Matrilineal empowers wheat pollen with haploid induction potency by triggering postmitosis reactive oxygen species activity.

Reactive oxygen species (ROS) play important roles during anther and pollen development. DNA damage may cause chromosome fragmentation that is considered to underlie chromosome elimination for haploid induction by matrilineal pollen, a key step in MATRILINEAL-based double haploid breeding technology. But when and how DNA damage occurs is unknown. We performed comparative studies of wheat pollens from the wild-type and the CRISPR/Cas9 edited matrilineal mutant (mMTL). Chemical assays detected a second wave of ROS in mMTL pollen at the three-nuclei-stage and subsequently, along with reduced antioxidant enzyme activities. RNA-seq analysis revealed disturbed expression of genes for fatty acid biosynthesis and ROS homoeostasis. Gas chromatography-mass spectrometry measurement identified abnormal fatty acid metabolism that may contribute to defective mMTL pollen walls as observed using electron microscopy, consistent with the function of MTL as a phospholipase. Moreover, DNA damage was identified using TdT-mediated dUTP nick-end labelling and quantified using comet assays. Velocity patterns showed that ROS increments preceded that of DNA damage over the course of pollen maturation. Our work hypothesises that mMTL-triggered later-stage-specific ROS causes DNA damage that may contribute to chromosome fragmentation and hence chromosome elimination during haploid induction. These findings may provide more ways to accelerate double haploid-based plant breeding.

Tacrolimus Protects Podocytes from Injury in Lupus Nephritis Partly by Stabilizing the Cytoskeleton and Inhibiting Podocyte Apoptosis.

OBJECTIVE: Several studies have reported that tacrolimus (TAC) significantly reduced proteinuria in lupus nephritis (LN) patients and mouse models. However, the mechanism for this effect remains undetermined. This study explored the mechanism of how TAC protects podocytes from injury to identify new targets for protecting renal function. METHODS: MRL/lpr mice were given TAC at a dosage of 0.1 mg/kg per day by intragastric administration for 8 weeks. Urine and blood samples were collected. Kidney sections (2 µm) were stained with hematoxylin-eosin (HE), periodic acid-Schiff base (PAS) and Masson's trichrome stain. Mouse podocyte cells (MPC5) were treated with TAC and/or TGF-ß1 for 48 h. The mRNA levels and protein expression of synaptopodin and Wilms' tumor 1 (WT1) were determined by real-time PCR, Western blotting and/or immunofluorescence, respectively. Flow cytometry was used to detect cell apoptosis with annexin V. Podocyte foot processes were observed under transmission electron microscopy. IgG and C3 deposition were assessed with immunofluorescence assays and confocal microscopy. RESULTS: Synaptopodin expression significantly decreased in MRL/lpr disease control mice, accompanied by increases in 24-h proteinuria, blood urea nitrogen, and serum creatinine. TAC, however, reduced proteinuria, improved renal function, attenuated renal pathology, restored synaptopodin expression and preserved podocyte numbers. In MPC5 cells, TGF-ß1 enhanced F-actin damage in podocytes and TAC stabilized it. TAC also decreased TGF-ß1-induced podocyte apoptosis in vitro and inhibited foot process fusion in MRL/lpr mice. In addition, our results also showed TAC inhibited glomerular deposition of IgG and C3. CONCLUSION: This study demonstrated that TAC reduced proteinuria and preserved renal function in LN through protecting podocytes from injury partly by stabilizing podocyte actin cytoskeleton and inhibiting podocyte apoptosis.