General relations of stomatal responses to xylem sap abscisic acid under stress in the rooting zone – A global perspective

A sensitivity factor that quantifies the responsiveness of stomata to xylem sap abscisic acid concentration ([ABA]_xyl) is described, using the relation between [ABA]_xyl and maximum leaf conductance (g_max). Plotting g_max against this factor results in a common linear relationship for woody and herbaceous species from boreal to (semi-) arid climates. The global distribution of the sensitivity factor reveals an unexpected pattern which is inverse to rainfall, i.e., plants in humid climates respond more sensitively to ABA than plants in arid areas. The implications for the response of natural vegetation and consequences for agriculture are discussed.     

Selective observation of the disordered import signal of a globular protein by in-cell NMR: The example of frataxins

We have exploited the capability of in-cell NMR to selectively observe flexible regions within folded proteins to carry out a comparative study of two members of the highly conserved frataxin family which are found both in prokaryotes and in eukaryotes. They all contain a globular domain which shares more than 50% identity, which in eukaryotes is preceded by an N-terminal tail containing the mitochondrial import signal. We demonstrate that the NMR spectrum of the bacterial ortholog CyaY cannot be observed in the homologous E. coli system, although it becomes fully observable as soon as the cells are lysed. This behavior has been observed for several other compact globular proteins as seems to be the rule rather than the exception. The NMR spectrum of the yeast ortholog Yfh1 contains instead visible signals from the protein. We demonstrate that they correspond to the flexible N-terminal tail indicating that this is flexible and unfolded. This flexibility of the N-terminus agrees with previous studies of human frataxin, despite the extensive sequence diversity of this region in the two proteins. Interestingly, the residues that we observe in in-cell experiments are not visible in the crystal structure of a Yfh1 mutant designed to destabilize the first helix. More importantly, our results show that, in cell, the protein is predominantly present not as an aggregate but as a monomeric species.     

Hepatic Xbp1 Gene Deletion Promotes Endoplasmic Reticulum Stress-induced Liver Injury and Apoptosis

Endoplasmic reticulum (ER) stress activates the unfolded protein response (UPR), a highly conserved signaling cascade that functions to alleviate stress and promote cell survival. If, however, the cell is unable to adapt and restore homeostasis, then the UPR activates pathways that promote apoptotic cell death. The molecular mechanisms governing the critical transition from adaptation and survival to initiation of apoptosis remain poorly understood. We aim to determine the role of hepatic Xbp1, a key mediator of the UPR, in controlling the adaptive response to ER stress in the liver. Liver-specific Xbp1 knockout mice (Xbp1^LKO) and Xbp1^fl/fl control mice were subjected to varying levels and durations of pharmacologic ER stress. Xbp1^LKO and Xbp1^fl/fl mice showed robust and equal activation of the UPR acutely after induction of ER stress. By 24 h, Xbp1^fl/fl controls showed complete resolution of UPR activation and no liver injury, indicating successful adaptation to the stress. Conversely, Xbp1^LKO mice showed ongoing UPR activation associated with progressive liver injury, apoptosis, and, ultimately, fibrosis by day 7 after induction of ER stress. These data indicate that hepatic XBP1 controls the adaptive response of the UPR and is critical to restoring homeostasis in the liver in response to ER stress.     

ERManI (Endoplasmic Reticulum Class I α-Mannosidase) Is Required for HIV-1 Envelope Glycoprotein Degradation via Endoplasmic Reticulum-associated Protein Degradation Pathway

Previously, we reported that the mitochondrial translocator protein (TSPO) induces HIV-1 envelope (Env) degradation via the endoplasmic reticulum (ER)-associated protein degradation (ERAD) pathway, but the mechanism was not clear. Here we investigated how the four ER-associated glycoside hydrolase family 47 (GH47) α-mannosidases, ERManI, and ER-degradation enhancing α-mannosidase-like (EDEM) proteins 1, 2, and 3, are involved in the Env degradation process. Ectopic expression of these four α-mannosidases uncovers that only ERManI inhibits HIV-1 Env expression in a dose-dependent manner. In addition, genetic knock-out of the ERManI gene MAN1B1 using CRISPR/Cas9 technology disrupts the TSPO-mediated Env degradation. Biochemical studies show that HIV-1 Env interacts with ERManI, and between the ERManI cytoplasmic, transmembrane, lumenal stem, and lumenal catalytic domains, the catalytic domain plays a critical role in the Env-ERManI interaction. In addition, functional studies show that inactivation of the catalytic sites by site-directed mutagenesis disrupts the ERManI activity. These studies identify ERManI as a critical GH47 α-mannosidase in the ER-associated protein degradation pathway that initiates the Env degradation and suggests that its catalytic domain and enzymatic activity play an important role in this process.     

Activation of the UPR Protects against Cigarette Smoke-induced RPE Apoptosis through Up-Regulation of Nrf2

Recent studies have revealed a role of endoplasmic reticulum (ER) stress-induced unfolded protein response (UPR) in the regulation of RPE cell activity and survival. Herein, we examined the mechanisms by which the UPR modulates apoptotic signaling in human RPE cells challenged with cigarette smoking extract (CSE). Our results show that CSE exposure induced a dose- and time-dependent increase in ER stress markers, enhanced reactive oxygen species (ROS), mitochondrial fragmentation, and apoptosis of RPE cells. These changes were prevented by the anti-oxidant NAC or chemical chaperone TMAO, suggesting a close interaction between oxidative and ER stress in CSE-induced apoptosis. To decipher the role of the UPR, overexpression or down-regulation of XBP1 and CHOP genes was manipulated by adenovirus or siRNA. Overexpressing XBP1 protected against CSE-induced apoptosis by reducing CHOP, p-p38, and caspase-3 activation. In contrast, XBP1 knockdown sensitized the cells to CSE-induced apoptosis, which is likely through a CHOP-independent pathway. Surprisingly, knockdown of CHOP reduced p-eIF2α and Nrf2 resulting in a marked increase in caspase-3 activation and apoptosis. Furthermore, Nrf2 inhibition increased ER stress and exacerbated cell apoptosis, while Nrf2 overexpression reduced CHOP and protected RPE cells. Our data suggest that although CHOP may function as a pro-apoptotic gene during ER stress, it is also required for Nrf2 up-regulation and RPE cell survival. In addition, enhancing Nrf2 and XBP1 activity may help reduce oxidative and ER stress and protect RPE cells from cigarette smoke-induced damage.     

Carbohydrate metabolism before and after dehiscence in the recalcitrant pollen of pumpkin (Cucurbita pepo L.)

Pumpkin (Cucurbita pepo L.) pollen is starchy, sucrose‐poor and recalcitrant, features opposite to those of several model species; therefore, some differences in carbohydrate metabolism could be expected in this species. By studying pumpkin recalcitrant pollen, the objective was to provide new biochemical evidence to improve understanding of how carbohydrate metabolism might be involved in pollen functioning in advanced stages. Four stages were analysed: immature pollen from 1 day before anthesis, mature pollen, mature pollen exposed to the environment for 7 h, and pollen rehydrated in a culture medium. Pollen viability, water and carbohydrate content and activity of enzymes involved in carbohydrate metabolism were quantified in each stage. Pollen viability and water content dropped quickly after dehiscence, as expected. The slight changes in carbohydrate concentration and enzyme activity during pollen maturation contrast with major changes recorded with ageing and rehydration. Pumpkin pollen seems highly active and closely related to its surrounding environment in all the stages analysed; the latter is particularly evident among insoluble sucrolytic enzymes, mainly wall‐bound acid invertase, which would be the most relevant for sucrose cleavage. Each stage was characterised by a particular metabolic/enzymatic profile; some particular features, such as the minor changes during maturation, fast sucrolysis upon rehydration or sharp decrease in insoluble sucrolytic activity with ageing seem to be related to the lack of dormancy and recalcitrant nature of pumpkin pollen.     

采用高效液相色谱技术分析烟草体内的维生素B6化合物

维生素B6 (VB6)是一类化合物的总称.近年来研究发现VB6在植物体内发挥抗逆作用.烟草作为模式植物其体内VB6的存在形态还未见报道.本研究采用高效液相色谱结合荧光检测技术对烟草体内VB6的存在形态进行了分析.结果表明:土壤栽培烟草叶、茎和根中VB6的含量依次为2.9、1.7、3.0 μg/g鲜重;组培烟草叶片的VB...