Novel (p)ppGpp0 suppressor mutations reveal an unexpected link between methionine catabolism and GTP synthesis in Bacillus subtilis

In bacteria, guanosine (penta)tetra-phosphate ([p]ppGpp) is essential for controlling intracellular metabolism that is needed to adapt to environmental changes, such as amino acid starvation. The (p)ppGpp⁰ strain of Bacillus subtilis, which lacks (p)ppGpp synthetase, is unable to form colonies on minimal medium. Here, we found suppressor mutations in the (p)ppGpp⁰ strain, in the purine nucleotide biosynthesis genes, prs, purF and rpoB/C, which encode RNA polymerase core enzymes. In comparing our work with prior studies of ppGpp⁰ suppressors, we discovered that methionine addition masks the suppression on minimal medium, especially of rpoB/C mutations. Furthermore, methionine addition increases intracellular GTP in rpoB suppressor and this effect is decreased by inhibiting GTP biosynthesis, indicating that methionine addition activated GTP biosynthesis and inhibited growth under amino acid starvation conditions in (p)ppGpp⁰ backgrounds. Furthermore, we propose that the increase in intracellular GTP levels induced by methionine is due to methionine derivatives that increase the activity of the de novo GTP biosynthesis enzyme, GuaB. Our study sheds light on the potential relationship between GTP homeostasis and methionine metabolism, which may be the key to adapting to environmental changes.     

Requirements for human embryonic stem cells

‘Requirements for Human Embryonic Stem Cells’ is the first set of guidelines on human embryonic stem cells in China, jointly drafted and agreed upon by experts from the Chinese Society for Stem Cell Research. This standard specifies the technical requirements, test methods, test regulations, instructions for use, labelling requirements, packaging requirements, storage requirements and transportation requirements for human embryonic stem cells, which is applicable to the quality control for human embryonic stem cells. It was originally released by the China Society for Cell Biology on 26 February 2019 and was further revised on 30 April 2020. We hope that publication of these guidelines will promote institutional establishment, acceptance and execution of proper protocols, and accelerate the international standardization of human embryonic stem cells for applications.     

Ultra‐high α‐linolenic acid accumulating developmental defective embryo was rescued by lysophosphatidic acid acyltransferase 2

For decades, genetic engineering approaches to produce unusual fatty acids (UFAs) in crops has reached a bottleneck, including reduced seed oil production and seed vigor. Currently, plant models in the field of research are primarily used to investigate defects in oil production and seedling development, while the role of UFAs in embryonic developmental defects remains unknown. In this study, we developed a transgenic Arabidopsis plant model, in which the embryo exhibits severely wrinkled appearance owing to α‐linolenic acid (ALA) accumulation. RNA‐sequencing analysis in the defective embryo suggested that brassinosteroid synthesis, FA synthesis and photosynthesis were inhibited, while FA degradation, endoplasmic reticulum stress and oxidative stress were activated. Lipidomics analysis showed that ultra‐accumulated ALA is released from phosphatidylcholine as a free FA in cells, inducing severe endoplasmic reticulum and oxidative stress. Furthermore, we identified that overexpression of lysophosphatidic acid acyltransferase 2 rescued the defective phenotype. In the rescue line, the pool capacity of the Kennedy pathway was increased, and the esterification of ALA indirectly to triacylglycerol was enhanced to avoid stress. This study provides a plant model that aids in understanding the molecular mechanism of embryonic developmental defects and generates strategies to produce higher levels of UFAs.     

Sl‐lncRNA15492 interacts with Sl‐miR482a and affects Solanum lycopersicum immunity against Phytophthora infestans

Long non‐coding RNAs (lncRNAs) are involved in the resistance of plants to infection by pathogens via interactions with microRNAs (miRNAs). Long non‐coding RNAs are cleaved by miRNAs to produce phased small interfering RNAs (phasiRNAs), which, as competing endogenous RNAs (ceRNAs), function as decoys for mature miRNAs, thus inhibiting their expression, and contain pre‐miRNA sequences to produce mature miRNAs. However, whether lncRNAs and miRNAs mediate other molecular mechanisms during plant resistance to pathogens is unknown. In this study, as a positive regulator, Sl‐lncRNA15492 from tomato (Solanum lycopersicum Zaofen No. 2) plants affected tomato resistance to Phytophthora infestans. Gain‐ and loss‐of‐function experiments and RNA ligase‐mediated 5′‐amplification of cDNA ends (RLM‐5′ RACE) also revealed that Sl‐miR482a was negatively involved in tomato resistance by targeting Sl‐NBS‐LRR genes and that silencing of Sl‐NBS‐LRR1 decreased tomato resistance. Sl‐lncRNA15492 inhibited the expression of mature Sl‐miR482a, whose precursor was located within the antisense sequence of Sl‐lncRNA15492. Further degradome analysis and additional RLM‐5′ RACE experiments verified that mature Sl‐miR482a could also cleave Sl‐lncRNA15492. These results provide a mechanism by which lncRNAs might inhibit precursor miRNA expression through antisense strands of lncRNAs, and demonstrate that Sl‐lncRNA15492 and Sl‐miR482a mutually inhibit the maintenance of Sl‐NBS‐LRR1 homeostasis during tomato resistance to P. infestans.     

Overexpression of the persimmon abscisic acid β‐glucosidase gene (DkBG1) alters fruit ripening in transgenic tomato

β‐Glucosidases (BG) are present in many plant tissues. Among these, abscisic acid (ABA) β‐glucosidases are thought to take part in the adjustment of cellular ABA levels, however the role of ABA‐BG in fruits is still unclear. In this study, through RNA‐seq analysis of persimmon fruit, 10 full‐length DkBG genes were isolated and were all found to be expressed. In particular, DkBG1 was highly expressed in persimmon fruits with a maximum expression 95 days after full bloom (DAFD). We verified that, in vitro, DkBG1 protein can hydrolyze ABA‐glucose ester (ABA‐GE) to release free ABA. Compared with wild‐type, tomato plants that overexpressed DkBG1 significantly upregulated the expression of ABA receptor PYL3/7 genes and showed typical symptoms of ABA hypersensitivity in fruits. DkBG1 overexpression (DkBG1‐OE) accelerated fruit ripening onset by 3–4 days by increasing ABA levels at the pre‐breaker stage and induced early ethylene release compared with wild‐type fruits. DkBG1‐OE altered the expression of ripening regulator NON‐RIPENING (NOR) and its target genes; this in turn altered fruit quality traits such as coloration. Our results demonstrated that DkBG1 plays an important role in fruit ripening and quality by adjusting ABA levels via hydrolysis of ABA‐GE.