Jin-Liang Qi

Comparative proteome analysis of differentially expressed proteins induced by Al toxicity in soybean.

Phytotoxic aluminum (Al) is a limiting factor for crop production on acid soils. The molecular mechanism, however, underlying Al toxicity and responses in plants is still not well understood. We report here the characterization of comparative proteome of aluminum-stress-responsive proteins in a known Al-resistant soybean cultivar, Baxi 10 (BX10). To investigate time-dependent responses, 1-week-old soybean seedlings were exposed to 50 microM AlCl3 for 24, 48 and 72 h, and total proteins extracted from roots were separated by two-dimensional electrophoresis. More than 1200 root proteins of the soybean BX10 seedling were reproducibly resolved on the gels. A total of 39 differentially expressed spots in abundance were identified by mass spectrometry, with 21 upregulated, 13 newly induced and 5 downregulated. The heat shock protein, glutathione S-transferase, chalcone-related synthetase, GTP-binding protein and ABC transporter ATP-binding protein were previously detected at the transcriptional or translational level in other plants. Other proteins, identified in this study, are new Al-induced proteins. Soybean BX10 roots under aluminum stress could be characterized by the cellular activities involved in stress/defense, signal transduction, transport, protein folding, gene regulation, and primary metabolisms, which are critical for plant survival under Al toxicity. This present study expands our understanding of differentially expressed proteins associated with aluminum stress on soybean BX10.

Nitric oxide regulates shikonin formation in suspension-cultured Onosma paniculatum cells.

Endogenously occurring nitric oxide (NO) is involved in the regulation of shikonin formation in Onosma paniculatum cells. NO generated after cells were inoculated into shikonin production medium reached the highest level after 2 d of culture, which was 16 times that at the beginning of the experiment, and maintained a high level for 6 d. A nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine (L-NNA), and a nitrate reductase (NR) inhibitor, sodium azide (SoA), consistent with their inhibition of NO biosynthesis, decreased shikonin formation significantly. This reduction could be alleviated or even abolished by exogenous NO supplied by sodium nitroprusside (SNP), suggesting that the inhibition of NO biosynthesis resulted in decreased shikonin formation. However, when endogenous NO biosynthesis was up-regulated by the elicitor from Rhizoctonia cerealis, shikonin production was enhanced further, showing a dependence on the elicitor-induced NO burst. Real-time PCR analysis showed that NO could significantly up-regulate the expression of PAL, PGT and HMGR, which encode key enzymes involved in shikonin biosynthesis. These results demonstrated that NO plays a critical role in shikonin formation in O. paniculatum cells.

Effect of Light on Gene Expression and Shikonin Formation in Cultured Onosma Paniculatum Cells

The cell culture in a production medium in the dark is capable of producing a large quantity of shikonin and its derivatives, which were completely inhibited when the cell cultures were irradiated with continuous white light and blue light and partially repressed under continuous red light. The expressions of PAL1, 4CL1, and CYP98A6 were induced under continuous red, blue and white light. Transcript levels of HMGR and LDI2 gradually were decreased in the dark and under continuous red light, while no transcriptional product of LDI2 was detected under blue and white light. C4H2 and LePGT1 genes were constantly expressed irrespective of different light conditions. Among the genes studied, the expression of LDI2 was critical for light-regulated shikonin formation. The inducible expression of PAL1, 4CL1, and CYP98A6 as well as the inhibitory transcription of LDI2 mediated by continuous red light irradiation were likely accounted for the reduced accumulation of shikonin.

LeERF-1, a novel AP2/ERF family gene within the B3 subcluster, is down-regulated by light signals in Lithospermum erythrorhizon.

We previously showed that ethylene might be involved in the process of shikonin biosynthesis regulated by light signals. Here, we cloned a full-length cDNA of LeERF-1, a putative ethylene response factor gene, from Lithospermum erythrorhizon using the RACE (rapid amplification of cDNA ends) method. Phylogenetic analysis revealed that LeERF-1 was classified in the B3 subfamily, together with ERF1 and ORA59 of Arabidopsis. Heterologous expression of LeERF-1 in Arabidopsis showed that LeERF-1:eGFP fusion protein was precisely localised to the nucleus, implying that it might function as a transcription factor. Detailed expression analysis with real-time PCR showed that LeERF-1 was significantly down-regulated by white, blue and red light, although the inhibitory effect of red light was relatively weak compared to other light conditions. Tissue-specific expression analysis also indicated that LeERF-1 was dominantly expressed in the roots, which grow in soil in darkness. These patterns are all consistent with the effects of different light signals on regulating formation of shikonin and its derivatives, indicating that LeERF-1 might be a crucial positive regulator, like other B3 subfamily proteins (such as ORCA3 and ORA59), in regulating biosynthesis of secondary metabolites.

Expression analysis of shikonin-biosynthetic genes in response to M9 medium and light in Lithospermum erythrorhizon cell cultures

When cells of Lithospermum erythrorhizon were transferred from B5 solid medium into M9 liquid medium and grown in the dark, they produced and accumulated shikonin and its derivatives; meanwhile, transcripts of PAL, 4CL, HMGR, PGT, and CYP98A6 were rapidly induced within 2xa0h, peaking within 6xa0h, and then decreasing over time. However, when L. erythrorhizon cells were cultured in M9 medium and grown under white light, a negative regulator for the biosynthesis of shikonin and its derivatives, the “stimulating effect” of the medium transition on the transcription of these genes was also displayed with similar patterns over 24xa0h of culture period. To investigate the time-dependent effect of light signals on regulating gene transcription, expression analysis of L. erythrorhizon dark-inducible genes LeDI-1–LeDI-5 was conducted. No obvious inhibitory effects of light signals on the transcription of LeDI-1–LeDI-5 were detected during the first 12xa0h, but these genes, in particular LeDI-2 and LeDI-4, were suppressed by light signals to some degree during longer culture periods, 12xa0h to 10xa0days. These findings suggest that light signals are likely to inhibit the transcription of the shikonin-biosynthetic genes indirectly or under long-term conditions.

Differential Responses of Anti-Oxidative Enzymes to Aluminum Stress in Tolerant and Sensitive Soybean Genotypes

ABSTRACT Seedlings of two soybean genotypes, BX10 [aluminum (Al)-tolerant] and BD2 (Al-sensitive), were treated with Al to evaluate the relative root growth (RRG), callose content, Al-sensitive zone, lipid peroxidation, and the anti-oxidative enzyme activities by histochemical and biochemical assays. Under Al toxicity, the RRG reduction of BD2 was more significant than that of BX10, while callose content displayed a contrary trend. The 2–5 mm zone of root apex was the main Al-sensitive zone for soybeans. The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX) were found to be higher in the two genotypes under Al toxicity than that of the controls. The SOD, POD, and CAT activities of BX10; however, were significantly lower than those of BD2. These results implied that producing low quantity of ROS may be one aspect of the Al-tolerant mechanism for soybeans, which in turn helps them adapt to Al stress.

Expression analysis of light-regulated genes isolated from a full-length-enriched cDNA library of Onosma paniculatum cell cultures

Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.

Sequence analysis and expression of the calmodulin gene, MCaM-3, in mulberry (Morus L.)

A full-length cDNA sequence encoding CaM from mulberry, which we designated MCaM-3 (GenBank accession No: GQ303247), was cloned based on mulberry expressed sequence tags(ESTs). Sequence analysis showed that MCaM-3 is 951 base pairs in length, encoding 149 amino acids with a predicted molecular weight of 16.85 kD and an isoelectric point of 3.95. Online SMART analysis showed that the MCaM-3 protein has four EFh functional domains, which can bind calcium. The expression level of MCaM-3at different developmental stages in mulberry leaves and flowers and in different tissues was investigated. The results showed that MCaM-3 transcripts are most abundantly expressed in mature tissues, such as mature female flowers and climax leaves, and the expression level of the mRNA could be increased significantly under low temperature, drought, and salt stress conditions compared to the normal growth environment. This research will help us understand the resistance mechanism of functional genes in mulberry.

Molecular cloning, characterization, and expression analysis of LeMYB1 from Lithospermum erythrorhizon

MYB transcription factors (TFs) are known to have important functions in regulating the biosynthesis of secondary metabolites in plants. In this study, LeMYB1, a member of the MYB gene family of Lithospermum erythrorhizon, was cloned via the rapid amplification of cDNA ends. The alignment of the predicted translations of LeMYB1 with other MYB proteins revealed that LeMYB1 contained an N-terminal R2R3 repeat and a high degree of amino acid identity to NtMYBJS1 which is involved in jasmonic acid signalling and phenylpropanoid biosynthetic pathway regulation. To determine the expression pattern of LeMYB1, its promoter was cloned and the sequence analysis was performed. The results revealed a number of potential regulatory motifs related to tissue-specific gene expression and abiotic and biotic stress responses. Real-time PCR results suggest that LeMYB1 was induced transiently during the early stage when L. erythrorhizon cells were transferred from a B5 growth medium to a M9 production medium for shikonin formation. Exogenous methyl jasmonate (MeJA), an effective inducer of shikonin biosynthesis, induced the rapid LeMYB1 expression. In contrast, a treatment with ibuprofen (IBU), an inhibitor of jasmonate biosynthesis, significantly inhibited the LeMYB1 expression. Another inhibitor of shikonin formation, 2,4-dichlorophenoxyacetic acid (2,4-D), also markedly repressed the expression of LeMYB1. Tissue-specific expression analysis showed that LeMYB1 mRNA was predominantly accumulated in roots where shikonin was synthesized. Thus, the LeMYB1 gene may be a valuable member of the R2R3-MYB family in L. erythrorhizon and is possibly involved in the regulation of shikonin biosynthesis.

RfaB, a galactosyltransferase, contributes to the resistance to detergent and the virulence of Salmonella enterica serovar Enteritidis

In this study, a deletion mutant of rfaB (ΔrfaB) was observed to be susceptible to sodium dodecyl sulfate and less tolerant to bile salts. In addition, pre-incubation in 10% bile salts increased bacterial tolerance to 30% bile salts. We also showed that the ΔrfaB mutant invaded HeLa cells less than the wild type and resulted in a lower ratio of intracellular bacteria. Competitive infection of mice showed that the ΔrfaB mutant was defective in the colonization of host organs and was cleared more quickly in fecal shedding. Transforming of a plasmid containing a wild-type allele of rfaB (pRB3-rfaB) partially rescued the defect of the ΔrfaB mutant. The results suggest that RfaB, which is responsible to add the glycosyl residue to the core lipopolysaccharide, contributes to the tolerance to detergent and the virulence of Salmonella enterica serovar Enteritidis.