Over-expression of BcFLC1 from non-heading Chinese cabbage enhances cold tolerance in Arabidopsis

A gene (named BcFLC1) homologous to the AtFLC gene, which encodes a floral repressor, was isolated from the nonheading Chinese cabbage (Brassica campestris L. ssp. chinensis) cv. NJ074. The gene showed high similarity to AtFLC. For studying the gene function, we designed to introduce the BcFLC1 gene into Arabidopsis thaliana. The results showed that BcFLC1 had effects on flowering time similar to AtFLC. We also found that Arabidopsis cold-tolerance was enhanced by BcFLC1 overexpression. Under low temperature stress, the BcFLC1 transgenic plants exhibited stronger growth than wild-type plants. The elevated cold tolerance of the BcFLC1 over-expressing plants was also confirmed by the changes of electrolyte leakage and malonyldialdehyde and proline content.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.     

Arabidopsis phosphomannose isomerase 1, but not phosphomannose isomerase 2, is essential for ascorbic acid biosynthesis

We studied molecular and functional properties of Arabidopsis phosphomannose isomerase isoenzymes (PMI1 and PMI2) that catalyze reversible isomerization between D-fructose 6-phosphate and D-mannose 6-phosphate (Man-6P). The apparent K(m) and V(max) values for Man-6P of purified recombinant PMI1 were 41.3+/-4.2 microm and 1.89 micromol/min/mg protein, respectively, whereas those of purified recombinant PMI2 were 372+/-13 microm and 22.5 micromol/min/mg protein, respectively. Both PMI1 and PMI2 were inhibited by incubation with EDTA, Zn(2+), Cd(2+), and L-ascorbic acid (AsA). Arabidopsis PMI1 protein was constitutively expressed in both vegetative and reproductive organs under normal growth conditions, whereas the PMI2 protein was not expressed in any organs under light. The induction of PMI1 expression and an increase in the AsA level were observed in leaves under continuous light, whereas the induction of PMI2 expression and a decrease in the AsA level were observed under long term darkness. PMI1 showed a diurnal expression pattern in parallel with the total PMI activity and the total AsA content in leaves. Moreover, a reduction of PMI1 expression through RNA interference resulted in a substantial decrease in the total AsA content of leaves of knockdown PMI1 plants, whereas the complete inhibition of PMI2 expression did not affect the total AsA levels in leaves of knock-out PMI2 plants. Consequently, this study improves our understanding of the molecular and functional properties of Arabidopsis PMI isoenzymes and provides genetic evidence of the involvement of PMI1, but not PMI2, in the biosynthesis of AsA in Arabidopsis plants.     

Manipulation of monoubiquitin improves salt tolerance in transgenic tobacco

Ubiquitin (Ub) is regarded as a stress protein involved in many stress responses. In this paper, sense and antisense transgenic tobacco plants, as well as the wild type and vector control, were used to study the role of Ub in salt tolerance of plants. In sense Ta-Ub2 transgenic tobacco plants, there was higher expression of Ub protein conjugates than in the wild type and vector control, but the reverse trend was observed in antisense Nt-Ub1 transgenic plants. The germination rate of tobacco seed, growth status and photosynthesis of the tobacco plants suggested that over-expressing Ub promoted the growth of transgenic tobacco plants and enhanced their salt tolerance, but the opposite effect was seen in plants with repressed Ub expression. Changes in antioxidant capacity may be one of the mechanisms underlying Ub-regulated salt tolerance. Furthermore, improved tolerance to a combination of stresses was also observed in the sense transgenic tobacco plants. These findings imply that Ub is involved in the tolerance of plants to abiotic stress.     

BcNRT1, a plasma membrane-localized nitrate transporter from non-heading Chinese cabbage

A nitrate transporter, BcNRT1, was isolated from non-heading Chinese cabbage (Brassica campestris ssp. chinensis Makino) cultivar 'Suzhouqing'. The full-length cDNA was obtained using the rapid amplification of cDNA ends technique and contains an open reading frame of 1,770?bp that predicts a protein of 589 acid residues that possesses 12 putative transmembrane domains. Using the GUS marker gene driven by the BcNRT1 promoter, we found BcNRT1 expression to be concentrated in primary and lateral root tips and in shoots of transgenic Arabidopsis plants. The YFP fused to BcNRT1 and transformed into cabbage protoplasts indicated that BcNRT1 was localized to the plasma membrane. The expression of BcNRT1 in roots was induced by exposure to 25?mM nitrate, and the BcNRT1 cRNA heterologously expressed in Xenopus laevis oocytes showed nitrate conductance when nitrate was included in the medium. Moreover, mutant chl1-5 plants harboring 35S::BcNRT1 showed sensitivity to chlorate treatment and exhibited restored nitrate uptake. In conclusion, the results indicate that BcNRT1 functions as a low affinity nitrate transporter in non-heading Chinese cabbage.     

Overexpression of the PtSOS2 gene improves tolerance to salt stress in transgenic poplar plants

In higher plants, the salt overly sensitive (SOS) signalling pathway plays a crucial role in maintaining ion homoeostasis and conferring salt tolerance under salinity condition. Previously, we functionally characterized the conserved SOS pathway in the woody plant Populus trichocarpa. In this study, we demonstrate that overexpression of the constitutively active form of PtSOS2 (PtSOS2TD), one of the key components of this pathway, significantly increased salt tolerance in aspen hybrid clone Shanxin Yang (Populus davidiana × Populus bolleana). Compared to the wild‐type control, transgenic plants constitutively expressing PtSOS2TD exhibited more vigorous growth and produced greater biomass in the presence of high concentrations of NaCl. The improved salt tolerance was associated with a decreased Na⁺ accumulation in the leaves of transgenic plants. Further analyses revealed that plasma membrane Na⁺/H⁺ exchange activity and Na⁺ efflux in transgenic plants were significantly higher than those in the wild‐type plants. Moreover, transgenic plants showed improved capacity in scavenging reactive oxygen species (ROS) generated by salt stress. Taken together, our results suggest that PtSOS2 could serve as an ideal target gene to genetically engineer salt‐tolerant trees.     

Expression of a calcineurin gene improves salt stress tolerance in transgenic rice

Calcineurin is a Ca²⁺- and calmodulin-dependent serine/threonine phosphatase and has multiple functions in animal cells including regulating ionic homeostasis. We generated transgenic rice plants that not only expressed a truncated form of the catalytic subunit of mouse calcineurin, but also were able to grow and fertilize normally in the field. Notably, the expression of the mouse calcineurin gene in rice resulted in its higher salt stress tolerance than the non-transgenic rice. Physiological studies have indicated that the root growth of transgenic plants was less inhibited than the shoot growth, and that less Na⁺ was accumulated in the roots of transgenic plants after a prolonged period of salt stress. These findings imply that the heterologous calcineurin plays a significant role in maintaining ionic homeostasis and the integrity of plant roots when exposed to salt. In addition, the calcineurin gene expression in the stems of transgenic plants correlated with the increased expression of the Rab16A gene that encodes a group 2-type late-embryogenesis-abundant (LEA) protein. Altogether our findings provide the first genetic and physiological evidence that expression of the mouse calcineurin protein functionally improves the salt stress tolerance of rice partly by limiting Na⁺ accumulation in the roots.     

Chloroplast elongation factor BcEF-Tu responds to turnip mosaic virus infection and heat stress in non-heading Chinese cabbage

Eukaryotic elongation factor Tu has been implicated in responses to heat stress and viral infection. In this study, the turnip mosaic virus (TuMV)-response gene BcLRK01, which encodes a leucine-rich repeat receptor-like kinase, was probed using the cDNA library of TuMV-infected leaves of non-heading Chinese cabbage (Brassica campestris ssp. chinensis). The BcEF-Tu gene, which encodes chloroplast elongation factor Tu, was obtained and verified by a yeast two-hybrid system to interact with the BcLRK01 gene. TuMV infection depressed the expression of this gene, whereas a heat stress induced its expression. Overexpression of BcEF-Tu enhanced the viability of Escherichia coli transformants under the heat stress. These results demonstrate that elongation factor BcEF-Tu responded to the TuMV infection and heat stress. This is the first report on chloroplast EF-Tu in non-heading Chinese cabbage which provides a theoretical basis for the functional research of EF-Tu.     

Cloning of a cDNA encoding the Saussurea medusa chalcone isomerase and its expression in transgenic tobacco.

Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.     

Karyotype of mitotic metaphase and meiotic diakinesis in non-heading Chinese cabbage

Non-heading Chinese cabbage [Brassica rapa L. ssp. chinensis (L.) Hanelt] is one of the most popular leafy vegetables. Despite the economic importance of non-heading Chinese cabbage, little attention has been given to its cytogenetic profile. This study reveals the karyotype of non-heading Chinese cabbage. Fluorescence in situ hybridization (FISH) with 45S and 5S rDNA probes was performed on mitotic metaphase complementary regions. We located 45S rDNA on the centromeric or adjacent region of chromosomes A1 and A2, with the largest on the satellite of chromosome A5. Meanwhile, 5S rDNA co-localized with 45S rDNA on chromosomes A2 and A5, and on the telomeric region of chromosome A10. We performed DAPI fluorescence banding on the same metaphase chromosomes to identify homologous chromosomes. The DAPI fluorescence pattern was observed mainly on the centromeric heterochromatin regions of each chromosome. However, the lengths of chromosomes A2 and A6 were completely stained, except for their telomeric regions. Meiotic diakinesis chromosomes as new substrates in FISH-developed karyotype were revealed for the first time. The karyotype of non-heading Chinese cabbage reveals that it contains eight submetacentric chromosomes, one subtelocentric chromosome (bearing satellite), and one telocentric chromosome. Diakinetic chromosome pairing can overcome the difficulty of unlabeled chromosome identification. This study provided valuable information for cytogenetic research and molecular breeding of non-heading Chinese cabbage by using the combination of FISH and DAPI fluorescence patterns on mitotic and meiotic chromosomes.     

RcMYBPA2 of Rosa chinensis functions in proanthocyanidin biosynthesis and enhances abiotic stress tolerance in transgenic tobacco

Plant Cell, Tissue and Organ Culture (PCTOC) - Proanthocyanidins (PAs) are major antioxidant flavonoids that play a key role in protecting plants against adverse environmental stress, but the...