16-237 Sigma-AldrichNormal Rabbit IgG, Alexa Fluor® 488 conjugate

Post-translational modifications of histone proteins play a crucial role in responding to environmental stresses. Histone deacetylases (HDACs) catalyze the removal of an acetyl group from histones and are generally believed to be a transcriptional repressor. In this paper, we report that cold treatment highly induces the up-regulation of HDACs, leading to global deacetylation of histones H3 and H4. Treatment of maize with the HDAC inhibitor trichostatin A (TSA) under cold stress conditions strongly inhibits induction of the maize cold-responsive genes ZmDREB1 and ZmCOR413. However, up-regulation of the ZmICE1 gene in response to cold stress is less affected. The expression of drought and salt induced genes, ZmDBF1 and rab17, is almost unaffected by TSA treatment. Thus, these observations show that HDACs may selectively activate transcription. The time course of TSA effects on the expression of ZmDREB1 and ZmCOR413 genes indicates that HDACs appear to directly activate the ZmDREB1 gene, which in turn modulates ZmCOR413 expression. After cold treatment, histone hyperacetylation and DNA demethylation occurs in the ICE1 binding region, accompanied by an increase in accessibility to micrococcal nuclease (MNase). The two regions adjacent to the ICE1 binding site remain hypoacetylated and methylated. However, during cold acclimation, TSA treatment increases the acetylation status and accessibility of MNase and decreases DNA methylation at these two regions. However, TSA treatment does not affect histone hyperacetylation and DNA methylation levels at the ICE1 binding regions of the ZmDREB1 gene. Altogether, our findings indicate that HDACs positively regulate the expression of the cold-induced ZmDREB1 gene through histone modification and chromatin conformational changes and that this activation is both gene and site selective.

Recent studies have suggested that nitric oxide (NO) may function as both an intracellular and intercellular signal that affects neural and immunological activity, vascular tone, platelet adhesion, and production of some hormones. Arginine analogs such as NG-monomethyl-L-arginine (L-NMMA) and N omega-nitro-L-arginine methyl ester (L-NAME) act to inhibit the intracellular formation of NO and have been used to study the effects of decreased NO formation on physiological systems. A single in vivo study has suggested that a similar analog, NG-nitro-L-arginine, increases serum testosterone (T), but the organ site and mechanism of action were not investigated. The present study was performed to investigate the effects of NO synthase inhibitors on Leydig cell function. L-NMMA and L-NAME, but not the inactive enantiomer (D-NMMA), increased both basal and human chorionic gonadotropin (hCG)-stimulated T production while decreasing guanosine 3':5'-cyclic monophosphate (cGMP). There was no effect on either adenosine 3':5'-cyclic monophosphate (cAMP) accumulation or specific hCG binding. These results suggest that NO formation, which is inhibited by L-NMMA and L-NAME, is important in the regulation of Leydig cell T production by interstitial cells of the testis, and that changes in cGMP levels might be involved in this process.