due to recruitment of extra initial cells, required to support the increase in phyllotactic complexity. The proportional increase in field size is interpreted as an adjustment in the balance between the central and peripheral zone of the SAM, accompanying the shift from leaf production to flower formation.

The frequency of plasmodesmata increases early in the whole shoot apical meristem of Sinapis alba L. during floral transition.

The frequency of plasmodesmata increases in the shoot apical meristem of plants of Sinapis alba L. induced to flower by exposure to a single long day. This increase is observed within all cell layers (L1, L2, L3) as well as at the interfaces between these layers, and it occurs in both the central and peripheral zones of the shoot apical meristem. The extra plasmodesmata are formed only transiently, from 28 to 48 h after the start of the long day, and acropetally since they are detectable in L3 4 h before they are seen in L1 and L2. These observations indicate that (i) in the Sinapis shoot apical meristem at floral transition, there is an unfolding of a single field with increased plasmodesmatal connectivity, and (ii) this event is an early effect of the arrival at this meristem of the floral stimulus of leaf origin. Since (i) the wave of increased frequency of plasmodesmata is 12 h later than the wave of increased mitotic frequency (A. Jacqmard et al. 1998, Plant cell proliferation and its regulation in growth and development, pp. 67 78; Wiley), and (ii) the increase in frequency of plasmodesmata is observed in all cell walls, including in walls not deriving from recent divisions (periclinal walls separating the cell layers), it is concluded that the extra plasmodesmata seen at floral transition do not arise in the forming cell plate during mitosis and are thus of secondary origin.

Transcripts and sequence elements suggest differential promoter usage within the ycf3-psaAB gene cluster on mustard (Sinapis alba L.) chloroplast DNA.

The mustard chloroplast DNA region spanning the ycf3 gene and part of the psaAB operon was investigated. The ycf3 gene reveals two class-II introns that are removed during processing to give a mature 0.7-kb transcript, but no RNA editing seems to be involved. RNase protection and RT-PCR experiments suggest cotranscription of ycf3 with the downstream psaA gene, possibly from a NEP promoter upstream of ycf3, whereas distinct ycf3 and psaA transcripts are each initiated from PEP promoters. This situation is reminiscent of that for the trnK-psbA gene region. The implications for light-regulated versus light-independent expression of photosystem core-protein genes are discussed.

The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.). Integration of a prokaryotic core into a larger complex with organelle-specific functions.

We previously identified two multisubunit plastid RNA polymerases termed A and B. The B enzyme has a bacterial-type polypeptide composition and is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif); the A enzyme has a more complex subunit structure and is Rif-resistant. Here we report results of N-terminal sequencing and MS carried out with the A enzyme, which establish that the latter contains rpo gene products and is structurally related to the B enzyme. Furthermore, evidence is provided that the A enzyme can be converted into a Rif-sensitive enzyme form in a phosphorylation-dependent manner in vitro by a treatment that results in depletion of a beta-like subunit. Database searches using sequence information derived from additional polypeptides that are present in purified A preparations revealed sequence similarity with chloroplast proteins involved in RNA processing and redox control. This proteomics approach thus points to the complexity of the chloroplast transcription apparatus and its interconnections with post-transcriptional and signalling mechanisms.