Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) with resistance to the mustard aphid (<Emphasis Type="Italic">Lipaphis erysimi</Emphasis> Kalt.)

. Wheat germ agglutinin (WGA), the chitin-binding lectin from wheat germ, has been shown to be antimetabolic, antifeedant and insecticidal to the mustard aphid (Lipaphis erysimi. Kalt). A cDNA encoding WGA was transferred to Indian mustard (Brassica juncea cv. RLM-198) through Agrobacterium-mediated transformation. Southern analysis of the transgenics showed the integration of the transgene, while Northern and Western analyses demonstrated that the transgene was expressed in the transgenics. Bioassays using leaf discs showed that feeding on transgenics induced high mortality and significantly reduced fecundity of aphids.     

Production of interspecific somatic hybrids between tuber mustard (<Emphasis Type="Italic">Brassica</Emphasis><Emphasis Type="Italic">juncea</Emphasis>) and red cabbage (<Emphasis Type="Italic">Brassica oleracea</Emphasis>)

In the present investigation, the interspecific somatic hybridization between tuber mustard and red cabbage was established in order to introduce valuable genes from red cabbage (Brassica oleracea) into Brassica juncea. Prior to fusion treatment, protoplasts of red cabbage were inactivated with 2 mM iodoacetamide to inhibit cell division. Micro-calluses were obtained at a frequency of 10.3% after approximately 5 weeks culture following protoplast fusion. Some of the fusion-derived calluses possessed red pigmented cells after being transferred to proliferation medium, and they were presumably considered to be somatic hybrid cell lines. Plantlets were regenerated from 12 cell lines, of which nine plantlets exhibited characteristics intermediate of both parents in terms of plant morphology. With the exception of common protein bands featured by two parents, there were unique banding patterns produced in the hybrids by using SDS-PAGE analysis. By chromosome countings, it was showed that they ranged approximately from 2n=30 to 42 in chromosome numbers. Their hybridity were further confirmed by RAPD analysis revealing that genes of both parents were partially incorporated into the hybrids. Positively, all these hybrids were capable of seed-setting. The pod-setting was 4.2 in somatic hybrid H₇ when backcrossed with tuber mustard.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) plants expressing an <Emphasis Type="Italic">Arabidopsis</Emphasis> phytochelatin synthase (<Emphasis Type="Italic">AtPCS1</Emphasis>) exhibit enhanced As and Cd tolerance

Phytochelatins (PCs) are post-translationally synthesized thiol reactive peptides that play important roles in detoxification of heavy metal and metalloids in plants and other living organisms. The overall goal of this study is to develop transgenic plants with increased tolerance for and accumulation of heavy metals and metalloids from soil by expressing an Arabidopsis thaliana AtPCS1 gene, encoding phytochelatin synthase (PCS), in Indian mustard (Brassica juncea L.). A FLAG-tagged AtPCS1 gDNA, under its native promoter, is expressed in Indian mustard, and transgenic pcs lines have been compared with wild-type plants for tolerance to and accumulation of cadmium (Cd) and arsenic (As). Compared to wild type plants, transgenic plants exhibit significantly higher tolerance to Cd and As. Shoots of Cd-treated pcs plants have significantly higher concentrations of PCs and thiols than those of wild-type plants. Shoots of wild-type plants accumulated significantly more Cd than those of transgenic plants, while accumulation of As in transgenic plants was similar to that in wild type plants. Although phytochelatin synthase improves the ability of Indian mustard to tolerate higher levels of the heavy metal Cd and the metalloid As, it does not increase the accumulation potential of these metals in the above ground tissues of Indian mustard plants.     

Intraspecific variability of cadmium tolerance in hydroponically grown Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern.) seedlings

Indian mustard (Brassica juncea (L.) Czern.) is a promising plant species for phytoremediation of heavy metal polluted soils. However, genetic variability of metal tolerance in Indian mustard has not been studied. We evaluated intraspecific variation of Cd tolerance of this species by screening 64 varieties in hydroponics. The tolerance index (TI), calculated as percentage of root length of Cd-treated (7 μM CdCl₂) over untreated control seedlings, significantly varied from 34 to 79%, depending on the variety. Information about phenotypic and economic traits of the studied varieties was taken from the literature and subjected to a cluster analysis. The varieties were distributed into three clusters and most of the varieties characterized by the highest TI values (TI > 65%) were grouped together in one cluster. Moreover, TI negatively correlated with the following characteristics: yellow seed colour (R = −0.35, P = 0.005), total oil content (R = −0.33, P = 0.008), oleic acid (R = −0.25, P = 0.047) and linoleic acid (R = −0.36, P = 0.004) contents in seeds. The results showed the presence of significant variability for Cd tolerance in Indian mustard. The knowledge about correlations between Cd tolerance and phenotypic characteristics of plants might be utilized for rapid selection of cultivars to be used for phytoremediation of polluted soils.     

Ectopic expression of cucumber (<Emphasis Type="Italic">Cucumis sativus</Emphasis> L.) <Emphasis Type="Italic">CsTIR</Emphasis>/<Emphasis Type="Italic">AFB</Emphasis> genes enhance salt tolerance in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis>

Auxin receptors TIR1/AFBs play an essential role in a series of signaling network cascades. These F-box proteins have also been identified to participate in different stress responses via the auxin signaling pathway in Arabidopsis. Cucumber (Cucumis sativus L.) is one of the most important crops worldwide, which is also a model plant for research. In the study herein, two cucumber homologous auxin receptor F-box genes CsTIR and CsAFB were cloned and studied for the first time. The deduced amino acid sequences showed a 78% identity between CsTIR and AtTIR1 and 76% between CsAFB and AtAFB2. All these proteins share similar characteristics of an F-box domain near the N-terminus, and several Leucine-rich repeat regions in the middle. Arabidopsis plants ectopically overexpressing CsTIR or CsAFB were obtained and verified. Shorter primary roots and more lateral roots were found in these transgenic lines with auxin signaling amplified. Results showed that expression of CsTIR/AFB genes in Arabidopsis could lead to higher seeds germination rates and plant survival rates than wild-type under salt stress. The enhanced salt tolerance in transgenic plants is probably caused by maintaining root growth and controlling water loss in seedlings, and by stabilizing life-sustaining substances as well as accumulating endogenous osmoregulation substances. We proposed that CsTIR/AFB-involved auxin signal regulation might trigger auxin mediated stress adaptation response and enhance the plant salt stress resistance by osmoregulation.     

Transgenic Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) expressing tomato glucanase leads to arrested growth of <Emphasis Type="Italic">Alternaria brassicae</Emphasis>

Brassica juncea is an important oilseed crop of the Indian sub-continent. Yield loss due to fungal disease alternaria leaf spot caused by Alternaria brassicae is a serious problem in cultivation of this crop. Nonavailability of resistance genes within crossable germplasms of Brassica necessitates use of genetic engineering strategies to develop genetic resistance against this pathogen. The pathogenesis related (PR) proteins are group of plant proteins that are toxic to invading fungal pathogens, but are present in plant in trace amount. Thus, overexpression of PR proteins leads to increased resistance to pathogenic fungi in several crops. The PR protein glucanase hydrolyzes a major cell-wall component, glucan, of pathogenic fungi and acts as a plant defense barrier. We report the expression of a class I basic glucanase gene, under the control of CaMV 35S promoter, in Indian mustard and its genetic resistance against alternaria leaf spot. Southern and Northern hybridization confirmed stable integration and expression of the glucanase gene in mustard transgenics. Several independent transgenics were screened in vitro and under poly house conditions for their resistance against Alternaria brassicae. In an in vitro antifungal assay, transgenics arrested hyphal growth of Alternaria brassicae by 15-54%. Under pathogen-challenged conditions in poly house, the transgenics showed restricted number, size and spread of lesions caused by Alternaria brassicae. Also, the onset of disease was delayed in transgenics compared to untransformed parent plants. The results demonstrate potentiality of a PR protein from a heterologous source in developing alternaria leaf spot resistance in Indian mustard.     

Antioxidant defense system in leaves of Indian mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>) and rape (<Emphasis Type="Italic">Brassica napus</Emphasis>) under cadmium stress

Plant species capable of hyper-accumulating heavy metals are of considerable interest for phytoremediation, and differ in their ability to accumulate metals from environment. Using two brassica species (Brassica juncea and Brassica napus), nutrient solution experiments were conducted to study variation in tolerance to cadmium (Cd) toxicity based on (1) lipid peroxidation and (2) changes in antioxidative defense system in leaves of both plants (i.e., superoxide dismutase (SOD EC 1.15.1.1), catalase (CAT EC 1.11.1.6), ascorbate peroxidase (APX EC 1.11.1.11), guaiacol peroxidase (GPX EC 1.11.1.7), glutathione reductase (GR EC 1.6.4.2), levels of phytochelatins (PCs), non-protein thiols (NP-SH), and glutathione. Plants were grown in nutrient solution under controlled environmental conditions, and subjected to increasing concentrations of Cd (0, 10, 25 and 50 μM) for 15 days. Results showed marked differences between both species. Brassica napus under Cd stress exhibited increased level of lipid peroxidation, as was evidenced by the increased malondialdehyde (MDA) content in leaves. However, in Brassica juncea treated plants, MDA content remained unchanged. In Brassica napus, with the exception of GPX, activity levels of some antioxidant enzymes involved in detoxification of reactive oxygen species (ROS), including SOD, CAT, GR, and APX, decreased drastically at high Cd concentrations. By contrast, in leaves of Brassica juncea treated plants, there was either only slight or no change in the activities of the antioxidative enzymes. Analysis of the profile of anionic isoenzymes of GPX revealed qualitative changes occurring during Cd exposure for both species. Moreover, levels of NP-SH and PCs, monitored as metal detoxifying responses, were much increased in leaves of Brassica juncea by increasing Cd supply, but did not change in Brassica napus. These results indicate that Brassica juncea plants possess the greater potential for Cd accumulation and tolerance than Brassica napus.     

Enhanced biomass and oxidative stress tolerance of <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 overexpressing the <Emphasis Type="Italic">DHAR</Emphasis> gene from <Emphasis Type="Italic">Brassica juncea</Emphasis>

Objectives

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H₂O₂, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

Results

Under H₂O₂ stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H₂O₂ level decreased. In addition, under H₂O₂ stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains.

Conclusion

By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H₂O₂ stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

     

Polymorphism of the <Emphasis Type="Italic">LEAFY</Emphasis> Gene in <Emphasis Type="Italic">Brassica</Emphasis> Plants

The arabidopsis gene LEAFY controls the induction of flowering and maintenance of the floral meristem identity. By comparing the primary structure of LEAFY and its homologs in other Brassicaceae species and beyond this family, we singled out four clusters corresponding to three systematically remote families of angiosperms, Brassicaceae, Solanaceae, and Poaceae, and to gymnosperms. Both structural and functional distinctions of LEAFY homologs from their arabidopsis prototype expanded in the range Brassicaceae—Solanaceae—Poaceae. A LEAFY homolog from B. juncea cloned in our laboratory was used as a hybridization probe to analyze the restriction fragment length polymorphism in six Brassica species comprising diploid (AA, BB, and CC) and allotetraploid (AABB, AACC, and BBCC) genomes. In this way we recognized LEAFY fragments specific of genomes A, B, and C; in contrast, the variations of the length and structure of the LEAFY intron 2 were not genome-specific. LEAFY polymorphism in the Brassica accessions comprising genome B was related to their geographic origin and apparently to the adaptation to day length.     

Polymorphism of the <Emphasis Type="Italic">CONSTANS</Emphasis> gene in <Emphasis Type="Italic">Brassica</Emphasis> plants

Using a direct amplification of genomic DNA from two Brassica rapa forms, we obtained two homologs of the CONSTANS gene, which controls the photoperiodic induction of flowering in Arabidopsis plants. The cloned fragments of B. rapa genome were identified as members of the CONSTANS-LIKE1 class. By aligning the nucleotide sequences of the CONSTANS gene and its homologs, three classes, CONSTANS, CONSTANS-LIKE1, and CONSTANS-LIKE2, were distinctly discerned by their primary structure. The pattern of restriction fragment length polymorphisms (RFLP) of the CONSTANS homologs in B. carinata, B. juncea, B. napus, B. nigra, B. oleracea, and B. rapa were genome-specific; in addition, the CONSTANS homologs were classified by plant geographic origin, and we assume that such classification is related to plant photoperiodic response.Translated from Fiziologiya Rastenii, Vol. 52, No. 2, 2005, pp. 274–281.Original Russian Text Copyright © 2005 by Martynov, Khavkin.This revised version was published online in April 2005 with a corrected cover date.     

Role of Arabidopsis <Emphasis Type="Italic">ABF1</Emphasis>/<Emphasis Type="Italic">3</Emphasis>/<Emphasis Type="Italic">4</Emphasis> during <Emphasis Type="Italic">det1</Emphasis> germination in salt and osmotic stress conditions

Key message

Arabidopsis det1 mutants exhibit salt and osmotic stress resistant germination. This phenotype requires HY5, ABF1, ABF3, and ABF4.

Abstract

While DE-ETIOLATED 1 (DET1) is well known as a negative regulator of light development, here we describe how det1 mutants also exhibit altered responses to salt and osmotic stress, specifically salt and mannitol resistant germination. LONG HYPOCOTYL 5 (HY5) positively regulates both light and abscisic acid (ABA) signalling. We found that hy5 suppressed the det1 salt and mannitol resistant germination phenotype, thus, det1 stress resistant germination requires HY5. We then queried publically available microarray datasets to identify genes downstream of HY5 that were differentially expressed in det1 mutants. Our analysis revealed that ABA regulated genes, including ABA RESPONSIVE ELEMENT BINDING FACTOR 3 (ABF3), are downregulated in det1 seedlings. We found that ABF3 is induced by salt in wildtype seeds, while homologues ABF4 and ABF1 are repressed, and all three genes are underexpressed in det1 seeds. We then investigated the role of ABF3, ABF4, and ABF1 in det1 phenotypes. Double mutant analysis showed that abf3, abf4, and abf1 all suppress the det1 salt/osmotic stress resistant germination phenotype. In addition, abf1 suppressed det1 rapid water loss and open stomata phenotypes. Thus interactions between ABF genes contribute to det1 salt/osmotic stress response phenotypes.

     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

Simple and efficient methods for <Emphasis Type="Italic">S</Emphasis> genotyping and <Emphasis Type="Italic">S</Emphasis> screening in genus <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis

In F₁ hybrid breeding of Brassica vegetables utilizing the self-incompatibility system, identification of S genotypes in breeding lines is required. In the present study, we developed S-tester lines of 87 S haplotypes, i.e., 42 S haplotypes in B. rapa and 45 S haplotypes in B. oleracea. With these materials, we established a simple, efficient, and reliable dot-blot technique for S genotyping for 40 S haplotypes of B. rapa and and 33 of B. oleracea using allele-specific oligonucleotide probes and allele-specific primer pairs designed from sequences of each SP11 allele. In this method, DNA fragments amplified using multiplex primer pairs with digoxigenin-dUTP were hybridized with dot-blotted allele-specific oligonucleotide probes with distinct signals. In addition, we developed a screening method for identification of plants harboring a particular S haplotype using a labeled allele-specific oligonucleotide probe. This method is considered to be useful for purity testing of F₁ hybrid seeds.     

Cadmium and zinc induced chlorosis in Indian mustard [<Emphasis Type="Italic">Brassica juncea</Emphasis> (L.) Czern] involves preferential loss of chlorophyll <Emphasis Type="Italic">b</Emphasis>

Plants of Indian mustard (Brassica juncea) were treated with either 50 μM Cd, 250 μM Zn, or 25 μM Cd+125 μM Zn and the progression of chlorosis in the mature leaves monitored. As relative chlorophyll (Chl) contents in the mature leaves decreased to 75, 50, and 25 % relative to controls, both mature and young leaves were harvested and the Chl pools extracted. The metal treatments caused a greater loss of Chl b than Chl a. As mature leaves underwent progressive chlorosis, the young leaves displayed a characteristic over-greening, due largely to increased content of Chl b. However, as the young leaves began to experience chlorosis, a greater loss of Chl b was also observed. Thus during metal induced chlorosis, there is a preferential turnover of the Chl b pool in mature and young leaves.     

Identification of <Emphasis Type="Italic">S</Emphasis> haplotypes in <Emphasis Type="Italic">Brassica</Emphasis> by dot-blot analysis of <Emphasis Type="Italic">SP11</Emphasis> alleles

A self-incompatibility system is used for F(1) hybrid breeding in Brassicaceae vegetables. The determinants of recognition specificity of self-incompatibility in Brassica are SRK in the stigma and SP11/SCR in the pollen. Nucleotide sequences of SP11 alleles are more highly variable than those of SRK. We analyzed the S haplotype specificity of SP11 DNA by Southern-blot analysis and dot-blot analysis using 16 S haplotypes in Brassica oleracea, and found that DNA fragments of a mature protein region of SP11 cDNA, SP11(m), of eight S haplotypes can detect only the SP11 alleles of the same S haplotypes. This specificity makes these methods useful for S haplotype identification. Therefore, we developed two methods of dot-blot analysis for SP11. One is dot blotting of DNA samples, i.e. plant genomic DNA probed with labeled SP11(m), and the other is dot blotting of SP11(m) DNA fragments probed with labeled DNA samples, i.e. the SP11 coding region labeled by PCR using a template of plant genomic DNA. The former is useful for testing many plant materials. The latter is suitable, if there is no previous information on the S haplotypes of plant materials.     

Distribution of similar self-incompatibility (<Emphasis Type="Italic">S</Emphasis>) haplotypes in different genera,<Emphasis Type="Italic"> Raphanus</Emphasis> and<Emphasis Type="Italic"> Brassica</Emphasis>

The nucleotide sequences of ten SP11 and nine SRK alleles in Raphanus sativus were determined, and deduced amino acid sequences were compared with those of Brassica SP11 and SRK. The amino acid sequence identity of class-I SP11s in R. sativus was about 30% on average, the highest being 52.2%, while that of the S domain of class-I SRK was 77.0% on average and ranged from 70.8% to 83.9%. These values were comparable to those of SP11 and SRK in Brassica oleracea and B. rapa. SP11 of R. sativus S-21 was found to be highly similar to SP11 of B. rapa S-9 (89.5% amino acid identity), and SRK of R. sativus S-21 was similar to SRK of B. rapa S-9 (91.0%). SP11 and SRK of R. sativus S-19 were also similar to SP11 and SRK of B. oleracea S-20, respectively. These similarities of both SP11 and SRK alleles between R. sativus and Brassica suggest that these S haplotype pairs originated from the same ancestral S haplotypes.     

Mitochondrial <Emphasis Type="Italic">atpA</Emphasis> gene is altered in a new <Emphasis Type="Italic">orf220</Emphasis>-type cytoplasmic male-sterile line of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis>)

The purpose of this research is to identify the probable mitochondrial factor associated with cytoplasmic male sterility (cms) by comparative analysis of cms and its isogenic maintainer lines in stem mustards. Dramatic variations in the morphology of floral organs were observed in cms stem mustard. Mitochondrial atpA gene was shown to be altered in cms compared with that in its maintainer line, of which mitochondrial atpA gene from its maintainer line was sequenced to encode 507 amino acids. It was indicative of high homology with mitochondrial atpA genes from other species, even as high as 94% in similarity with Oryza sativa in terms of amino acid constituents. However, only 429 amino acids were deduced in cms showing 83% similarity with atpA gene from its maintainer line. Two copies were observed in its maintainer line, but only one was found in cms. Such numerous differences of mitochondrial atpA gene between cms and its maintainer lines may not be the results of evolutionary divergence but the rearrangements of mitochondria. Expression of mitochondrial atpA gene was shown to be down-regulated in cms by using Northern blot. Consequently, mitochondrial ATP synthesis was severely decreased more than one fold in cms stem mustard indicating deficiency in mitochondrial ATP synthesis in this type of cms. Therefore, we deduced that mitochondrial atpA gene altered in cms could be associated with male-sterility in this type of cms. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Jing-Hua Yang and Yan Huai contributed equally to this work.