OsLDDT1, encoding a transmembrane structural DUF726 family protein, is essential for tapetum degradation and pollen formation in rice.

Formation of the pollen wall, which is mainly composed of lipid substances secreted by tapetal cells, is important to ensure pollen development in rice. Although several regulatory factors related to lipid biosynthesis during pollen wall formation have been identified in rice, the molecular mechanisms controlling lipid biosynthesis are unclear. In this study, we isolated the male-sterile rice mutant oslddt1 (leaked and delayed degraded tapetum 1). oslddt1 plants show complete pollen abortion resulting from delayed degradation of the tapetum and blocked formation of Ubisch bodies and pollen walls. OsLDDT1 (LOC_Os03g02170) encodes a DUF726 containing protein of unknown function with highly conserved transmembrane and α/ß Hydrolase domains. OsLDDT1 localizes to the endoplasmic reticulum and the gene is highly expressed in rice panicles. Genes involved in regulating fatty acid synthesis and formation of sporopollenin and pollen exine during anther development showed significantly different expression patterns in oslddt1 plants. Interestingly, the wax and cutin contents in mature oslddt1-1 anthers were decreased by 74.07 % and 72.22 % compared to WT, indicating that OsLDDT1 is involved in fatty acid synthesis and affects formation of the anther epidermis. Our results provide as deeper understanding of the role of OsLDDT1 in regulating male sterility and also provide materials for hybrid rice breeding.

The MYB transcription factor Baymax1 plays a critical role in rice male fertility.

Key message Rice male fertility gene Baymax1, isolated through map-based cloning, encodes a MYB transcription factor and is essential for rice tapetum and microspore development.Abstract The mining and characterization of male fertility gene will provide theoretical and material basis for future rice production. In Arabidopsis, the development of male organ (namely anther), usually involves the coordination between MYB (v-myb avian myeloblastosis viral oncogene homolog) and bHLH (basic helix-loop-helix) members. However, the role of MYB proteins in rice anther development remains poorly understood. In this study, we isolated and characterized a male sterile mutant (with normal vegetative growth) of Baymax1 (BM1), which encodes a MYB protein. The bm1 mutant exhibited slightly lagging meiosis, aborted transition of the tapetum to a secretory type, premature tapetal degeneration, and abnormal pollen exine formation, leading to ultimately lacks of visible pollens in the mature white anthers. Map-based cloning, complementation and targeted mutagenesis using CRISPR/Cas9 technology demonstrated that the mutated LOC_Os04g39470 is the causal gene in bm1. BM1 is preferentially expressed in rice anthers from stage 5 to stage 10. Phylogenetic analysis indicated that rice BM1 and its homologs in millet, maize, rape, cabbage, and pigeonpea are evolutionarily conserved. BM1 can physically interacts with bHLH protein TIP2, EAT1, and PHD (plant homeodomain)-finger member TIP3, respectively. Moreover, BM1 affects the expression of several known genes related to tapetum and microspore development. Collectively, our results suggest that BM1 is one of key regulators for rice male fertility and may serve as a potential target for rice male-sterile line breeding and hybrid seed production.

TDR INTERACTING PROTEIN 3, encoding a PHD-finger transcription factor, regulates Ubisch bodies and pollen wall formation in rice.

Male reproductive development involves a complex series of biological events and precise transcriptional regulation is essential for this biological process in flowering plants. Several transcriptional factors have been reported to regulate tapetum and pollen development, however the transcriptional mechanism underlying Ubisch bodies and pollen wall formation remains less understood. Here, we characterized and isolated a male sterility mutant of TDR INTERACTING PROTEIN 3 (TIP3) in rice. The tip3 mutant displayed smaller and pale yellow anthers without mature pollen grains, abnormal Ubisch body morphology, no pollen wall formation, as well as delayed tapetum degeneration. Map-based cloning demonstrated that TIP3 encodes a conserved PHD-finger protein and further study confirmed that TIP3 functioned as a transcription factor with transcriptional activation activity. TIP3 is preferentially expressed in the tapetum and microspores during anther development. Moreover, TIP3 can physically interact with TDR, which is a key component of the transcriptional cascade in regulating tapetum development and pollen wall formation. Furthermore, disruption of TIP3 changed the expression of several genes involved in tapetum development and degradation, biosynthesis and transport of lipid monomers of sporopollenin in tip3 mutant. Taken together, our results revealed an unprecedented role for TIP3 in regulating Ubisch bodies and pollen exine formation, and presents a potential tool to manipulate male fertility for hybrid rice breeding.

OsMS1 functions as a transcriptional activator to regulate programmed tapetum development and pollen exine formation in rice.

KEY MESSAGE: OsMS1 functions as a transcriptional activator and interacts with known tapetal regulatory factors through its plant homeodomain (PHD) regulating tapetal programmed cell death (PCD) and pollen exine formation in rice. The tapetum, a hallmark tissue in the stamen, undergoes degradation triggered by PCD during post-meiotic anther development. This degradation process is indispensable for anther cuticle and pollen exine formation. Previous study has shown that PTC1 plays a critical role in the regulation of tapetal PCD. However, it remained unclear how this occurs. To further investigate the role of this gene in rice, we used CRISPR/Cas9 system to generate the homozygous mutant named as osms1, which showed complete male sterility with slightly yellow and small anthers, as well as invisible pollen grains. In addition, cytological observation revealed delayed tapetal PCD, defective pollen exine formation and a lack of DNA fragmentation according to a TUNEL analysis in the anthers of osms1 mutant. OsMS1, which encodes a PHD finger protein, was located in the nucleus of rice protoplasts and functioned as a transcription factor with transcriptional activation activity. Y2H and BiFC assays demonstrated that OsMS1 can interact with OsMADS15 and TDR INTERACTING PROTEIN2 (TIP2). It has been reported that TIP2 coordinated with TDR to modulate the expression of EAT1 and further regulated tapetal PCD in rice. Results of qPCR suggested that the expression of the genes associated with tapetal PCD and pollen wall biosynthesis, such as EAT1, AP37, AP25, OsC6 and OsC4, were significantly reduced in osms1 mutant. Taken together, our results demonstrate that the interaction of OsMS1 with known tapetal regulatory factors through its PHD finger regulates tapetal PCD and pollen exine formation in rice.

OsGPAT3 Plays a Critical Role in Anther Wall Programmed Cell Death and Pollen Development in Rice.

In flowering plants, ideal male reproductive development requires the systematic coordination of various processes, in which timely differentiation and degradation of the anther wall, especially the tapetum, is essential for both pollen formation and anther dehiscence. Here, we show that OsGPAT3, a conserved glycerol-3-phosphate acyltransferase gene, plays a critical role in regulating anther wall degradation and pollen exine formation. The gpat3-2 mutant had defective synthesis of Ubisch bodies, delayed programmed cell death (PCD) of the inner three anther layers, and abnormal degradation of micropores/pollen grains, resulting in failure of pollen maturation and complete male sterility. Complementation and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) experiments demonstrated that OsGPAT3 is responsible for the male sterility phenotype. Furthermore, the expression level of tapetal PCD-related and nutrient metabolism-related genes changed significantly in the gpat3-2 anthers. Based on these genetic and cytological analyses, OsGPAT3 is proposed to coordinate the differentiation and degradation of the anther wall and pollen grains in addition to regulating lipid biosynthesis. This study provides insights for understanding the function of GPATs in regulating rice male reproductive development, and also lays a theoretical basis for hybrid rice breeding.

Genomic Regions Analysis of Seedling Root Traits and Their Regulation in Responses to Phosphorus Deficiency Tolerance in CSSL Population of Elite Super Hybrid Rice.

Phosphorus (P) is the essential macro-element supporting rice productivity. Quantitative trait loci (QTL) underlying related traits at the seedling stage under two different phosphorus levels was investigated in rice using a population of 76 Chromosomal Sequence Substitution Lines (CSSLs) derived from a cross between the maintainer variety XieqingzaoB (P stress tolerant) and the restorer variety Zhonghui9308 (P stress sensitive); the parents of super hybrid rice Xieyou9308. A genetic linkage map with 120 DNA marker loci was constructed. At logarithmic odd (LOD) value of 2.0, a total of seven QTLs were detected for studied traits under two P levels and their relative ratio. The LOD values ranged from 2.00 to 3.32 and explaining 10.82% to 18.46% of phenotypic variation. Three QTLs were detected under low phosphorus (P-), one under normal (P⁺) and three under their relative ratio (P-/P⁺) on the rice chromosomes 3, 5, 6, 8 and 10. No significant QTLs were found for shoot dry weight (SDW) and total dry weight (TDW). The pleiotropic QTLs influencing root number (qRN5) and root dry weight (qRDW5) as novel QTLs under P- level were detected near marker RM3638 on chromosome 5, which considered to directly contributing to phosphorus deficiency tolerance in rice. These QTLs need further analysis, including the fine mapping and cloning, which may use in molecular marker assisted breeding.

Identification of cyp703a3-3 and analysis of regulatory role of CYP703A3 in rice anther cuticle and pollen exine development.

Anther cuticle and pollen exine are two elaborated lipid-soluble barriers protecting pollen grains from environmental and biological stresses. However, less is known about the mechanisms underlying the synthesis of these lipidic polymers. Here, we identified a no-pollen male-sterility mutant cyp703a3-3 from the indica restorer line Zhonghui 8015 (Zh8015) mutant library treated with 60Coγ-ray radiation. Histological analysis indicated that cyp703a3-3 underwent abnormal tapetal cells development, produced few orbicules and secreted less sporopollenin precursors to anther locule, as well as cutin monomers on anther. Genetic analysis revealed that cyp703a3-3 was controlled by a single recessive gene. Map-based cloning was performed to narrow down the mutant gene to a 47.78-kb interval on the chromosome 8 between two markers S15-29 and S15-30. Sequence analysis detected three bases (GAA) deletion in the first exon of LOC_Os08g03682, annotated as CYP703A3 with homologous sequences related to male sterility in Arabidopsis, causing the Asparagine deletion in the mutant site. Moreover, we transformed genomic fragment of CYP703A3 into cyp703a3-3, which male-sterility phenotype was recovered. Both the wild-type and cyp703a3-3 mutant 3D structure of CYP703A3 protein were modeled. Results of qPCR suggested CYP703A3 mainly expressed in anthers with greatest abundance at microspore stage, and genes involved in sporopollenin precursors formation and transportation, such as GAMYB, TDR, CYP704B2, DPW2, OsABCG26 and OsABCG15, were significantly reduced in cyp703a3-3. Collectively, our results further elaborated CYP703A3 plays vital role in anther cuticle and pollen exine development in rice (Oryza sativa L.).

Involvement of norepinephrine and serotonin system in antidepressant-like effects of oleoylethanolamide in the mice models of behavior despair.

Oleoylethanolamide (OEA) is an endocannabinoid analogy that belongs to a family of endogenous acylethanolamides. Increasing evidence suggests that OEA may act as an endogenous neuroprotective factor and participate in the control of mental disorder-related behaviors. In the present study, we investigated the antidepressant- like potential of OEA in mice in comparison with clomipramine (Cp). 50 mice were randomly divided into 5 groups, and treated with a vehicle (0.3% methyl cellulose, 20 mL/kg, p.o.), OEA (2.5, 5-10mg/kg, p.o.), or Cp (10mg/kg, p.o.) for 7 days. The immobility was used to evaluate depressive-like behaviors in tail suspension test (TST) and forced swimming test (FST). ELISA detected changes in cerebral noradrenaline (NE) and serotonin (5-HT) levels. Likewise, in the drug-induced model of depression, OEA was given once daily at 10mg/kg (p.o.) for 7 consecutive days. Then, the mice received reserpine (4 mg/kg, i.p.) and the rectal temperature was measured at different time points. Consequently, head twitch behavior induced by intraperitoneal injection of 5-hydroxy-tryptophan (5-HTP; 300 mg/kg) were determined. The experimental data showed that OEA (2.5-10mg/kg) treatment significantly decreased the immobility as compared to the control group, and OEA (10mg/kg) treatment significantly increased 5-HTP-induced head twitch behavior and reversed reserpine-induced hypothermia and increased cerebral levels of NE and 5-HT. Thus, the antidepressant effects of OEA may be related to regulating central monoamine neurotransmitters.

Organized intrasylvian subarachnoid hematoma in post-traumatic child with dyskinesia for 8 years: a case report and review of the literature.

OBJECT: The authors present their experience with an organized intrasylvian subarachnoid hematoma (OISH) in a post-traumatic pediatric patient with dyskinesia for nearly 8 years. METHODS: An 11-year-old Chinese boy was admitted to the authors' hospital because of dyskinesia in his right upper and lower extremities. When he was 18 months old, he fell down from a trolley and then his mouth drooped to a right angle. The brain computer tomography (CT) revealed a space-occupying lesion in his left temporoparietal region. The symptom improved after 20 days of acupuncture therapy in local hospital. Two years later when he was 4 years old, his right lower limb became lame gradually with sensorial deficit. A concealed arteriovenous malformation was suggested by the brain magnetic resonance imaging and magnetic resonance angiography at that time. The child had been treated with ginkgo biloba leaf extract from 2001 to 2007 and the symptom improved gradually during that period. However, the symptom of his right upper and lower extremities deteriorated continually since January 2007. He fell down again when he was walking 1 month before he was admitted to the authors' department in July 2007. An enlarged left pterional craniotomy was performed to remove the lesion. Histopathology diagnosis was compatible with an organized hematoma with remote hemorrhage and gliosis. The child is presently healthy after 1 year's follow-up. CONCLUSION: The rarity of an OISH in a post-traumatic pediatric patient with dyskinesia for nearly 8 years makes this case very peculiar. This is the first reported pediatric case of OISH found in the literature.

Effect of salvia miltiorrhiza Bge on left ventricular hypertrophy and the expression of tumor necrosis factor-alpha in spontaneously hypertensive rats.

The effects of salvia miltiorrhiza Bge (SMB) on left ventricular hypertrophy (LVH) and the expression of tumor necrosis factor-alpha (TNF-alpha) in the left ventricle of spontaneously hypertensive rats and the action mechanism were investigated. Normal Wistar-kyoto (WKY) rats were used as negative control, and spontaneously hypertensive rats (SHR) were randomly assigned to receive placebo or SMB. SMB (1 g/kg.d) was injected intraperitoneally for 12 weeks. Systolic blood pressure (SBP) and left ventricular mass index (LVMI) were measured. HE, VG and immunohistochemical staining combined with computed morphometry were employed to evaluate the cardiomyocyte size, diameter, the collagen volume fraction (CVF), perivascular circumferential area (PVCA), and tumor necrosis factor-alpha (TNF-alpha) expression in the left ventricular tissue. The results showed, as compared with WKY rats, the SBP, LVMI, cardiomyocyte size, diameter, CVF, PCVA, and TNF-alpha expression were increased markedly in the 20-week-old spontaneously hypertensive rats. SMB decreased LVMI (P<0.01), size of cardiomyocytes (P<0.01), collagen volume fraction (P<0.01), perivascular circumferential area (P<0.01), and TNF-alpha expression (P<0.01), but had no effect on SBP (P>0.05). It was suggested that chronic administration of SMB could inhibit and reverse the development of LVH in spontaneously hypertensive rats independent of BP. TNF-alpha may be involved in the reversal mechanism of LVH by SMB.

Protective effects of Hippophae rhamnoides L. juice on lead-induced neurotoxicity in mice.

We examined the effect of Hippophae rhamnoides L. (HRL) juice on lead-induced memory impairment and neuronal damage in the brains of adult mice. Kunming mice were exposed to lead acetate 10 mg/kg body weight for 20 d. Twenty percent and 40% HRL prevented the lead-induced decrease in step-through latency. In the water maze test, the swimming time was lengthened in mice treated with lead acetate, but this time was decreased in mice that received 20% and 40% HRL. The malondialdehyde (MDA) levels were increased in lead-treated mice, which were reduced by 20% and 40% HRL in dose-dependent manner. The activities of acetylcholinesterase (AchE) and monoamine oxidase-A and -B were significantly increased in the lead-treated group, which were decreased by 40% HRL but not by 20% HRL. The levels of norepinephrine, serotonin, and 5-hydroxyindole acetic acid were decreased significantly in the lead-treated mice, and the decreases were antagonized by 40% HRL, except for than in dopamine, but 20% HRL had no effect on this change. These data suggest that the different doses of the HRL juice protect against the lead acetate-induced deficits in learning and memory and changes in neurobiochemical parameters.