Saussurea involucrata PIP2;4 improves growth and drought tolerance in Nicotiana tabacum by increasing stomatal density and sensitivity.

Aquaporins, the major facilitators of water transport across membranes, are involved in growth and development and adaptation to drought stress in plants. In this study, a plasma membrane intrinsic protein (SiPIP2;4) was cloned from Saussurea involucrata, a cold-tolerant hardy herb. The expression of SiPIP2;4 increased the stomatal density and sensitivity of tobacco (Nicotiana tabacum), thus, affecting the plant's growth and resistance to the diverse water environment. The higher stomatal density under well-watered conditions effectively promoted the photosynthetic rate, which led to the rapid growth of transgenic lines. The stomata in the transgenic lines responded more sensitively to the vapor pressure deficit than the wild-type under different levels of ambient humidity. Their stomatal apertures positively correlated with the ambient humidity. Under drought conditions, the overexpression of SiPIP2;4 promoted rapid stomatal closure, reduced water dissipation, and enhanced drought tolerance. These results indicate that SiPIP2;4 regulates the density and sensitivity of plant stomata, thus, playing an important role in balancing plant growth and stress tolerance. This suggests that SiPIP2;4 has the potential to serve as a genetic resource for crop improvement.

Overexpression of Saussurea involucrata dehydrin gene SiDHN promotes cold and drought tolerance in transgenic tomato plants.

Dehydrins are late embryogenesis abundant proteins that help regulate abiotic stress responses in plants. Overexpression of the Saussurea involucrata dehydrin gene SiDHN has previously been shown to improve water-use efficiency and enhance cold and drought tolerance of transgenic tobacco. To understand the mechanism by which SiDHN exerts its protective function, we transformed the SiDHN gene into tomato plants (Solanum lycopersicum L.) and assessed their response to abiotic stress. We observed that in response to stresses, the SiDHN transgenic tomato plants had increased contents of chlorophyll a and b, carotenoid and relative water content compared with wild-type plants. They also had higher maximal photochemical efficiency of photosystem II and accumulated more proline and soluble sugar. Compared to those wild-type plants, malondialdehyde content and relative electron leakage in transgenic plants were not significantly increased, and H2O2 and O2- contents in transgenic tomato plants were significantly decreased. We further observed that the production of stress-related antioxidant enzymes, including superoxide dismutase, ascorbate peroxidase, peroxidase, and catalase, as well as pyrroline-5-carboxylate synthetase and lipid transfer protein 1, were up-regulated in the transgenic plants under cold and drought stress. Based on these observations, we conclude that overexpression of SiDHN gene can promote cold and drought tolerance of transgenic tomato plants by inhibiting cell membrane damage, protecting chloroplasts, and enhancing the reactive oxygen species scavenging capacity. The finding can be beneficial for the application of SiDHN gene in improving crop tolerance to abiotic stress and oxidative damage.

A novel cold-regulated protein isolated from Saussurea involucrata confers cold and drought tolerance in transgenic tobacco (Nicotiana tabacum).

Adverse environmental conditions, such as cold and drought, can inhibit plant growth, development, and productivity. The isolation and characterization of stress response genes from stress-tolerant plants can provide a better understanding of the underlying adaptive mechanisms. In this study, a novel cold-regulated gene, SikCOR413PM1, was isolated from Saussurea involucrata Kar. et Kir., which is a plant that survives at the high altitudes and in the low temperatures of alpine slopes in northwestern China. SikCOR413PM1 was induced in response to cold and drought in S. involucrata, and phylogenetic analysis revealed that the gene groups with a COR gene encoding a COR413PM protein family member. Subcellular localization of a SikCOR413PM1-green fluorescent fusion protein showed that SikCOR413PM1 was localized to the plasma membrane. A transgenic tobacco (Nicotiana tabacum) system was employed to investigate the possible role of SikCOR413PM1 in cold and drought tolerance. Analyses of growth, germination and survival rates, relative water content, malondialdehyde content, relative electrolyte leakage, and maximal photochemical efficiency of photosystem II showed that transgenic tobacco plants expressing SikCOR413PM1 were more tolerant to cold and drought stresses than WT plants. SikCOR413PM1 overexpression was also accompanied by constitutive activation of NtDREB1 and NtDREB3, two cold-responsive transcription factor genes, and NtERD10A and NtERD10B, two cold-induced genes. The expression levels of downstream transcription factor genes NtDREB3, NtERD10C, NtERD10D, and NtLEA5 were also induced in SikCOR413PM1-expressing transgenic plants under drought conditions. Our results suggest that the overexpression of SikCOR413PM1 induces changes in tobacco plants, and facilitates enhanced tolerance to cold and drought stresses.

Cloning and characterization of SiDHN, a novel dehydrin gene from Saussurea involucrata Kar. et Kir. that enhances cold and drought tolerance in tobacco.

Saussurea involucrata Kar. et Kir. is a hardy, dicotyledonous plant featured by strong tolerance to severe stress conditions. In a previous study, we created a cDNA library to characterize genetic factors associated with the cold acclimation response of S. involucrata. The full-length cDNA of one dehydrin-like gene, SiDHN, was obtained by RT-PCR and the SiDHN gene was further characterized in this study. The full-length SiDHN cDNA consists of 703 base pairs (bp) with a 333bp open reading frame encoding a protein comprising 111 amino acid residues. The alignment of the deduced amino acid sequence showed that SiDHN protein shared 36% identity with one homolog in Helianthus annuus. To evaluate its biological functions, 35S:SiDHN recombinant plasmid was introduced into tobacco using Agrobacterium tumefaciens and the resistance of transgenic plants to freezing and drought stresses were analyzed. Compared with the wild-type, transgenic tobacco plants showed greater resistance to freezing and drought stresses. In this study, we provided evidence that SiDHN can enhance plant cold and drought resistance, suggesting that SiDHN could be potentially used to genetically improve plant tolerance to abiotic stresses.

[Cloning of flavone synthase (FNSII) gene and expression in three cell lines of Saussurea medusa].

Saussurea medusa is a rare traditional Chinese medicinal herb, of which luteolin is the niain active medicinal compound for cancer prevention and treatment. A full-length FNSII gene, namely SmFNSII (GenBank Accession No. KF170286), was obtained from green cell line of Saussurea medusa by RT-PCR and RACE-PCR. Sequence analysis indicated that SmFNSII is 1 710 bp in full length, containing a 34 bp 5'-untranslated region (5'-UTR), a 125 bp 3'-UTR, and a 1 551 bp open reading frame (ORF) encoding 516 amino acid residues. Amino acid sequence analysis indicated that SmFNSII belonged to subfamily CYP93B of plant cytochrome P450. Sequence alignment and phylogenetic analysis revealed that amino acid sequences of SmFNSII shared 87% homology with the protein in Hieracium pilosella. Quantitative real-time PCR analysis indicated that SmFNSII expression is the highest in red cell line and the lowest in white cell line, corresponding to quantitative analysis of luteolin concentration. pET-SmFNSII, a prokaryotic expression recombinant plasmid, was constructed and transferred into Escherichia coli, and the expressed protein band was the same size with predicted protein. Saussurea medusa cultivars with high anti-inflammatory, anti-cancer activities and health care function would be cultivated through filtering cell lines and plants with high expression level of FNSII gene and luteolin accumulation.

Cloning and characterization of a novel dehydrin gene, SiDhn2, from Saussurea involucrata Kar. et Kir.

Saussurea involucrata Kar. et Kir. is a hardy dicotyledonous plant capable of tolerating severe abiotic stress conditions. In a previous study, we created a cDNA library to determine what factors are associated with the cold acclimation response in S. involucrata. From this, a full-length cDNA of a dehydrin-like gene (SiDhn2) was obtained by RT-PCR. The SiDhn2 gene was characterized in this study. The full-length SiDhn2 cDNA comprised 693 bp containing an open reading frame of 345 bp specifying a protein of 115 amino acids. An alignment of the deduced amino acid sequence showed that SiDhn2 shared 55 % identity with two Brassica dehydrins. Agrobacterium tumefaciens was used to transform RD29A:SiDhn2 and 35S:SiDhn2 constructs into tobacco to investigate the germination and resistance to freezing and drought stress of transgenic plants. The RD29A:SiDhn2 transgenic plants showed greater resistance to freezing and drought stress than 35S:SiDhn2 transgenic plants or the wild-type. This study demonstrates that SiDhn2 confers cold hardiness and drought resistance, and may be a candidate resistance gene for genetic improvement of crops to increase stress resistance.

Cloning and expression analysis of a hsp70 gene from Saussurea medusa.

Members of the 70 kD heat-shock gene family are highly conserved across a wide range of organisms. In an effort to learn more about the evolution and possible functions of extreme environment plant Saussurea medusa Maxim hsp70, we isolated a cDNA clone encoding a putative cytosolic member (Smhsp70) of this family of proteins from a cDNA library of S. medusa cell cultures. The cDNA clone was 2224 bp in length and contained a 1941 bp open reading frame (ORF) encoding a polypeptide of 647 amino acid residues with a predicted molecular mass of 70,794 Da. The predicted protein was found to contain a C-terminal amino acid motif of "PKIEEVD" indicating that Smhsp70 was related to cytosolic members of the hsp70 family in higher plant. The secondary and three-dimensional structures of Smhsp70 were analyzed by molecular modeling. The genomic structure of Smhsp70 included one intron of 1134 bp in length. The deduced Smhsp70 protein has 93.7 and 93.2% similarity with the hsp70 of tobacco and tomato, 73.2% with the hsp70 of human, and 43.7% with DnaK of Escherichia. coli, respectively. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analyses indicated that the cytosolic Smhsp70 protein was constitutively expressed and markedly increased after relatively short periods of heat shock (37 degrees C) as well as by low temperature (4 degree C) treatments.

Overexpression of the Saussurea medusa chalcone isomerase gene in S. involucrata hairy root cultures enhances their biosynthesis of apigenin.

Saussurea involucrata is a medicinal plant well known for its flavonoids, including apigenin, which has been shown to significantly inhibit tumorigenesis. Since naturally occurring apigenin is in very low abundance, we took a transgenic approach to increase apigenin production by engineering the flavonoid pathway. A construct was made to contain the complete cDNA sequence of the Saussurea medusa chalcone isomerase (CHI) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. Using an Agrobacterium rhizogenes-mediated transformation system, the chi overexpression cassette was incorporated into the genome of S. involucrata, and transgenic hairy root lines were established. CHI converts naringenin chalcone into naringenin that is the precursor of apigenin. We observed that transgenic hairy root lines grew faster and produced higher levels of apigenin and total flavonoids than wild-type hairy roots did. Over a culture period of 5 weeks, the best-performing line (C46) accumulated 32.1 mgL(-1) apigenin and 647.8 mgL(-1) total flavonoids, or 12 and 4 times, respectively, higher than wild-type hairy roots did. The enhanced productivity corresponded to elevated CHI activity, confirming the key role that CHI played for total flavonoids and apigenin synthesis and the efficiency of the current metabolic engineering strategy.

[Cloning and sequence analysis of MYB transcriptional regulator SmP gene of Saussurea medusa Maxim].

A full-length cDNA encoding a MYB-related regulatory gene was isolated from a cDNA library prepared from mRNAs of the red line callus of S. medusa by TD-PCR. The cDNA, designated SmP, is 969 nucleotides long and has an open reading frame of 771 bp with a deduced amino acid sequence of 256 residues. The putative protein of SmP has two typical conversed R2R3-Myb DNA-binding domains in N-terminal and displays a rather high degree of similarity to OsMYB from rice and LBMI from tobacco, showing 73% and 70% identity within the DNA-binding domains. However, the C-terminal domain of the SmP protein does not show obvious similarity to any other known protein sequence. It is rich in hydrophilic amino acids, especially in serine residues (18.38%), partly organized in homopolymeric stretches, a feature often found in activation domain of transcription factors.