Late embryogenesis abundant (LEA) gene family in Salvia miltiorrhiza: identification, expression analysis, and response to drought stress.

Late embryogenesis abundant (LEA) proteins play important roles in plant defense response to drought stress. However, genome-wide identification of the LEA gene family was not revealed in Salvia miltiorrhiza. In this study, 61 SmLEA genes were identified from S. miltiorrhiza and divided into 7 subfamilies according to their conserved domains and phylogenetic relationships. SmLEA genes contained the LEA conserved motifs and few introns. SmLEA genes of the same subfamilies had similar gene structures and predicted subcellular locations. Our results indicated that the promoters of SmLEA genes contained various cis-acting elements associated with abiotic stress response. In addition, RNA-seq and real-time PCR results suggested that SmLEA genes are specifically expressed in different tissue, and most SmLEA genes can be induced by drought stress. These results provide a valuable foundation for future functional investigations of SmLEA genes and drought stress-resistant breeding of S. miltiorrhiza.

Systematic analysis of SmWD40s, and responding of SmWD40-170 to drought stress by regulation of ABA- and H2O2-induced stomal movement in Salvia miltiorrhiza bunge.

WD40 proteins play crucial roles in response to abiotic stress. By screening the genome sequences of Salvia miltiorrhiza Bunge, 225 SmWD40 genes were identified and divided into 9 subfamilies (I-IX). Physiological, biochemical, gene structure, conserved protein motif and GO annotation analyses were performed on SmWD40 family members. The SmWD40-170 was found in 110 SmWD40 genes that contain drought response elements, SmWD40-170 was one of these genes whose response in terms of expression under drought was significant. The expression of SmWD40-170 was also up-regulated by ABA and H2O2. Through observed the stomatal phenotype of SmWD40-170 transgenic lines, the stomatal closure was abolished under dehydration, ABA and H2O2 treatment in SmWD40-170 knockdown lines. Abscisic acid (ABA), as the key phytohormone, elevates reactive oxygen species (ROS) levels under drought stress. The ABA-ROS interaction mediated the generation of H2O2 and the activation of anion channel in guard cells. The osmolality alteration of guard cells further accelerated the stomatal closure. As a second messenger, nitric oxide (NO) regulated ABA signaling, the NO stimulated protein kinase activity inhibited the K+ influx which result in stomatal closure. These NO-relevant events were essential for ABA-induced stomatal closure. The reduction of NO production was also observed in the guard cells of SmWD40-170 knockdown lines. The abolished of stomatal closure attributed to the SmWD40-170 deficiency induced the reduction of NO content. In general, the SmWD40-170 is a critical drought response gene in SmWD40 gene family and regulates ABA- and H2O2-induced stomatal movement by affecting the synthesis of NO.

Physiological and transcriptome analysis reveal molecular mechanism in Salvia miltiorrhiza leaves of near-isogenic male fertile lines and male sterile lines.

BACKGROUND: Our previous study finds that male sterility in Salvia miltiorrhiza could result in stunted growth and reduced biomass, but their molecular mechanisms have not yet been revealed. In this article, we investigate the underlying mechanism of male sterility and its impact on plant growth and metabolic yield by using physiological analysis and mRNA sequencing (RNA-Seq). RESULTS: In this study, transcriptomic and physiological analysis were performed to identify the mechanism of male sterility in mutants and its impact on plant growth and metabolic yield. Through Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, it is found that the pathways are mainly enriched in processes including organ development, primary metabolic process and secondary metabolic process. Physiological analysis show that the chloroplast structure of male sterile mutants of S. miltiorrhiza is abnormally developed, which could result in decrease in leaf gas exchange (A, E and gs), chlorophyll fluorescence (Fv, Fm and Fv/Fm), and the chlorophyll content. Expression level of 7 differentially expressed genes involved in photosynthesis-related pathways is downregulated in male sterile lines of S. miltiorrhiza, which could explain the corresponding phenotypic changes in chlorophyll fluorescence, chlorophyll content and leaf gas exchange. Transcriptomic analysis establishes the role of disproportionating enzyme 1 (DPE1) as catalyzing the degradation of starch, and the role of sucrose synthase 3 (SUS3) and cytosolic invertase 2 (CINV2) as catalyzing the degradation of sucrose in the S. miltiorrhiza mutants. The results also confirm that phenylalanine ammonialyase (PAL) is involved in the biosynthesis of rosmarinic acid and salvianolic acid B, and flavone synthase (FLS) is an important enzyme catalyzing steps of flavonoid biosynthesis. CONCLUSIONS: Our results from the physiological and transcriptome analysis reveal underlying mechanism of plant growth and metabolic yield in male sterile mutants, and provide insight into the crop yield of S. miltiorrhiza.

Transcriptome Analyses from Mutant Salvia miltiorrhiza Reveals Important Roles for SmGASA4 during Plant Development.

Salvia miltiorrhiza (S. miltiorrhiza) is an important Chinese herb that is derived from the perennial plant of Lamiaceae, which has been used to treat neurasthenic insomnia and cardiovascular disease. We produced a mutant S. miltiorrhiza (MT), from breeding experiments, that possessed a large taproot, reduced lateral roots, and defective flowering. We performed transcriptome profiling of wild type (WT) and MT S. miltiorrhiza using second-generation Illumina sequencing to identify differentially expressed genes (DEGs) that could account for these phenotypical differences. Of the DEGs identified, we investigated the role of SmGASA4, the expression of which was down-regulated in MT plants. SmGASA4 was introduced into Arobidopsis and S. militiorrhiza under the control of a CaMV35S promoter to verify its influence on abiotic stress and S. miltiorrhiza secondary metabolism biosynthesis. SmGASA4 was found to promote flower and root development in Arobidopsis. SmGASA4 was also found to be positively regulated by Gibberellin (GA) and significantly enhanced plant resistance to salt, drought, and paclobutrazol (PBZ) stress. SmGASA4 also led to the up-regulation of the genes involved in salvianolic acid biosynthesis, but inhibited the expression of the genes involved in tanshinone biosynthesis. Taken together, our results reveal SmGASA4 as a promising candidate gene to promote S. miltiorrhiza development.

Salvia castanea Hairy Roots are More Tolerant to Phosphate Deficiency than Salvia miltiorrhiza Hairy Roots Based on the Secondary Metabolism and Antioxidant Defenses.

Salvia miltiorrhiza is a well-known traditional Chinese herb which is used to treat heart disease. Salvia castanea is a substitute product for S. miltiorrhiza in the medicinal field. Previous study has shown that phosphate (Pi) deficiency could promote the accumulation of secondary metabolism in herbs, and it has also developed a strategy for saving Pi resources and increasing the yield of active substances in herbs. In the present study, the hairy roots of S. miltiorrhiza and S. castanea were used to identify the Pi deficiency response mechanisms of these two Salvia species. The results showed that Pi deficiency increased the accumulation of specifically secondary metabolites, such as phenolic acids and tanshinones, which were caused by promoting the expression levels of key enzyme genes. In addition, Pi deficiency promoted the antioxidant activity in these two Salvia species. The data demonstrated that Pi deficiency increased the quality of the medicinal material in the plant. The hairy roots of S. castanea were more adaptive to Pi deficiency than those of S. miltiorrhiza in terms of biomass, secondary metabolism, and antioxidant activity. The results of this study provide insights into breeding herbs that are better adapted to Pi deficiency, which could increase the yield of active ingredients in herbs and save Pi resources.

SmLEA2, a gene for late embryogenesis abundant protein isolated from Salvia miltiorrhiza, confers tolerance to drought and salt stress in Escherichia coli and S. miltiorrhiza.

Abiotic stresses, such as drought and high salinity, are major factors that limit plant growth and productivity. Late embryogenesis abundant (LEA) proteins are members of a diverse, multigene family closely associated with tolerance to abiotic stresses in numerous organisms. We examined the function of SmLEA2, previously isolated from Salvia miltiorrhiza, in defense responses to drought and high salinity. Phylogenetic analysis indicated that SmLEA2 belongs to the LEA_2 subfamily. Its overexpression in Escherichia coli improved growth performance when compared with the control under salt and drought stresses. We further characterized its roles in S. miltiorrhiza through overexpression and RNAi-mediated silencing. In response to drought and salinity treatments, transgenic plants overexpressing SmLEA2 exhibited significantly increased superoxide dismutase activity, reduced levels of lipid peroxidation, and more vigorous growth than empty-vector control plants did. However, transgenic lines in which expression was suppressed showed the opposite results. Our data demonstrate that SmLEA2 plays an important role in the abiotic stress response and its overexpression in transgenic S. miltiorrhiza improves tolerance to excess salt and drought conditions.

Ectopic Expression of DREB Transcription Factor, AtDREB1A, Confers Tolerance to Drought in Transgenic Salvia miltiorrhiza.

Drought decreases crop productivity more than any other type of environmental stress. Transcription factors (TFs) play crucial roles in regulating plant abiotic stress responses. The Arabidopsis thaliana gene DREB1A/CBF3, encoding a stress-inducible TF, was introduced into Salvia miltiorrhiza Ectopic expression of AtDREB1A resulted in increased drought tolerance, and transgenic lines had higher relative water content and Chl content, and exhibited an increased photosynthetic rate when subjected to drought stress. AtDREB1A transgenic plants generally displayed lower malondialdehyde (MDA), but higher superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) activities under drought stress. In particular, plants with ectopic AtDREB1A expression under the control of the stress-induced RD29A promoter exhibited more tolerance to drought compared with p35S::AtDREB1A transgenic plants, without growth inhibition or phenotypic aberrations. Differential gene expression profiling of wild-type and pRD29A::AtDREB1A transgenic plants following drought stress revealed that the expression levels of various genes associated with the stress response, photosynthesis, signaling, carbohydrate metabolism and protein protection were substantially higher in transgenic plants. In addition, the amount of salvianolic acids and tanshinones was significantly elevated in AtDREB1A transgenic S. miltiorrhiza roots, and most of the genes in the related biosynthetic pathways were up-regulated. Together, these results demonstrated that inducing the expression of a TF can effectively regulate multiple genes in the stress response pathways and significantly improve the resistance of plants to abiotic stresses. Our results also suggest that genetic manipulation of a TF can improve production of valuable secondary metabolites by regulating genes in associated pathways.

Arabidopsis DREB1B in transgenic Salvia miltiorrhiza increased tolerance to drought stress without stunting growth.

Multiple stress response genes are controlled by transcription factors in a coordinated manner; therefore, these factors can be used for molecular plant breeding. CBF1/DREB1B, a known stress-inducible gene, was isolated from Arabidopsis thaliana and introduced into Salvia miltiorrhiza under the control of the CaMV35S or RD29A promoter. Under drought stress, relative water content, chlorophyll content, and the net photosynthetic rate were observed to be higher in the transgenic lines than in the wild type (WT). Moreover, O2(-) and H2O2 accumulation was observed to be lower in the transgenic lines. Additional analyses revealed that the AtDREB1B transgenic plants generally displayed lesser malondialdehyde (MDA) but higher superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities than the WT under drought stress. Quantitative real-time polymerase chain reaction of a subset of genes involved in photosynthesis, stress response, carbohydrate metabolism, and cell protection further verified that AtDREB1B could enhance tolerance to drought by activating different downstream DREB/CBF genes in the transgenic plants. Furthermore, no growth inhibition was detected in transgenic S. miltiorrhiza plants that expressed AtDREB1B driven by either the constitutive CaMV35S promoter or the stress-inducible RD29A promoter. Together, these results suggest that AtDREB1B is a good candidate gene for increasing drought tolerance in transgenic S. miltiorrhiza.

Production of herbicide-resistant medicinal plant Salvia miltiorrhiza transformed with the bar gene.

In this study, we successfully performed Agrobacterium-mediated genetic transformation of Salvia miltiorrhiza and produced herbicide-resistant transformants. Leaf discs of S. miltiorrhiza were infected with Agrobacterium tumefaciens EHA105 harboring pCAMBIA 3301. The pCAMBIA 3301 includes an intron-containing gus reporter and a bar selection marker. To increase stable transformation efficiency, a two-step selection was employed which consists of herbicide resistance and gus expression. Here, we put more attention to the screening step of herbicide resistance. The current study provides an efficient screening system for the transformed plant of S. miltiorrhiza harboring bar gene. To determine the most suitable phosphinothricin concentration for plant selection, non-transformed leaf discs were grown on selection media containing six different phosphinothricin concentrations (0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/l). Based on the above results of non-transformed calluses, the sensitivity of phosphinothricin (0, 0.4, 0.8, 1.2, 1.6 mg/l) was tested in the screening of transgenic S. miltiorrhiza. We identified that 0.6 mg/l phosphinothricin should be suitable for selecting putatively transformed callus because non-transformed callus growth was effectively inhibited under this concentrations. When sprayed with Basta, the transgenic S. miltiorrhiza plants were tolerant to the herbicide. Hence, we report successful transformation of the bar gene conferring herbicide resistance to S. miltiorrhiza.

Regulation of water-soluble phenolic acid biosynthesis in Salvia miltiorrhiza Bunge.

Salvia miltiorrhiza Bunge (Lamiaceae) root, generally called Danshen, is an important herb in Chinese medicine widely used for treatment of various diseases. Phenolic acids in S. miltiorrhiza, as important effective compounds, have become a new research focus in plant secondary metabolism in recent years. This review summarizes the recent advances in the regulation of water-soluble phenolic acid biosynthesis in S. miltiorrhiza via regulators at molecular level, such as the phenylalanine ammonia-lyase gene (PAL), cinnamic acid 4-hydroxylase gene (C4H), 4-coumarate-CoA ligase gene (4CL), tyrosine aminotransferase gene (TAT), 4-hydroxyphenylpyruvate reductase gene (HPPR), 4-hydroxyphenylpyruvated dioxygenase gene (HPPD), hydroxycinnamoyl-CoA:hydroxyphenyllactate hydroxycinnamoyl transferase-like gene (RAS-like), and v-myb avian myeloblastosis viral oncogene homolog 4 gene (MYB4), and production of anthocyanin pigmentation 1 gene (AtPAP1), and via regulators at cell level, such as methyl jasmonate, salicylic acid, abscisic acid, polyamines, metal ions, hydrogen peroxide (H2O2), ultraviolet-B radiation, and yeast elicitor.

A dominant gene for male sterility in Salvia miltiorrhiza Bunge.

A natural male sterile mutant of Salvia miltiorrhiza (Labiatae, Sh-B) was found during field survey in 2002. Our objective was to analyze its genetic mechanism for producing F1 hybrid seeds and to develop a molecular marker linked to male sterile gene for selection of a hybrid parent line. The segregation ratios of male sterile plants to fertile plants in the progenies of both testcross and backcross were 1:1 in continuous experiments conducted in 2006-2009. The male sterile Sh-B was heterozygous (Msms). The male sterile plants could capture most pollen (2 granule/cm(2)·24 h) with row ratio (female:male 2:1) within 45-cm distance and harvest the largest amount of 6495 g hybrid seeds per hectare. We also developed DNA markers linked to the male sterile gene in a segregating population using bulked segregant analysis (BSA) and amplified fragment length polymorphism (AFLP) techniques. The segregating population was subjected to BSA-AFLP with 128 primer combinations. One out of fourteen AFLP markers (E11/M4208) was identified as tightly linked to the dominant male sterile gene with a recombination frequency of 6.85% and at a distance of 6.89 cM. This marker could be converted to PCR-based assay for large-scale selection of fertile plants in MAS (marker-assisted selection) at the seedling stage. Blastn analysis indicated that the male sterile gene sequence showed higher identity with nucleotides in Arabidopsis chromosome 1-5, and was more likely to encode S-adenosylmethionine-dependent methyltransferase, in which DNA methylation regulated the development of plant gametogenesis.

Molecular characterization and expression of three galactinol synthase genes that confer stress tolerance in Salvia miltiorrhiza.

To adapt to changes in their growing environment, plants express several stress-responsive genes. For example, the products of galactinol synthase (Gols) genes play a key role in regulating the levels of raffinose family oligosaccharides and conferring resistance to stress. We cloned and characterized three Gols genes in Salvia miltiorrhiza. Their expression followed three distinct patterns. Compared with the control, SmGols1 was up-regulated by temperature changes but was suppressed by exposure to methyl jasmonate or short-term drought. This gene had the greatest abundance of transcripts and was assigned a general function of carbon storage. SmGols2 responded to all stress and hormone treatments, and transcripts were maintained at a high level. Finally, expression of SmGols3 was weaker than the other two genes, but was increased significantly under different treatments. Over the experimental period, its expression declined to normal levels in response to all treatments except exposure to 100 µM ABA, long-term drought, heat (42 °C), or chilling (8 °C). Based on our finding of cis-elements in the 5' flanking regions, we concluded that these genes seem to be regulated by several HSF transcription factors. We also targeted their 90-bp conserved sequences and used them for RNA interference analysis. Some were knocked down to various extents in our transgenic lines. Fluctuations in their malondialdehyde contents under different stress treatments, as well as the rate of water loss in transformed plants, suggested that lipid peroxidation was more likely to occur in the transgenics than in the control. These results indicate that SmGols genes could have a main function in responding to cold or heat. Therefore, we believe that it is important to investigate this mechanism for tolerance in S. miltiorrhiza and to examine how expression of these SmGols and other homologs are influenced by abiotic stresses.

[Effects of drought stress on leaf gas exchange and chlorophyll fluorescence of Salvia miltiorrhiza].

Taking the seedlings of Salvia miltiorrhiza cv. Sativa (SA) and S. miltiorrhiza cv. Silcestris (SI) as test materials, this paper studied the effects of drought stress on their leaf gas exchange and chlorophyll fluorescence parameters. After 15 days of drought stress, the net photosynthetic rate (P(n)) and the maximal photochemical efficiency of PS II (F(v)/F(m)) of SA were decreased by 66.42% and 10.98%, whereas those of SI were decreased by 29.32% and 5.47%, respectively, compared with the control, suggesting that drought stress had more obvious effects on the P(n) and F(v)/F(m) of SA than of SI. For SI, the reduction of P, under drought stress was mainly due to stomatal limitation; while for SA, it was mainly due to non-stomatal limitation. Drought led to a decrease of leaf stomatal conductance (G(s)), but induced the increase of water use efficiency (WUE), non-photochemical quenching coefficient (q(N)), and the ratio of photorespiration rate to net photosynthetic rate (P(r)/P(n)), resulting in the enhancement of drought resistance. The increment of WUE, q(N), and P(r)/P(n) was larger for SI than for SA, indicating that SI had a higher drought resistance capacity than SA.

[Pollen viability and stigma receptivity of Salvia miltiorrhiza and its relative].

OBJECTIVE: To provide the basal data for artificial cross breeding of Chinese herb Salvia miltiorrhiza from 7 provinces in China and its 4 relatives. METHOD: The pollen viability was evaluated by TTC (2, 3, 5-triphenylte trazolium chloride) test and the stigma receptivity was evaluated by benzidine-H2O2 method. RESULT: The pollen viability of S. miltiorrhiza from 6 provinces in China and its 4 relatives deceased during time of pollen shedding. Their highest pollen viability was in 2 or 3 days after blooming. But the pollen viability of S. miltiorrhiza (wild and culture) from Hean province in China declined with time after blooming. The most obvious variation of the pollen viability was in S. miltiorrhiza from Shanxi province (RSD 71.3% ) and the least was in wild S. miltiorrhiza from Henan province (RSD 12.4%). The highest average pollen viability was wild S. miltiorrhiza (72.3%) from Henan province while the lowest was S. yunnanensis (38.8%). The stigmas of all the accessions had receptivity when blooming. The stigma receptivity of S. brevilabra was strong in 2 to 4 days after blooming, while the others had less change after blooming. The life span of pollen grains and stigmas could be maintained from 3 to 5 days. CONCLUSION: The optimum artificial pollination time of S. miltiorrhiza and its relatives was 2 to 3 days after blooming.

[Study on seed testing for Salvia miltiorrhiza].

OBJECTIVE: To establish a seed testing methods for Salvia miltiorrhiza. METHOD: Referring to the International Seed Testing Rules made by ISTA and the Seed Testing for Crops (GB/T3543. 1-1995) issued by China. RESULT: The seeds are selected by winnowing; the seed purity is about 50%-60%; 100 grain weight is used to determine the quality of the seed; the seed moisture content is determined by air drying, the drying hour is 3 h. Seed viability is tested by TFC method.

Salt and drought tolerance of transgenic salvia miltiorrhiza Bunge with the TaLEA1 gene.

TaLEA1, a gene encoding a late-embryogenesis-abundant protein, was cloned from wheat and was transformed into Salvia Miltiorrhiza Bunge by Agrobacterium-mediated leaf-disk transformation method. Seven transgenic lines were obtained after kanamycin (50 mg/L) screening. Six positive lines were obtained by PCR amplification, and after four additional generations, one stable line was obtained by Southern hybridization. Transgenic plants had better growth states than control plants on mediums containing 1% NaCl and 8% PEG6000, which demonstrated that TaLEA1 played an important role in increasing the salt and drought tolerance of S. miltiorrhiza.

[Study on configuration fabric and germinative conditions of Salvia miltiorrhizy seeds].

OBJECTIVE: To provide theoretic warrant and technical reference for Salvia miltiorrhizr standardization planting, by carrying out various systemic studies such as observation of seeds configuration fabric, idiosyncrasy of water absorption and groping germinating conditions. METHOD: In the study of configuration fabric, seeds were observed and taken photos by scanning electronic microscope, and heft method was used for measuring changes of water absorption velocity and dehydration velocity. Seeds germination conditions were probed into under the national test regulations for crop seeds and related prescription from international standards. RESULT: (1) There was a layer of slime about 10-20 microm thickness covering epicarp of Danshen seeds. The slime formed as diamond meshwork (reseau) and the weight of it was 8%-10% of total seeds weight. (2) The speed of water absorption of seeds was extremely rapid. The weight of seeds could increase above 10 times as original while the dehydration velocity was quite low. (3) The optimal temperature for the seeds germination is around 25 degrees C, and the germination rate of the new seeds gained yearly was above 75%, but the rate would decrease sharply as years went by. It was also found that the seeds germination power and exponent of vigor were quite high under the temperature transformation between 23 degrees C, 28 degrees C. Such treatments as pre-cool, PEG treatment and infusing with GA3 could increase the rate of seeds germination capacity obviously.