Transcriptome Analysis of Eggplant under Salt Stress: AP2/ERF Transcription Factor SmERF1 Acts as a Positive Regulator of Salt Stress

Salt stress, a type of abiotic stress, impedes plant growth and development and strongly reduces crop yield. The molecular mechanisms underlying plant responses to salt stress remain largely unclear. To characterize the enriched pathways and genes that were affected during salt treatment, we performed mRNA sequencing (mRNA-seq) in eggplant roots and identified 8509 differentially expressed genes (DEGs) between the mock and 24 h under salt stress. Among these DEGs, we found that the AP2/ERF transcription factor family member SmERF1 belongs to the plant–pathogen interaction pathway, which was significantly upregulated by salt stress. We found that SmERF1 localizes in the nuclei with transcriptional activity. The results of the virus-induced gene silencing assay showed that SmERF1 silencing markedly enhanced the susceptibility of plants to salt stress, significantly downregulated the transcript expression levels of salt stress defense-related marker genes (9-cis-epoxycarotenoid dioxygenase [SmNCED1, SmNCED2], Dehydrin [SmDHN1], and Dehydrin (SmDHNX1), and reduced the activity of superoxide dismutase and catalase. Silencing SmERF1 promoted the generation of H2O2 and proline. In addition, the transient overexpression of SmERF1 triggered intense cell death in eggplant leaves, as assessed by the darker diaminobenzidine and trypan blue staining. These findings suggest that SmERF1 acts as a positive regulator of eggplant response to salt stress. Hence, our results suggest that AP2/ERF transcription factors play a vital role in the response to salt stress

Stathmin mediates hepatocyte resistance to death from oxidative stress by down regulating JNK.

Stathmin 1 performs a critical function in cell proliferation by regulating microtubule polymerization. This proliferative function is thought to explain the frequent overexpression of stathmin in human cancer and its correlation with a bad prognosis. Whether stathmin also functions in cell death pathways is unclear. Stathmin regulates microtubules in part by binding free tubulin, a process inhibited by stathmin phosphorylation from kinases including c-Jun N-terminal kinase (JNK). The involvement of JNK activation both in stathmin phosphorylation, and in hepatocellular resistance to oxidative stress, led to an examination of the role of stathmin/JNK crosstalk in oxidant-induced hepatocyte death. Oxidative stress from menadione-generated superoxide induced JNK-dependent stathmin phosphorylation at Ser-16, Ser-25 and Ser-38 in hepatocytes. A stathmin knockdown sensitized hepatocytes to both apoptotic and necrotic cell death from menadione without altering levels of oxidant generation. The absence of stathmin during oxidative stress led to JNK overactivation that was the mechanism of cell death as a concomitant knockdown of JNK1 or JNK2 blocked death. Hepatocyte death from JNK overactivation was mediated by the effects of JNK on mitochondria. Mitochondrial outer membrane permeabilization occurred in stathmin knockdown cells at low concentrations of menadione that triggered apoptosis, whereas mitochondrial β-oxidation and ATP homeostasis were compromised at higher, necrotic menadione concentrations. Stathmin therefore mediates hepatocyte resistance to death from oxidative stress by down regulating JNK and maintaining mitochondrial integrity. These findings demonstrate a new mechanism by which stathmin promotes cell survival and potentially tumor growth

Relationships between X-ray Diffraction Peaks, Molecular Components, and Heat Properties of C-Type Starches from Different Sweet Potato Varieties

C-type starches with different proportions of A- and B-type crystallinities have different intensities and crystallinities of X-ray diffraction peaks. In this study, the intensities and crystallinities of X-ray diffraction peaks, molecular components and heat properties of C-type starches were investigated in seven sweet potato varieties, and their relationships were analyzed. The intensity and crystallinity of a diffraction peak at 5.6° were significantly positively correlated to the DP6-12 branch-chains of amylopectin and significantly negatively correlated to the true amylose content (TAC) determined by concanavalin A precipitation, gelatinization temperature, gelatinization enthalpy, water solubility at 95 °C, and pasting temperature. The intensity of diffraction peaks at 15° and 23° were significantly positively correlated to the gelatinization temperature and pasting temperature and significantly negatively correlated to the pasting peak viscosity. The significantly positive relationships were detected between the crystallinity of a diffraction peak at 15° and the DP13-24 branch-chains of amylopectin, gelatinization conclusion temperature and water solubility, between the crystallinity of diffraction peak at 17–18° and the TAC, gelatinization onset temperature, water solubility and pasting temperature, between the crystallinity of a diffraction peak at 23° and the gelatinization conclusion temperature and pasting peak time, and between the total crystallinity and the TAC, gelatinization conclusion temperature, water solubility and pasting temperature. The score plot of principle component analysis showed that the molecular components and heat property parameters could differentiate the C-type starches and agreed with their characteristics of X-ray diffraction peaks. This study provides some references for the utilizations of C-type starches

Working model of the regulation by stathmin of JNK-dependent hepatocyte death from oxidative stress.

<p>Increased superoxide generation triggers phosphorylation of MKK4 which then phosphorylates and activates JNK. If activated for a long enough period of time, JNK compromises mitochondrial integrity leading to cytochrome c (Cyt c) release and apoptosis or ATP depletion and necrosis. However, JNK also phosphorylates stathmin which acts through a negative feedback loop to suppress phosphorylation of MKK4 and its downstream substrate JNK to promote cell survival.</p

shRNA Sequences.

<p>shRNA Sequences.</p

Menadione-induced stathmin phosphorylation is JNK dependent.

<p>(a) Wild-type cells were pretreated with dimethyl sulfoxide (DMSO) as vehicle control or SP600125, treated with 40 µM menadione for the indicated times, and their total protein isolated and immunoblotted with the antibodies shown. (b–e) Densitometric scanning of immunoblot band intensities for total stathmin (b), and stathmin phosphorylated at Ser-16 (c), Ser-25 (d) and Ser-38 (e) (*<i>P</i><0.01, as compared to cells treated with DMSO; n = 3–6).</p

Loss of stathmin leads to JNK overactivation in response to oxidant stress.

<p>(a) RALA hepatocytes were treated with 40 µM menadione for the indicated number of hours. Total protein was isolated and immunoblotted for phospho- (P-JNK) and total (JNK) JNK, phospho- (P-c-Jun) and total (c-Jun) c-Jun, phospho- (P-ERK1/2) and total (ERK1/2) ERK1/2, phospho-MKK4 (P-MKK4) and tubulin. (b) Immunoblots of protein from cells treated with actinomycin D+TNF for the indicated number of hours. (c) siStath cells were infected with the control virus AdLacZ or the catalase-expressing virus AdCat, treated with 40 µM menadione for number of hours indicated and cells harvested for total protein isolation and immunoblotting with the antibodies shown.</p

Stathmin blocks death from menadione-induced oxidant stress.

<p>(a) Total protein was isolated from RALA hepatocytes stably infected with a lentivirus containing vector alone (VEC), or expressing either of two shRNAs for stathmin (siStath #1 and #2), and immunoblotted with antibodies for stathmin and β-actin. (b) Percentage cell death by MTT assay in VEC and stathmin knockdown cells treated with the indicated concentrations of menadione for 24 h (*<i>P</i><0.0001 as compared to VEC cells treated with the same concentration of menadione; n = 6). (c) Percentage cell death at 24 h in cells cultured under transformed conditions at 33°C and treated with the menadione concentrations shown (*<i>P</i><0.0001 as compared to VEC cells treated with the same concentration of menadione; n = 5). (d) VEC and siStath cells were untreated or treated for 8 h with 40 (M40) or 50 (M50) µM menadione, costained with acridine orange/ethidium bromide, and the numbers of necrotic and apoptotic cells determined by fluorescence microscopy (*<i>P</i><0.004 as compared to VEC cells treated with the same concentration of menadione; n = 4). (e) Total protein was isolated from VEC and siStath cells treated for 24 h with 40 or 50 µM menadione (Men) or actinomycin D+TNF (ActD/TNF) and immunoblotted with antibodies to caspase 3 or 7 and tubulin as a loading control. The procaspase (Pro) and cleaved caspase 3 (p17) and caspase 7 (p19) forms are indicated. (f) Percentage cell death by MTT assay in VEC and siStath cells after 24 h of treatment with actinomycin D (ActD) or actinomycin D+TNF (ActD/TNF) (*<i>P</i><0.003 as compared to VEC cells with the same treatment; n = 4).</p

Cell death is blocked by caspase inhibition and Bcl-2.

<p>(a) Percentage cell death by MTT assay 24 h after treatment with vehicle dimethyl sulfoxide (DMSO) or the caspase inhibitor Q-VD-OPh (QVD) (*<i>P</i><0.001, ^#<i>P</i><0.01 as compared to VEC cells; n = 4). (b) Percentage cell death in siStath cells infected with the control adenovirus AdLacZ or a Bcl-2-expressing adenovirus and treated with 40 or 50 µM menadione for 24 h (*<i>P</i><0.001, ^#<i>P</i><0.02 as compared to VEC cells; n = 9–10). (c) Levels of hydrogen peroxide (H₂O₂) generated in VEC and siStath cells that were untreated controls (Con) or treated with 50 µM menadione for 1 or 2 h (n = 4–6).</p

Menadione induces stathmin phosphorylation.

<p>(a) Wild-type RALA hepatocytes were treated with 40 or 50 µM menadione (Men) for the indicated times. Total cellular protein was isolated and immunoblotted for total stathmin (Stath), the indicated phospho-serine (P-) stathmin forms, and β-actin as a loading control. (b) Relative immunoblot band intensities for the indicated stathmin proteins normalized to the signal for β-actin as quantified by scanning densitometry in cells untreated or treated with 40 µM menadione for the indicated times (*<i>P</i><0.02, ^#<i>P</i><0.003 as compared to untreated cells; n = 5–7). (c) Densitometry scanning of immunoblots for cells treated with 50 µM menadione (*<i>P</i><0.03, ^#<i>P</i><0.005 as compared to untreated cells; n = 4–7).</p