Comparative transcriptome analysis of two contrasting wolfberry genotypes during fruit development and ripening and characterization of the LrMYB1 transcription factor that regulates flavonoid biosynthesis.

BACKGROUND: Lycium barbarum and L. ruthenicum have been used as traditional medicinal plants in China and other Asian counties for centuries. However, the molecular mechanisms underlying fruit development and ripening, as well as the associated production of medicinal and nutritional components, have been little explored in these two species. RESULTS: A competitive transcriptome analysis was performed to identify the regulators and pathways involved in the fruit ripening of red wolfberry (L. barbarum) and black wolfberry (L. ruthenicum) using an Illumina sequencing platform. In total, 155,606 genes and 194,385 genes were detected in red wolfberry (RF) and black wolfberry (BF), respectively. Of them, 20,335, 24,469, and 21,056 genes were differentially expressed at three different developmental stages in BF and RF. Functional categorization of the differentially expressed genes revealed that phenylpropanoid biosynthesis, flavonoid biosynthesis, anthocyanin biosynthesis, and sugar metabolism were the most differentially regulated processes during fruit development and ripening in the RF and BF. Furthermore, we also identified 38 MYB transcription factor-encoding genes that were differentially expressed during black wolfberry fruit development. Overexpression of LrMYB1 resulted in the activation of structural genes for flavonoid biosynthesis and led to an increase in flavonoid content, suggesting that the candidate genes identified in this RNA-seq analysis are credible and might offer important utility. CONCLUSION: This study provides novel insights into the molecular mechanism of Lycium fruit development and ripening and will be of value to novel gene discovery and functional genomic studies.

[Study on membrane type leaf water evaporation inhibitors for improving effect of preventing diseases and pest controlling of Lycium barbarum].

Through indoor and field comparative experiments, the properties of membrane type leaf evaporation inhibitors and its effects on photosynthesis of Lycium barbarum and compatibility and synergistic of pesticide were studied. The evaporation inhibitors and L. barbarum were chosen to investigate the suppression of water evaporation and the compatibility with pesticides. The effect of evaporation inhibitors on photosynthesis of L. barbarum leaves was determined by the chlorophyll fluorescence imaging system. The results showed that water evaporation of L. barbarum leaves of different leaf age were evidently suppressed after treated with evaporation inhibitor. The inhibitor was well compatible with pesticide and effectively improved the pesticide efficacy，and had no significant effect on chlorophyll fluorescence parameters. It is concluded that the evaporation inhibitor has good compatibility with the pesticide, and has remarkable effect of restraining moisture evaporation, which make it can be used for reducing the dosage and improving the efficacy of the pesticide in the field of L. barbarum.

Influence of Rhizoglomus irregulare on nutraceutical quality and regeneration of Lycium barbarum leaves under salt stress.

Whether arbuscular mycorrhizal fungi augment the nutraceutical quality of crops under salt stress is critical as a potential agronomic practice in salinized farmland. To evaluate the effect of Rhizoglomus irregulare on the nutraceutical quality of Lycium barbarum leaves under salt stress, we analyzed growth parameters and the rutin, polysaccharide, acidic polysaccharide, and amino acids contents of 2 harvests. Inoculation of R. irregulare significantly increased the regenerated bud number (partial eta squared (PES) = 0.577, P < 0.0001) and rutin concentration (PES = 0.544, P < 0.001) of L. barbarum leaves, with and without salt stress. The biomass of the 2nd harvest (PES = 0.355, P = 0.0091) and acidic polysaccharide (PES = 0.518, P = 0.001) of L. barbarum leaves were notably enhanced by R. irregulare under 200 mmol/L salt level. Rhizoglomus irregulare had insignificant effect on polysaccharide (PES = 0.092, P = 0.221) and amino acids levels (PES = 0.263, P = 0.130) in the leaves of L. barbarum. However, inoculation by R. irregulare decreased proline level (PES = 0.761, P = 0.001) in the leaves of L. barbarum when subjected to salt stress. Taken together, these results indicate that R. irregulare significantly improved the nutraceutical quality and facilitated the sustainable production of L. barbarum leaves exposed to salt stress.

Characterization of six PHT1 members in Lycium barbarum and their response to arbuscular mycorrhiza and water stress.

Phosphorus (P) is vitally important for most plant processes. However, the P available to plants is present in the soil in the form of inorganic phosphate (Pi), and is often present in only limited amounts. Water stress further reduces Pi availability. Previous studies have highlighted the important roles of members of the PHOSPHATE TRANSPORTER 1 (PHT1) family and arbuscular mycorrhizal (AM) associations for Pi acquisition by plants growing in various environments. In order to understand the Pi uptake of Lycium barbarumL., a drought-tolerant ligneous species belonging to the Solanaceae family, we cloned and characterized six L. barbarum genes encoding transporter proteins belonging to the PHT1 family, and investigated their transcriptional response to AM associations and water stress. The six cloned PHT1 genes of L. barbarum had a similar evolutionary history to that of PHT1 genes found in other Solanaceae species. Three of these genes (LbPT3, LbPT4 and LbPT5) were AM-induced; the other three genes (LbPT1, LbPT2 and LbPT7) played distinct roles in Pi acquisition, translocation and remobilization in roots and leaves. AM-induced PHT1 genes maintained their function under water stress, while moderate and severe water stress upregulated non-AM-induced PHT1 genes in roots and leaves, respectively. Moreover, although LbPT1 was upregulated in AM roots under water stress, LbPT2 and LbPT7 were inhibited in AM roots, which suggested that an AM association satisfied the demand for Pi in roots under water stress and that LbPT1 may play a role in translocating Pi from roots to shoots in this situation.

Phenolic compounds as indicators of drought resistance in shrubs from Patagonian shrublands (Argentina).

UNLABELLED: Plants exposed to drought stress, as usually occurs in Patagonian shrublands, have developed different strategies to avoid or tolerate the lack of water during their development. Production of phenolic compounds (or polyphenols) is one of the strategies used by some native species of adverse environments to avoid the oxidative damage caused by drought. In the present study the relationship between phenolic compounds content, water availability and oxidative damage were evaluated in two native shrubs: Larrea divaricata (evergreen) and Lycium chilense (deciduous) of Patagonian shrublands by their means and/or by multivariate analysis. Samples of both species were collected during the 4 seasons for the term of 1 year. Soil water content, relative water content, total phenols, flavonoids, flavonols, tartaric acid esters, flavan-3-ols, proanthocyanidins, antioxidant capacity and lipid peroxidation were measured. According to statistical univariate analysis, L. divaricata showed high production of polyphenols along the year, with a phenolic compound synthesis enhanced during autumn (season of greatest drought), while L. chilense has lower production of these compounds without variation between seasons. The variation in total phenols along the seasons is proportional to the antioxidant capacity and inversely proportional to lipid peroxidation. Multivariate analysis showed that, regardless their mechanism to face drought (avoidance or tolerance), both shrubs are well adapted to semi-arid regions and the phenolic compounds production is a strategy used by these species living in extreme environments. The identification of polyphenol compounds showed that L. divaricata produces different types of flavonoids, particularly bond with sugars, while L. chilense produces high amount of non-flavonoids compounds. SYNTHESIS: These results suggest that flavonoid production and accumulation could be a useful indicator of drought tolerance in native species.

Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium.

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

[Effect of exogenous calcium on seed germination and seedling physiological characteristics of Lycium ruthenium].

OBJECTIVE: In order to get the method for improving the salt resistance of Lycium ruthenium seeds and seedlings under NaCl stress, the seed germination and physiological characteristics of L. ruthenium seedlings was studied. METHOD: Several physiological indexes of L. ruthenium seeds under NaCl stress, such as the germination rate (Gr), germination vigor (Gv), germination index (Gi), vigor index (Vi), and relative salt damage rate were measured. Other indexes of the seedlings like relative water contents (RWC) , chlorophyll contents, soluble protein contents, electrolyte leakage, the contents of malondialdehyde (MDA), and peroxidase (POD) were also measured. RESULT: NaCl at lower concentration could promote the seed germination but inhibit the seed germination at higher concentration. After the treatment by CaCl2 at the different concentrations, all germination indexes were increased. With the increase of salt concentration, the relative water contents and the contents of chlorophyll were decreased, the content of MDA and electrolyte leakage were increased. The change trend of POD activity showed the first increase and then decrease with the increase of salt concentration, which was similar to that of the soluble protein. After the treatment by CaCl2, relative water contents, chlorophyll and POD activities were decreased more slowly, and also electrolyte leakage and MDA contents increased slowly. CONCLUSION: The CaCl2 could significantly alleviate the damages to the seeds and seedlings of L. ruthenium under NaCl stress, and promote the salt resistance to the seeds and seedlings of L. ruthenium.

[Comparative study of growth potential on hardwood cutting of Lycium barbarum strains].

OBJECTIVE: To lay foundation of strains selection through primary selection of 44 strains of Lycium barbarum. METHODS: Used the single plant selection. RESULTS: Screened out 22 strains of Lycium barbarum by preliminary determination of germination rates and seeding rates of hardwood cutting, the growth potential of 2009-26, 2009-21, 2009-17, 2009-29, 2009-2 were better than those of others strains. CONCLUSION: 2009-26 has better growth potential except the length of root, the result provides a basis for further screening can focus on research for 2009-26.

[Effects of covering on growth potential and chlorophyll content of hardwood cutting of Lycium barbarum].

OBJECTIVE: To study the effect of plastic film covering, straw covering, plastic film covering in greenhouse and no-covering cutting on growth and Chlorophyll content of Lycium barbarum hardwood cutting seedlings. METHODS: Single factor randomized block design method was used in this research. RESULTS: The effect of different covering treatments on germination rate, seedling rate and growth potential were all plastic film covering in greenhouse > plastic film covering > straw covering > no-covering cutting. Chlorophyll a, Chlorophyll b, Chlorophyll a + b content and Ca/Cb were significantly different with different coverings in different periods. CONCLUSION: The optimal covering on hardwood cutting of Lycium barbarum is plastic film covering in greenhouse. Both plastic film and straw covering are better than no-covering cutting.

[Effects of irrigation amount on leaf structure, photosynthetic physiology, and fruit yield of Lycium barbarum in arid area].

Lycium barbarum is an important traditional medicinal plant in China. Under controlled condition, a field experiment was conducted to study the effects of different monthly irrigation quota on the leaf structure, photosynthetic physiology, and fruit yield of L. barbarum, aimed to determine an appropriate irrigation amount for the plant. When the monthly irrigation quota was less than 900 m3 x hm(-2), the leaf area, leaf thickness, palisade tissue thickness, cell tense ratio (CTR), net photosynthetic rate (Pn), intrinsic water use efficiency (WUE), stomatal limitation value (Ls), and fruit yield of L. barbarum all increased significantly with monthly irrigation quota, while leaf stoma density and intercellular CO2 concentration (Ci) showed a reverse trend. When the irrigation quota was more than 900 m3 x hm(-2), the Ci increased with irrigation quota, the leaf area, stoma density, and fruit yield had no obvious change, whereas the other indices showed a reverse trend. The leaf transpiration rate and Gs were the highest at irrigation quota 450 m3 x hm(-2), being 8.02 and 324 mmol x m(-2) x s(-1), respectively; whereas at other irrigation quota, these two indices were lower than the control. In terms of saving water, the monthly irrigation quota 900 m3 x hm(-2) was more appropriate for Lycium barbarum.