A new illudane sesquiterpene from the edible fungus Pholiota nameko.

Fungi have different genetic expression abilities and biosynthetic pathways under different cultivation conditions, which can produce various secondary metabolites. The "one strain many compounds" strategy is used to activate silent biosynthetic genes of fungi to produce various compounds, which is an effective method. In order to discover various new compounds in the edible fungus Pholiota nameko, a fermentation strategy involving precursor feeding and enzyme inhibitor addition has been employed. A new illudane sesquiterpene (1), along with one known indole diterpenoid alkaloid, cladosporine A (2) were isolated from the extracts of liquid culture of P. nameko. The new compound was identified by combination of 1D and 2D NMR, MS, optical rotation, and ECD calculations. We conducted experiments on the cytotoxicity of all isolated compounds on three cancer cell lines, but we did not observe any significant cytotoxicity (IC50 > 40 µM).

Molecular characterization reveals that OsSAPK3 improves drought tolerance and grain yield in rice.

BACKGROUND: Many data suggest that the sucrose non-fermenting 1-related kinases 2 (SnRK2s) are very important to abiotic stress for plants. In rice, these kinases are known as osmotic stress/ABA-activated protein kinases (SAPKs). Osmotic stress/ABA-activated protein kinase 3 (OsSAPK3) is a member of SnRK2II in rice, but its function is still unclear. RESULTS: The expression of OsSAPK3 was up regulated by drought, NaCl, PEG and ABA. OsSAPK3 mutated seedings (sapk3-1 and sapk3-2) showed reduced hypersensitivity to exogenous ABA. In addition, under drought conditions, sapk3-1 and sapk3-2 showed more intolerance to drought, including decreased survival rate, increased water loss rate, increased stomatal conductance and significantly decreased expression levels of SLAC1 and SLAC7. Physiological and metabolic analyses showed that OsSAPK3 might play an important role in drought stress signaling pathway by affecting osmotic adjustment and osmolytes, ROS detoxification and expression of ABA dependent and independent dehydration-responsive genes. All gronomic traits analyses demonstrated that OsSAPK3 could improve rice yield by affecting the regulation of tiller numbers and grain size. CONCLUSION: OsSAPK3 plays an important role in both ABA-dependent and ABA-independent drought stress responses. More interestingly, OsSAPK3 could improve rice yield by indirectly regulating tiller number and grain size. These findings provide new insight for the development of drought-resistant rice.

A new chamigrane sesquiterpene from the basidiomycete Antrodiella albocinnamomea.

One new chamigrane sesquiterpene, antroalbol A (1), was isolated from the cultures of the higher fungus Antrodiella albocinnamomea. Its structure was established by means of spectroscopic methods, and the absolute configuration of 1 was confirmed by single crystal x-ray diffraction analysis. The compound was evaluated for its cytotoxicity against five human cancer cell lines, but no significant cytotoxicity was found.

Production of Minor Ginsenosides from Panax notoginseng Flowers by Cladosporium xylophilum.

Panax notoginseng flowers have the highest content of saponins compared to the other parts of Panax notoginseng, but minor ginsenosides have higher pharmacological activity than the main natural ginsenosides. Therefore, this study focused on the transformation of the main ginsenosides in Panax notoginseng flowers to minor ginsenosides using the fungus of Cladosporium xylophilum isolated from soil. The main ginsenosides Rb1, Rb2, Rb3, and Rc and the notoginsenoside Fa in Panax notoginseng flowers were transformed into the ginsenosides F2 and Rd2, the notoginsenosides Fd and Fe, and the ginsenoside R7; the conversion rates were 100, 100, 100, 88.5, and 100%, respectively. The transformation products were studied by TLC, HPLC, and MS analyses, and the biotransformation pathways of the major ginsenosides were proposed. In addition, the purified enzyme of the fungus was prepared with the molecular weight of 66.4 kDa. The transformation of the monomer ginsenosides by the crude enzyme is consistent with that by the fungus. Additionally, three saponins were isolated from the transformation products and identified as the ginsenoside Rd2 and the notoginsenosides Fe and Fd by NMR and MS analyses. This study provided a unique and powerful microbial strain for efficiently transformating major ginsenosides in P. notoginseng flowers to minor ginsenosides, which will help raise the functional and economic value of the P. notoginseng flower.

SAPK2 contributes to rice yield by modulating nitrogen metabolic processes under reproductive stage drought stress.

BACKGROUND: The sucrose non-fermenting 1-related kinases 2 (SnRK2s) play important roles in osmotic stress responses in A. thaliana and rice (Oryza sativa L.). Osmotic stress/ABA-activated protein kinase 2 (SAPK2) is a member of SnRK2s subclass II in rice, but its function in rice yield under drought stress is unclear. RESULTS: Compared with wild-type (Oryza.Sativa L.spp.japonica, WT) plants, the sapk2 rice mutant lines were shorter and produced fewer grains per panicle, smaller grains and lower grain yield under reproductive stage drought stress (RDS). Subsequent analysis suggested that SAPK2 considerably influences the nitrogen, phosphorus, and potassium contents of rice grains. The examination of rice seedling growth and development under nutrient-deprived conditions (-N, -K, and - P) proved that SAPK2 can significantly affect rice seedling growth and root development in hydroponic cultures lacking N and K. Moreover, the NO3- influx rate and nitrate concentration analysis indicated that SAPK2 promotes nitrate uptake and assimilation by regulating nitrate-related transporters. CONCLUSION: These results suggest that SAPK2 could enhance grain production by regulating nitrogen utilization efficiency under RDS. Our work provided insights to breeding drought tolerant rice with high nutrient uptake.

One new sterpurane sesquiterpene from cultures of the basidiomycete Pholiota nameko.

One new sterpurane sesquiterpene (1), named (3R,6S,7S,8R,10S)-3,7,14-trihydroxy-1-sterpurene was isolated from cultures of the basidiomycete Pholiota nameko. The structure of new compound was elucidated by extensive spectroscopic. Additionally, a single crystal X-ray diffraction not only confirmed the structure, but also determined the absolute configuration of the new compound. The compound was evaluated for cytotoxicity against five human cancer cell lines, but no significant cytotoxicity were found (IC50 values > 40 µM).

The sucrose non-fermenting-1-related protein kinases SAPK1 and SAPK2 function collaboratively as positive regulators of salt stress tolerance in rice.

BACKGROUND: The sucrose non-fermenting-1-related protein kinase 2 family (SnRK2s) unifies different abiotic stress signals in plants. To date, the functions of two rice SnRK2s, osmotic stress/ABA-activated protein kinase 1 (SAPK1) and SAPK2, have been unknown. We investigated their roles in response to salt stress by generating loss-of-function lines using the CRISPR/Cas9 system and by overexpressing these proteins in transgenic rice plants. RESULTS: Expression profiling revealed that SAPK1 and SAPK2 expression were strongly induced by drought, NaCl, and PEG treatment, but not by ABA. SAPK2 expression was highest in the leaves, followed by the roots, whereas SAPK1 was highest expressed in roots followed by leaves. Both proteins were localized to the nucleus and the cytoplasm. Under salt stress, sapk1, sapk2 and, in particular, sapk1/2 mutants, exhibited reduced germination rates, more severe growth inhibition, more distinct chlorosis, reduced chlorophyll contents, and reduced survival rates in comparison with the wild-type plants. In contrast, SAPK1- and SAPK2-overexpression lines had increased germination rates and reduced sensitivities to salt; including mild reductions in growth inhibition, reduced chlorosis, increased chlorophyll contents and improved survival rates in comparison with the wild-type plants. These results suggest that SAPK1 and SAPK2 may function collaboratively as positive regulators of salt stress tolerance at the germination and seedling stages. We also found that SAPK1 and SAPK2 affected the osmotic potential following salt stress by promoting the generation of osmotically active metabolites such as proline. SAPK1 and SAPK2 also improved reactive oxygen species (ROS) detoxification following salt stress by promoting the generation of ROS scavengers such as ascorbic acid, and by increasing the expression levels of proteins such as superoxide dismutase (SOD) and catalase (CAT). SAPK1 and SAPK2 may function collaboratively in reducing Na+ toxicity by affecting the Na+ distribution between roots and shoots, Na+ exclusion from the cytoplasm, and Na+ sequestration into the vacuoles. These effects may be facilitated through the expression of Na+-and K+-homeostasis-related genes. CONCLUSION: SAPK1 and SAPK2 may function collaboratively as positive regulators of salt stress tolerance at the germination and seedling stages in rice. SAPK1 and SAPK2 may be useful to improve salt tolerance in crop plants.

OsSAPK2 Confers Abscisic Acid Sensitivity and Tolerance to Drought Stress in Rice.

SNF 1-RELATED PROTEIN KINASE 2 (SnRK2) is a family of plant-specific protein kinases which is the key regulator of hyper-osmotic stress signaling and abscisic acid (ABA)-dependent development in various plants. Among the rice subclass-I and -II SnRK2s, osmotic stress/ABA-activated protein kinase 2 (SAPK2) may be the primary mediator of ABA signaling. However, SAPK2 has not been comprehensively characterized. In this study, we elucidated the functional properties of SAPK2 using loss-of-function mutants produced with the CRISPR/Cas9 system. The SAPK2 expression level was strongly upregulated by drought, high-salinity, and polyethylene glycol (PEG) treatments. The sapk2 mutants exhibited an ABA-insensitive phenotype during the germination and post-germination stages, suggesting that SAPK2 had a pivotal role related to ABA-mediated seed dormancy. The sapk2 mutants were more sensitive to drought stress and reactive oxygen species (ROS) than the wild-type plants, indicating that SAPK2 was important for responses to drought conditions in rice. An additional investigation revealed that SAPK2 increased drought tolerance in the following two ways: (i) by reducing water loss via the accumulation of compatible solutes, promoting stomatal closure, and upregulating the expression levels of stress-response genes such as OsRab16b, OsRab21, OsbZIP23, OsLEA3, OsOREB1 and slow anion channel (SLAC)-associated genes such as OsSLAC1 and OsSLAC7; (ii) by inducing the expression of antioxidant enzyme genes to promote ROS-scavenging abilities that will ultimately decrease ROS damages. Moreover, we also observed that SAPK2 significantly increased the tolerance of rice plants to salt and PEG stresses. These findings imply that SAPK2 is a potential candidate gene for future crop improvement studies.

The DELLA-CONSTANS Transcription Factor Cascade Integrates Gibberellic Acid and Photoperiod Signaling to Regulate Flowering.

Gibberellin (GA) and photoperiod pathways have recently been demonstrated to collaboratively modulate flowering under long days (LDs). However, the molecular mechanisms underlying this collaboration remain largely unclear. In this study, we found that GA-induced expression of FLOWERING LOCUS T (FT) under LDs was dependent on CONSTANS (CO), a critical transcription factor positively involved in photoperiod signaling. Mechanistic investigation revealed that DELLA proteins, a group of crucial repressors in GA signaling, physically interacted with CO. The DELLA-CO interactions repressed the transcriptional function of CO protein. Genetic analysis demonstrated that CO acts downstream of DELLA proteins to regulate flowering. Disruption of CO rescued the earlier flowering phenotype of the gai-t6 rga-t2 rgl1-1 rgl2-1 mutant (dellap), while a gain-of-function mutation in GA INSENSITIVE (GAI, a member of the DELLA gene) repressed the earlier flowering phenotype of CO-overexpressing plants. In addition, the accumulation of DELLA proteins and mRNAs was rhythmic, and REPRESSOR OF GA1-3 protein was noticeably decreased in the long-day afternoon, a time when CO protein is abundant. Collectively, these results demonstrate that the DELLA-CO cascade inhibits CO/FT-mediated flowering under LDs, which thus provide evidence to directly integrate GA and photoperiod signaling to synergistically modulate flowering under LDs.

Selection of highly efficient sgRNAs for CRISPR/Cas9-based plant genome editing.

The CRISPR/Cas9-sgRNA system has been developed to mediate genome editing and become a powerful tool for biological research. Employing the CRISPR/Cas9-sgRNA system for genome editing and manipulation has accelerated research and expanded researchers' ability to generate genetic models. However, the method evaluating the efficiency of sgRNAs is lacking in plants. Based on the nucleotide compositions and secondary structures of sgRNAs which have been experimentally validated in plants, we instituted criteria to design efficient sgRNAs. To facilitate the assembly of multiple sgRNA cassettes, we also developed a new strategy to rapidly construct CRISPR/Cas9-sgRNA system for multiplex editing in plants. In theory, up to ten single guide RNA (sgRNA) cassettes can be simultaneously assembled into the final binary vectors. As a proof of concept, 21 sgRNAs complying with the criteria were designed and the corresponding Cas9/sgRNAs expression vectors were constructed. Sequencing analysis of transgenic rice plants suggested that 82% of the desired target sites were edited with deletion, insertion, substitution, and inversion, displaying high editing efficiency. This work provides a convenient approach to select efficient sgRNAs for target editing.