Arabidopsis Novel Microgametophyte Defective Mutant 1 Is Required for Pollen Viability via Influencing Intine Development in Arabidopsis.

The pollen intine layer is necessary for male fertility in flowering plants. However, the mechanisms behind the developmental regulation of intine formation still remain largely unknown. Here, we identified a positive regulator, Arabidopsis novel microgametophyte defective mutant 1 (AtNMDM1), which influences male fertility by regulating intine formation. The AtNMDM1, encoding a pollen nuclei-localized protein, was highly expressed in the pollens at the late anther stages, 10-12. Both the mutations and the knock-down of AtNMDM1 resulted in pollen defects and significantly lowered the seed-setting rates. Genetic transmission analysis indicated that AtNMDM1 is a microgametophyte lethal gene. Calcofluor white staining revealed that abnormal cellulose distribution was present in the aborted pollen. Ultrastructural analyses showed that the abnormal intine rather than the exine led to pollen abortion. We further found, using transcriptome analysis, that cell wall modification was the most highly enriched gene ontology (GO) term used in the category of biological processes. Notably, two categories of genes, Arabinogalactan proteins (AGPs) and pectin methylesterases (PMEs) were greatly reduced, which were associated with pollen intine formation. In addition, we also identified another regulator, AtNMDM2, which interacted with AtNMDM1 in the pollen nuclei. Taken together, we identified a novel regulator, AtNMDM1 that affected cellulose distribution in the intine by regulating intine-related gene expression; furthermore, these results provide insights into the molecular mechanisms of pollen intine development.

Emerin anchors Msx1 and its protein partners at the nuclear periphery to inhibit myogenesis.

BACKGROUND: Previous studies have shown that in myogenic precursors, the homeoprotein Msx1 and its protein partners, histone methyltransferases and repressive histone marks, tend to be enriched on target myogenic regulatory genes at the nuclear periphery. The nuclear periphery localization of Msx1 and its protein partners is required for Msx1's function of preventing myogenic precursors from pre-maturation through repressing target myogenic regulatory genes. However, the mechanisms underlying the maintenance of Msx1 and its protein partners' nuclear periphery localization are unknown. RESULTS: We show that an inner nuclear membrane protein, Emerin, performs as an anchor settled at the inner nuclear membrane to keep Msx1 and its protein partners Ezh2, H3K27me3 enriching at the nuclear periphery, and participates in inhibition of myogenesis mediated by Msx1. Msx1 interacts with Emerin both in C2C12 myoblasts and mouse developing limbs, which is the prerequisite for Emerin mediating the precise location of Msx1, Ezh2, and H3K27me3. The deficiency of Emerin in C2C12 myoblasts disturbs the nuclear periphery localization of Msx1, Ezh2, and H3K27me3, directly indicating Emerin functioning as an anchor. Furthermore, Emerin cooperates with Msx1 to repress target myogenic regulatory genes, and assists Msx1 with inhibition of myogenesis. CONCLUSIONS: Emerin cooperates with Msx1 to inhibit myogenesis through maintaining the nuclear periphery localization of Msx1 and Msx1's protein partners.

The homeoprotein Msx1 cooperates with Pkn1 to prevent terminal differentiation in myogenic precursor cells.

The homeoprotein Msx1 plays a critical role in embryonic patterning, particularly to maintain myogenic progenitor cell fate. However, the mechanisms underlying Msx1-mediated inhibition of myogenesis remain largely unknown. Here, we show that Msx1 cooperates with the protein kinase C-related kinase 1 (Pkn1), a member of the protein kinase C-related kinase family, to prevent the terminal differentiation of myogenic precursor cells. In mouse C2C12 cells, Pkn1 knockout partly impaired Msx1-mediated inhibition of myogenic differentiation, indicating a role for Pkn1 in this process. Furthermore, we found that Pkn1 was required for Msx1 enrichment at the promoter of Myf5, a myogenic regulatory gene. In Pkn1 knockout cells, this reduced Msx1 enrichment at the Myf5 promoter coincided with attenuated repression of Myf5 transcription. Together with our observation that Msx1 and Pkn1 were associated in a protein complex, these findings strongly suggest that Msx1 cooperates with Pkn1 to down-regulate Myf5 and, therefore, prevent the differentiation of myogenic precursor cells. Collectively, our data provide key insights into the mechanisms underlying Msx1 function in the prevention of myogenic differentiation.

The Arabidopsis CALLOSE DEFECTIVE MICROSPORE1 gene is required for male fertility through regulating callose metabolism during microsporogenesis.

During angiosperm microsporogenesis, callose serves as a temporary wall to separate microsporocytes and newly formed microspores in the tetrad. Abnormal callose deposition and dissolution can lead to degeneration of developing microspores. However, genes and their regulation in callose metabolism during microsporogenesis still remain largely unclear. Here, we demonstrated that the Arabidopsis (Arabidopsis thaliana) CALLOSE DEFECTIVE MICROSPORE1 (CDM1) gene, encoding a tandem CCCH-type zinc finger protein, plays an important role in regulation of callose metabolism in male meiocytes and in integrity of newly formed microspores. First, quantitative reverse transcription PCR and in situ hybridization analyses showed that the CDM1 gene was highly expressed in meiocytes and the tapetum from anther stages 4 to 7. In addition, a transfer DNA insertional cdm1 mutant was completely male sterile. Moreover, light microscopy of anther sections revealed that microspores in the mutant anther were initiated, and then degenerated soon afterward with callose deposition defects, eventually leading to male sterility. Furthermore, transmission electron microscopy demonstrated that pollen exine formation was severely affected in the cdm1 mutant. Finally, we found that the cdm1 mutation affected the expression of callose synthesis genes (CALLOSE SYNTHASE5 and CALLOSE SYNTHASE12) and potential callase-related genes (A6 and MYB80), as well as three other putative ß-1,3-glucanase genes. Therefore, we propose that the CDM1 gene regulates callose metabolism during microsporogenesis, thereby promoting Arabidopsis male fertility.

Analysis of Arabidopsis genome-wide variations before and after meiosis and meiotic recombination by resequencing Landsberg erecta and all four products of a single meiosis.

Meiotic recombination, including crossovers (COs) and gene conversions (GCs), impacts natural variation and is an important evolutionary force. COs increase genetic diversity by redistributing existing variation, whereas GCs can alter allelic frequency. Here, we sequenced Arabidopsis Landsberg erecta (Ler) and two sets of all four meiotic products from a Columbia (Col)/Ler hybrid to investigate genome-wide variation and meiotic recombination at nucleotide resolution. Comparing Ler and Col sequences uncovered 349,171 Single Nucleotide Polymorphisms (SNPs), 58,085 small and 2315 large insertions/deletions (indels), with highly correlated genome-wide distributions of SNPs, and small indels. A total of 443 genes have at least 10 nonsynonymous substitutions in protein-coding regions, with enrichment for disease-resistance genes. Another 316 genes are affected by large indels, including 130 genes with complete deletion of coding regions in Ler. Using the Arabidopsis qrt1 mutant, two sets of four meiotic products were generated and analyzed by sequencing for meiotic recombination, representing the first tetrad analysis with whole-genome sequencing in a nonfungal species. We detected 18 COs, six of which had an associated GC event, and four GCs without COs (NCOs), and revealed that Arabidopsis GCs are likely fewer and with shorter tracts than those in yeast. Meiotic recombination and chromosome assortment events dramatically redistributed genome variation in meiotic products, contributing to population diversity. In particular, meiosis provides a rapid mechanism to generate copy-number variation (CNV) of sequences that have different chromosomal positions in Col and Ler.

[The effect of pressure on binary solution phase transition and fermi resonance--comparing pressure effect for binary solution and pure liquid].

The Raman spectra of binary solution (CCl4 and C6 H6) and pure liquid were measured up to pressures of 11 GPa. The results show that pressure effect on binary solution is different from that on pure liquid: When mixing two liquids, owing to the changes in the density of the solution, intermolecular distance decreases and interaction energy increases, the frequency shift (blue shift) of spectral bands increases, and the frequency shift of binary liquid is faster than pure liquid frequency shift. Phase transitions (spectral bands splitting) change earlier and natural frequency difference delta0 increases with increasing pressure, while the Fermi resonance bands nu1 + nu6 and nu8 of benzene and nu1 + nu4 and nu3 of CCl4 disappear as the pressure decreases gradually, the spectral bands with different compressibility have different speed, whereas CCl4 has smaller density, longer bond, smaller force constant and larger compressibility and is easy to compress, C6H6 has larger density, smaller bond, larger force constant, smaller compressibility and is hard to compress. The frequency shift of CCl4 is faster than benzene. In this paper we provide good reference on Raman bands assignment and certification under high pressure, and also provide methods and ideas for study of the different environment of high pressure effect, intermolecular interaction and solvent effect.

[Effect of the change in fundamental Raman scattering coefficient with the concentration on Fermi resonance in binary solution].

The Raman scattering coefficients of the fundamental v1 (992 cm(-1)) of C6 H6 and the fundamental v1 (656 cm(-1) ) of CS2 changed dramatically with relative concentration of the binary solution of CS2 and C6 H6. The Raman spectra of the binary solution with different relative concentrations were measured. The results show that both the v1 fundamentals intensities changed dramatically with the relative concentration of the solution and the fundamental v1 of C6 H6 has little effect on the Fermi resonance v1 +v6-v8. On the contrary, the change in the v1 fundamental intensity of the CS2 changed which has more effect not only on the Fermi resonance v1 - 2v2 but also on the v2 fundamental. In this report, the experimental results were analyzed based on the J. F. Bertran theory and group theory.

[Investigation of hydrophobic effect on the hydrogen-bond formation of 1,1,3,3-tetramethylurea by Raman methods].

The binary systems of 1,1,3,3-tetramethylurea (TMU) with water, TMU with methanol, TMU with ethanol and N,N-dimethylformamide(DMF) with water were measured by Raman method. With the analysis of the frequency changes of stretching vibration of carboxyl with concentration the authors found that the frequency shift underwent two processes: first, the frequency of stretching vibration of carboxyl down shifts with the increase in hydrogen-bond acceptor concentration; secondly, when the concentration of binary system surpasses a critical value, the wave number remains almost constant, and only the rela tive intensity changes. Through this critical volume ratio, the authors found that the large self-associated water molecule was in volved in the TMU aqueous binary system; while the small self-associated molecule or dimer formation was present in other binary systems.

[Lycopene and beta-carotene content in tomato analyzed by the second harmonic].

Lycopene and beta-carotene are two important nutritional components in tomato. The main Raman spectrum group of lycopene and beta-carotene abundant in tomato is identical and difficult to be distinguished through fundamental frequency. With excitation wavelength of 514.5 nm, the excited light was just present in the half width range of the main absorption bands of Lycopene and beta-carotene, so the resonance Raman effect can occur. Based on resonance Raman spectra, by on-body measuring the second harmonic of stretching vibration of carbon-carbon conjugated double bond in lycopene and beta-carotene, the content of lycopene and beta-carotene can be obtained according to the integrated intensity of each component calculated by software. And this provides a method for on-body determining the content of the components with the homologous group.

3',5'-cyclic adenosine monophosphate response element binding protein up-regulated cytochrome P450 lanosterol 14alpha-demethylase expression involved in follicle-stimulating hormone-induced mouse oocyte maturation.

Cytochrome P450 lanosterol 14alpha-demethylase (CYP51) is a key enzyme in sterols and steroids biosynthesis that can induce meiotic resumption in mouse oocytes. The present study investigated the expression mechanism and function of CYP51 during FSH-induced mouse cumulus oocyte complexes (COCs) meiotic resumption. FSH increased cAMP-dependent protein kinase (PKA) RIIbeta level and induced cAMP response element-binding protein (CREB) phosphorylation and CYP51 expression in cumulus cells before oocyte meiotic resumption. Moreover, CYP51 and epidermal growth factor (EGF)-like factor [amphiregulin (AR)] expression were blocked by (2)-naphthol-AS-Ephosphate (KG-501) (a drug interrupting the formation of CREB functional complex). KG-501 and RS21607 (a specific inhibitor of CYP51 activity) inhibited oocyte meiotic resumption, which can be partially rescued by progesterone. These two inhibitors also inhibited FSH-induced MAPK phosphorylation. EGF could rescue the suppression by KG-501 but not RS21607. Furthermore, type II PKA analog pairs, N(6)-monobutyryl-cAMP plus 8-bromo-cAMP, increased PKA RIIbeta level and mimicked the action of FSH, including CREB phosphorylation, AR and CYP51 expression, MAPK activation, and oocyte maturation. All these data suggest that CYP51 plays a critical role in FSH-induced meiotic resumption of mouse oocytes. CYP51 and AR gene expression in cumulus cells are triggered by FSH via a type II PKA/CREB-dependent signal pathway. Our study also implicates that CYP51 activity in cumulus cells participates in EGF receptor signaling-regulated oocyte meiotic resumption.

PDE5 modulates oocyte spontaneous maturation via cGMP-cAMP but not cGMP-PKG signaling.

Phosphodiesterase type 5 (PDE5), a cGMP specific, cGMP binding phosphodiesterase, specifically hydrolyzes cGMP to 5'-GMP. Here, we examine the distribution of PDE5 in mouse ovary and its effects on spontaneous maturation of mouse oocytes. PDE5 is present in oocytes and cumulus cells of big, antral follicles. Inhibition of activity of PDE5 significantly and reversibly inhibits spontaneous maturation of cumulus-oocyte complexes (COCs). Suppressive effect of PDE5 on spontaneous maturation of COCs is not blocked by the inhibitor of cGMP-dependent protein kinase (PKG). While Sildenafil, an inhibitor of PDE5, has a poor effect on cGMP levels, it significantly increases cAMP levels. These results suggest that the activity of PDE5 plays a role in regulating spontaneous maturation of mouse oocytes and imply that an interaction between cGMP and cAMP signal is involved in this process.

Luteinizing hormone receptors expression in cumulus cells closely related to mouse oocyte meiotic maturation.

In the process of oocyte maturation, gonadotrophins are believed as main stimulators for oocyte meiosis resumption. However, which gonadotrophin (i.e. FSH or LH) is the key hormone in this process is still unknown. This study indicated a close relationship between LH and FSH on activating meiotic maturation of oocyte in vitro. FSH efficiently induced oocyte meiosis at concentration of 50 IU/L, while LH alone had no effect on oocyte meiotic initiation. Using RT-PCR and in situ hybridization, a tight corelationship between FSH-stimulated oocyte meiotic resumption and LHR mRNA expression in cumulus cells was found. LHR mRNA was present in cumulus cells of oocyte-cumulus cell complexes. Except the expression of LHRs was present in cumulus cells surrounding all maturing oocytes, low level expression was also detected in cells associated with oocytes that were still at GV stage. Its expression was enhanced by FSH stimulation before oocyte maturation. However, LHRs did not express in cumulus cells associated with oocytes that were completely arrested at GV stage by IBMX. Furthermore, increased progesterone concentration was found in the medium in which CEOs were cultured with FSH and LH, but not in those with FSH or LH alone. LHR expression in cumulus cells increases with time in culture, and the levels of expression are enhanced in the presence of FSH. Oocyte meiotic resumption may create conditions favorable for LHR expression. LHR expression may be considered as a potential marker for oocytes maturation initiation.