Transcriptome profiling reveals characteristics of hairy root and the role of AhGLK1 in response to drought stress and post-drought recovery in peanut.

BACKGROUND: HR (hairy root) has emerged as a valuable tissue for the rapid characterization of plant gene function and enzyme activity in vivo. AhGLK1 (Arachis hypogaea L. golden2-like 1) is known to play a role in post-drought recovery. However, it is unclear (a) whether HR has properties that are distinct from those of PR (primary root); and (b) which gene networks are regulated by AhGLK1 in response to drought stress and recovery in peanut. RESULTS: We found that cells of the root tip cortex were larger in HR than in PR, while a total of 850 differentially expressed genes (DEGs) were identified in HR compared to PR. Eighty-eight of these DEGs, relating to chlorophyll and photosynthesis, were upregulated in HR. In addition, AhGLK1-OX (AhGLK1-overexpressing) HR showed a green phenotype, and had a higher relative water content than 35 S::eGFP (control) HR during drought stress. RNA-seq analysis showed that 74 DEGs involved both in the drought response and the post-drought recovery process were significantly enriched in the galactose metabolism pathway. GO terms enrichment analysis revealed that 59.19%, 29.79% and 17.02% of the DEGs mapped to the 'biological process' (BP), 'molecular function' (MF) and 'cellular component' (CC) domains, respectively. Furthermore, 20 DEGs involved in post-drought recovery were uniquely expressed in AhGLK1-OX HR and were significantly enriched in the porphyrin metabolism pathway. GO analysis showed that 42.42%, 30.30% and 27.28% of DEGs could be assigned to the BP, MF and CC domains, respectively. Transcription factors including bHLH and MYB family members may play a key role during drought stress and recovery. CONCLUSION: Our data reveal that HR has some of the characteristics of leaves, indicating that HR is suitable for studying genes that are mainly expressed in leaves. The RNA-seq results are consistent with previous studies that show chlorophyll synthesis and photosynthesis to be critical for the role of AhGLK1 in improving post-drought recovery growth in peanut. These findings provide in-depth insights that will be of great utility for the exploration of candidate gene functions in relation to drought tolerance and/or post-drought recovery ability in peanut.

Transcriptome profiling reveals histone deacetylase 1 gene overexpression improves flavonoid, isoflavonoid, and phenylpropanoid metabolism in Arachis hypogaea hairy roots.

BACKGROUND: The peanut (Arachis hypogaea) is a crop plant of high economic importance, but the epigenetic regulation of its root growth and development has not received sufficient attention. Research on Arabidopsis thaliana has shown that histone deacetylases (HDACs) are involved in cell growth, cell differentiation, and stress response. Few studies have focused on the role of HDACs in the root development of other plants, particularly crop plants. In earlier studies, we found large accumulations of A. hypogaea histone deacetylase 1 (AhHDA1) mRNA in peanut roots. However, we did not explore the role of AhHDA1 in peanut root development. METHODS: In this paper, we investigated the role of the peanut AhHDA1 gene and focused on the effect of altered AhHDA1 expression in hairy roots at both the phenotypic and transcriptional levels. We analyzed the transformation of A. hypogaea hairy roots using Agrobacterium rhizogenes and RNA sequencing to identify differentially expressed genes that were assigned to specific metabolic pathways. Transgenic hairy roots were used as experimental material to analyze the downstream genes expression and histone acetylation levels. To thoroughly understand AhHDA1 function, we also simultaneously screened the AhHDA1-interacting proteins using a yeast two-hybrid system. RESULTS: AhHDA1-overexpressing hairy roots were growth-retarded after 20 d in vitro cultivation, and they had a greater accumulation of superoxide anions and hydrogen peroxide than the control and RNAi groups. AhHDA1 overexpression in hairy roots accelerated flux through various secondary synthetic metabolic pathways, as well as inhibited the primary metabolism process. AhHDA1 overexpression also caused a significant upregulation of genes encoding the critical enzyme chalcone synthase (Araip.B8TJ0, CHS) in the flavonoid biosynthesis pathway, hydroxyisoflavanone synthase (Araip.0P3RJ) in the isoflavonoid biosynthesis pathway, and caffeoyl-CoA O-methyltransferase (Aradu.M62BY, CCoAOMT) in the phenylpropanoid biosynthesis pathway. In contrast, ferredoxin 1 (Araip.327XS), the polypeptide of the oxygen-evolving complex of photosystem II (Araip.N6ZTJ), and ribulose bisphosphate carboxylase (Aradu.5IY98) in the photosynthetic pathway were significantly downregulated by AhHDA1 overexpression. The expression levels of these genes had a positive correlation with histone acetylation levels. CONCLUSION: Our results revealed that the relationship between altered gene metabolism activities and AhHDA1 overexpression was mainly reflected in flavonoid, isoflavonoid, and phenylpropanoid metabolism. AhHDA1 overexpression retarded the growth of transgenic hairy roots and may be associated with cell metabolism status. Future studies should focus on the function of AhHDA1-interacting proteins and their effect on root development.

Expression of AhDREB1, an AP2/ERF Transcription Factor Gene from Peanut, Is Affected by Histone Acetylation and Increases Abscisic Acid Sensitivity and Tolerance to Osmotic Stress in Arabidopsis.

Drought stress negatively affects plant growth and development. An increasing number of reports have revealed the involvement of APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription factors (TFs) in biotic and abiotic stress regulation in plants. However, research on these TFs in the peanut plant (Arachis hypogaea) has been limited. Here, we isolated a full-length coding sequence (CDS) of the AP2/ERF family gene AhDREB1 from the peanut plant and showed that its expression was induced by Polyethylene Glycol (PEG) 6000 and exogenous abscisic acid (ABA) treatment. When overexpressed in Arabidopsis, AhDREB1 increased both ABA levels and ABA sensitivity, affected the ABA signaling pathway and increased the expression of downstream drought stress-related genes RD29A, P5CS1, P5CS2 and NCED1. These results demonstrate that AhDREB1 can improve tolerance to drought via the ABA-dependent pathway in Arabidopsis. In the peanut plant, the specific histone deacetylases (HDACs) inhibitor trichostatin A (TSA) promotes AhDREB1 transcription and the enrichment level of H3ac was increased in regions of the AhDREB1 gene during TSA and PEG treatment. In summary, histone acetylation can affect the expression of AhDREB1 under osmotic stress conditions, thereby improving plant drought resistance.

AhGLK1 affects chlorophyll biosynthesis and photosynthesis in peanut leaves during recovery from drought.

Peanut is an important edible oil crop plant whose quality and yield are greatly affected by drought. The process and molecular mechanisms of recovery from drought are also critical to its productivity, but are currently poorly characterized. Here, we investigate the involvement of peanut AhGLK1 in recovery from drought, and in particular its relationship with AhPORA, which encodes a key enzyme in chlorophyll biosynthesis. We found that chlorophyll content, chlorophyll fluorescence, AhPORA protein level and genes related to chlorophyll biosynthesis and photosynthesis declined markedly under drought conditions, but all increased during recovery. Consistent with this, AhGLK1 expression decreased during water stress and increased when the stress was removed. When AhGLK1 was transformed into Arabidopsis glk1glk2 mutant, it increased the survival rate of the mutant during recovery from drought and fully rescued the mutant's pale-green phenotype. In addition, chlorophyll content and fluorescence, and the expression of genes related to chlorophyll biosynthesis and photosynthesis, were all increased. Bioinformatics analysis and experimental evidence suggested that AhGLK1 augments the expression of AhPORA by binding to its promoter. Our findings confirm that AhGLK1 plays a role as a transcription factor that upregulates expression of AhPORA during post-drought recovery, thereby stimulating chlorophyll biosynthesis and photosynthesis.

Negative feedback regulation of ABA biosynthesis in peanut (Arachis hypogaea): a transcription factor complex inhibits AhNCED1 expression during water stress.

Abscisic acid (ABA), a key plant stress-signaling hormone, is produced in response to drought and counteracts the effects of this stress. The accumulation of ABA is controlled by the enzyme 9-cis-epoxycarotenoid dioxygenase (NCED). In Arabidopsis, NCED3 is regulated by a positive feedback mechanism by ABA. In this study in peanut (Arachis hypogaea), we demonstrate that ABA biosynthesis is also controlled by negative feedback regulation, mediated by the inhibitory effect on AhNCED1 transcription of a protein complex between transcription factors AhNAC2 and AhAREB1. AhNCED1 was significantly down-regulated after PEG treatment for 10 h, at which time ABA content reached a peak. A ChIP-qPCR assay confirmed AhAREB1 and AhNAC2 binding to the AhNCED1 promoter in response to ABA. Moreover, the interaction between AhAREB1 and AhNAC2, and a transient expression assay showed that the protein complex could negatively regulate the expression of AhNCED1. The results also demonstrated that AhAREB1 was the key factor in AhNCED1 feedback regulation, while AhNAC2 played a subsidiary role. ABA reduced the rate of AhAREB1 degradation and enhanced both the synthesis and degradation rate of the AhNAC2 protein. In summary, the AhAREB1/AhNAC2 protein complex functions as a negative feedback regulator of drought-induced ABA biosynthesis in peanut.

CAPE suppresses migration and invasion of prostate cancer cells via activation of non-canonical Wnt signaling.

Prostate cancer (PCa) was the fifth most common cancer overall in the world. More than 80% of patients died from PCa developed bone metastases. Caffeic acid phenethyl ester (CAPE) is a main bioactive component of honeybee hive propolis. Transwell and wound healing assays demonstrated that CAPE treatment suppressed the migration and invasion of PC-3 and DU-145 PCa cells. Gelatin zymography and Western blotting indicated that CAPE treatment reduced the abundance and activity of MMP-9 and MMP-2. Analysis using Micro-Western Array (MWA), a high-throughput antibody-based proteomics platform with 264 antibodies detecting signaling proteins involved in important pathways indicated that CAPE treatment induced receptor tyrosine kinase-like orphan receptor 2 (ROR2) in non-canonical Wnt signaling pathway but suppressed abundance of ß-catenin, NF-κB activity, PI3K-Akt signaling, and epithelial-mesenchymal transition (EMT). Overexpression or knockdown of ROR2 suppressed or enhanced cell migration of PC-3 cells, respectively. TCF-LEF promoter binding assay revealed that CAPE treatment reduced canonical Wnt signaling. Intraperitoneal injection of CAPE reduced the metastasis of PC-3 xenografts in tail vein injection nude mice model. Immunohistochemical staining demonstrated that CAPE treatment increased abundance of ROR2 and Wnt5a but decreased protein expression of Ki67, Frizzle 4, NF-κB p65, MMP-9, Snail, ß-catenin, and phosphorylation of IκBα. Clinical evidences suggested that genes affected by CAPE treatment (CTNNB1, RELA, FZD5, DVL3, MAPK9, SNAl1, ROR2, SMAD4, NFKBIA, DUSP6, and PLCB3) correlate with the aggressiveness of PCa. Our study suggested that CAPE may be a potential therapeutic agent for patients with advanced PCa.

Isolation and characterization of an osmotic stress and ABA induced histone deacetylase in Arachis hygogaea.

Histone acetylation, which together with histone methylation regulates gene activity in response to stress, is an important epigenetic modification. There is an increasing research focus on histone acetylation in crops, but there is no information to date in peanut (Arachis hypogaea). We showed that osmotic stress and ABA affect the acetylation of histone H3 loci in peanut seedlings by immunoblotting experiments. Using RNA-seq data for peanut, we found a RPD3/HDA1-like superfamily histone deacetylase (HDAC), termed AhHDA1, whose gene is up-regulated by PEG-induced water limitation and ABA signaling. We isolated and characterized AhHDA1 from A. hypogaea, showing that AhHDA1 is very similar to an Arabidopsis HDAC (AtHDA6) and, in recombinant form, possesses HDAC activity. To understand whether and how osmotic stress and ABA mediate the peanut stress response by epigenetics, the expression of AhHDA1 and stress-responsive genes following treatment with PEG, ABA, and the specific HDAC inhibitor trichostatin A (TSA) were analyzed. AhHDA1 transcript levels were enhanced by all three treatments, as was expression of peanut transcription factor genes, indicating that AhHDA1 might be involved in the epigenetic regulation of stress resistance genes that comprise the responses to osmotic stress and ABA.

Elevation of soluble guanylate cyclase suppresses proliferation and survival of human breast cancer cells.

Nitric oxide (NO) is an essential signaling molecule in biological systems. Soluble guanylate cyclase (sGC), composing of α1 and ß1 subunit, is the receptor for NO. Using radioimmunoassay, we discovered that activation of sGC by treatment with bradykinin or sodium nitroprusside (SNP) is impaired in MCF-7 and MDA-MB-231 breast cancer cells as compared to normal breast epithelial 184A1 cells. The 184A1 cells expressed both sGC α1 and sGCß1 mRNAs. However, levels of sGCß1 mRNAs were relatively lower in MCF-7 cells while both mRNA of sGC subunits were absent in MDA-MB-231 cells. Treatment with DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-dC) increased mRNA levels of both sGCα1 and sGCß1 in MDA-MB-231 cells but only sGCß1 mRNAs in MCF-7 cells. The 5-aza-dC treatment increased the SNP-induced cGMP production in MCF-7 and MDA-MB-231, but not in 184A1 cells. Bisulfite sequencing revealed that the promoter of sGCα1 in MDA-MB-231 cells and promoter of sGCß1 in MCF-7 cells were methylated. Promoter hypermethylation of sGCα1 and sGCß1 was found in 1 out of 10 breast cancer patients. Over-expression of both sGC subunits in MDA-MB-231 cells induced apoptosis and growth inhibition in vitro as well as reduced tumor incidence and tumor growth rate of MDA-MB-231 xenografts in nude mice. Elevation of sGC reduced protein abundance of Bcl-2, Bcl-xL, Cdc2, Cdc25A, Cyclin B1, Cyclin D1, Cdk6, c-Myc, and Skp2 while increased protein expression of p53. Our study demonstrated that down-regulation of sGC, partially due to promoter methylation, provides growth and survival advantage in human breast cancer cells.