The role of D-GADD45 in oxidative,thermal and genotoxic stress resistance

There is a relationship between various cellular stress factors and aging. In earlier studies, we demonstrated that overexpression of the D-GADD45 gene increases the life span of Drosophila melanogaster. In this study, we investigate the relationship between D-GADD45 activity and resistance to oxidative, genotoxic and thermal stresses as well as starvation. In most cases, flies with constitutive and conditional D-GADD45 overexpression in the nervous system were more stress-resistant than ones without overexpression. At the same time, most of the studied stress factors increased D-GADD45 expression in the wild-type strain. The lifespan-extending effect of D-GADD45 overexpression was also retained after exposure to chronic and acute gamma-irradiation, with doses of 40 сGy and 30 Gy, respectively. However, knocking out D-GADD45 resulted in a significant reduction in lifespan, lack of radiation hormesis and radioadaptive response. A dramatic decrease in the spontaneous level of D-GADD45 expression was observed in the nervous system as age progressed, which may be one of the causes of the age-related deterioration of organismal stress resistance. Thus, D-GADD45 expression is activated by most of the studied stress factors, and D-GADD45 overexpression resulted in an increase of stress resistance.     

Modulation of methuselah expression targeted to Drosophila insulin‐producing cells extends life and enhances oxidative stress resistance

Ubiquitously reduced signaling via Methuselah (MTH), a G‐protein‐coupled receptor (GPCR) required for neurosecretion, has previously been reported to extend life and enhance stress resistance in flies. Whether these effects are due to reduced MTH signalling in specific tissues remains unknown. We determined that reduced expression of mth targeted to the insulin‐producing cells (IPCs) of the fly brain was sufficient to extend life and enhance oxidative stress resistance. Paradoxically, we discovered that overexpression of mth targeted to the same cells has similar phenotypic effects to reduced expression due to MTH's interaction with β‐arrestin, which uncouples GPCRs from their G‐proteins. We confirmed the functional relationship between MTH and β‐arrestin by finding that IPC‐targeted overexpression of β‐arrestin alone mimics the longevity phenotype of reduced MTH signaling. As reduced MTH signaling also inhibits insulin secretion from the IPCs, the most parsimonious mechanistic explanation of its longevity and stress‐resistance enhancement might be through reduced insulin/IGF signaling (IIS). However, examination of phenotypic features of long‐lived IPC‐mth modulated flies as well as several downstream IIS targets implicates enhanced activity of the JNK stress‐resistance pathway more directly than insulin signaling in the longevity and stress‐resistance phenotypes.     

The Expression of Race-Specific Resistance of Wheat Seedlings to Puccinia recondita

A range of wheat cultivars with resistance factors effective against Puccinia recondita f. sp. tritici was studied to investigate the mode of action and expression of resistance at first and third seedling leaf stages. In most cultivars, resistance to isolate 74/2 resulted in extremely low levels of infection, apparently linked with a predominantly hypersensitive response by the host. In seedlings of cultivars Mans Fundin and Sterna, however, race-specific resistance was expressed as increased pathogen latent period and intermediate infection levels.     

Proteasome dysfunction in Drosophila signals to an Nrf2‐dependent regulatory circuit aiming to restore proteostasis and prevent premature aging

The ubiquitin–proteasome system is central to the regulation of cellular proteostasis. Nevertheless, the impact of in vivo proteasome dysfunction on the proteostasis networks and the aging processes remains poorly understood. We found that RNAi‐mediated knockdown of 20S proteasome subunits in Drosophila melanogaster resulted in larval lethality. We therefore studied the molecular effects of proteasome dysfunction in adult flies by developing a model of dose‐dependent pharmacological proteasome inhibition. Impaired proteasome function promoted several ‘old‐age’ phenotypes and markedly reduced flies' lifespan. In young somatic tissues and in gonads of all ages, loss of proteasome activity induced higher expression levels and assembly rates of proteasome subunits. Proteasome dysfunction was signaled to the proteostasis network by reactive oxygen species that originated from malfunctioning mitochondria and triggered an Nrf2‐dependent upregulation of the proteasome subunits. RNAi‐mediated Nrf2 knockdown reduced proteasome activities, flies' resistance to stress, as well as longevity. Conversely, inducible activation of Nrf2 in transgenic flies upregulated basal proteasome expression and activity independently of age and conferred resistance to proteotoxic stress. Interestingly, prolonged Nrf2 overexpression reduced longevity, indicating that excessive activation of the proteostasis pathways can be detrimental. Our in vivo studies add new knowledge on the proteotoxic stress‐related regulation of the proteostasis networks in higher metazoans. Proteasome dysfunction triggers the activation of an Nrf2‐dependent tissue‐ and age‐specific regulatory circuit aiming to adjust the cellular proteasome activity according to temporal and/or spatial proteolytic demands. Prolonged deregulation of this proteostasis circuit accelerates aging.     

TaCIPK10 interacts with and phosphorylates TaNH2 to activate wheat defense responses to stripe rust

Calcineurin B‐like interacting protein kinase (CIPKs) has been shown to be required for biotic stress tolerance of plants in plant‐pathogen interactions. However, the roles of CIPKs in immune signalling of cereal crops and an in‐depth knowledge of substrates of CIPKs in response to biotic stress are under debate. In this study, we identified and cloned a CIPK homologue gene TaCIPK10 from wheat. TaCIPK10 was rapidly induced by Puccinia striiformis f. sp. tritici (Pst) inoculation and salicylic acid (SA) treatment. In vitro phosphorylation assay demonstrated that the kinase activity of TaCIPK10 is regulated by Ca²⁺ and TaCBL4. Knockdown TaCIPK10 significantly reduced wheat resistance to Pst, whereas TaCIPK10 overexpression resulted in enhanced wheat resistance to Pst by the induction of defense response in different aspects, including hypersensitive cell death, ROS accumulation and pathogenesis‐relative genes expression. Moreover, TaCIPK10 physically interacted with and phosphorylated TaNH2, which was homologous to AtNPR3/4. Silencing of TaNH2 in wheat resulted in enhanced susceptibility to the avirulent Pst race, CYR23, indicating its positive role in wheat resistance. Our results demonstrate that TaCIPK10 positively regulate wheat resistance to Pst as molecular links between of Ca²⁺ and downstream components of defense response and TaCIPK10 interacts with and phosphorylates TaNH2 to regulate wheat resistance to Pst.     

Enhancing blast disease resistance by overexpression of the calcium‐dependent protein kinase OsCPK4 in rice

Rice is the most important staple food for more than half of the human population, and blast disease is the most serious disease affecting global rice production. In this work, the isoform OsCPK4 of the rice calcium‐dependent protein kinase family is reported as a regulator of rice immunity to blast fungal infection. It shows that overexpression of OsCPK4 gene in rice plants enhances resistance to blast disease by preventing fungal penetration. The constitutive accumulation of OsCPK4 protein prepares rice plants for a rapid and potentiated defence response, including the production of reactive oxygen species, callose deposition and defence gene expression. OsCPK4 overexpression leads also to constitutive increased content of the glycosylated salicylic acid hormone in leaves without compromising rice yield. Given that OsCPK4 overexpression was known to confer also salt and drought tolerance in rice, the results reported in this article demonstrate that OsCPK4 acts as a convergence component that positively modulates both biotic and abiotic signalling pathways. Altogether, our findings indicate that OsCPK4 is a potential molecular target to improve not only abiotic stress tolerance, but also blast disease resistance of rice crops.     

Cloning and Drought Resistance Analysis of Soybean <i>GmHsps_p23-like</i> Gene

Soybean (Glycine max (L.) Merr.) is an important cultivated crop, which requires much water during its growth, and drought seriously affects soybean yields. Studies have shown that the expression of small heat shock proteins can enhance drought resistance, cold resistance and salt resistance of plants. In this experiment, soybean GmHsps_p23-like gene was successfully cloned by RT-PCR, the protein encoded by the GmHsps_p23-like gene was subjected to bioinformatics analysis, and the pCAMBIA3301-GmHsps_p23-like overexpression vector and pCBSG015-GmHsps_p23-like gene editing vector were constructed. Agrobacterium-mediated method was used to transform soybeans to obtain positive plants. RT-PCR detection, rehydration experiment and drought resistance physiological and biochemical index detection were performed on the T₂ generation positive transgenic soybean plants identified by PCR and Southern hybridization. The results showed that the overexpression vector plant GmHsps_p23-like gene expression increased. After rehydration, the transgenic overexpression plants returned to normal growth, and the damage to the plants was low. After drought stress, the SOD and POD activities and the PRO content of the transgenic overexpression plants increased, while the MDA content decreased. The reverse was true for soybean plants with genetically modified editing vectors. The drought resistance of the overexpressed soybeans under drought stress was higher than that of the control group, and had a stronger drought resistance. It showed that the expression of soybean GmHsps_p23-like gene can improve the drought resistance of soybean. The cloning and functional verification of soybean GmHsps_p23-like gene had not been reported yet. This is the first time that PCR technology has been used to amplify the soybean GmHsps_p23-like gene and construct an expression vector for this gene. This research has laid the foundation for transgenic technology to improve plant drought resistance and cultivate new drought-resistant transgenic soybean varieties.     

Overexpression of Mouse Estrogen Receptor-β Decreases but Its Transactivation and Ligand Binding Domains Increase the Growth Characteristics of <Emphasis Type="Italic">E. coli</Emphasis>

Escherichia coli is one of the most common and widely used prokaryotic hosts for the expression of recombinant proteins. The overexpression of recombinant proteins occasionally increases bacterial growth but sometimes reduces it and becomes lethal to the host cells. Here, we report the overexpression of mouse ER-β and its domains in the prokaryotic expression system and its opposite effect on the growth characteristics of E. coli. ER-β protein was immunologically detected as a 53 kDa his-tag protein in the pellet of the bacterial lysate. Its overexpression, as reflected by the total protein content and expression pattern, resulted in the decrease of bacterial growth. However, the overexpression of ER-β transactivation domain (TAD) using pIVEX and ligand binding domain (LBD) using pRSETA in E. coli BL21 (DE3) show opposite pattern. TAD was immunologically detected as 20 kDa and LBD as 22 kDa protein in the supernatant of the bacterial lysate and their overexpression increased the bacterial growth.     

Effects ofin Planta gamma-irradiation on growth, photosynthesis, and antioxidative capacity of red pepper (Capsicum annuum L.) plants

We investigated the effects of low-dose inplanta irradiation on red pepper plants treated with gamma rays of 2, 4, 8, and 16 Gy. Growth was stimulated at 2 and 4 Gy but inhibited at 8 and 16 Gy. Photochemical quenching (qP) increased slightly in all treatment groups for 1 d after irradiation (DAl), whereas non-photochemical quenching (NPQ) decreased more noticeably. These changes in qP and NPQ were transient and had almost recovered to the control level by 2 DAl. Although carotenoid pigments also fluctuated during the experimental period, chlorophylls were almost entirely insensitive to the gamma rays. Irradiation also partially protected leaves from a decrease in photochemical efficiency (Fv/Fm) under conditions of UV-B (2.2 W m^-2) and high light intensity (800 μmol m^-2 s^-1). This enhanced stress resistance could be partly explained by higher levels of SOD and APX activities, as well as ascorbate content. Our results demonstrate for the first time that the carotenoid pigments are the most radio-sensitive and fastest recovering compounds in plants, and that SOD, APX, and ascorbate are important inducible factors for improving stress resistance through the use ofin planta gamma-irradiation.     

Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development

Gadd45 proteins have been implicated in the cellular response to physiological or environmental stress and the accompanying cell cycle arrest, DNA repair, cell survival and senescence or apoptosis. Although their molecular function is well studied, the expression and role of Gadd45 genes during embryonic development in mice is largely unknown. Here we provide a comprehensive comparison of Gadd45a, Gadd45b and Gadd45g expression during mouse embryonic development. In situ hybridizations on sectioned and whole mouse embryos show most prominent Gadd45a expression in the tip of the closing neural tube, the cranial and dorsal root ganglia and the somites. Mouse Gadd45b is expressed strongly in the chorion, but only weakly in the embryo proper, including somites and limb buds. Murine Gadd45g expression strongly resembles Xenopus and medaka fish expression in primary neuron precursors and post-mitotic neurons, indicating a conserved role for Gadd45g in vertebrate neurogenesis. Additionally, Gadd45 genes show conserved expression during somitogenesis. In summary, Gadd45 genes are expressed in evolutionary conserved, but also divergent domains, which predominantly encompass areas of cell differentiation, consistent with their established function in growth arrest and DNA demethylation.     

铁皮石斛SPL膜结合(STM)转录因子的全基因组鉴定及表达分析

SPL转录因子广泛参与植物生长发育、胁迫响应等过程。目前,没有关于铁皮石斛SPL膜结合(SPL with transmembrane motif)转录因子即STM转录因子的研究。为了探究STM转录因子在铁皮石斛生长发育及胁迫响应等方面的作用,该文在铁皮石斛全基因组水平鉴定出4个STM转录因子,并对DoSTM基因家族成员进行生物信息学分析,又利用逆转录PCR研究了DoSTM在不同组织部位及不同逆境处理下的表达情况。结果表明:(1)DoSTM1-4为亲水蛋白,均具有SBP保守结构域和一些激素响应位点。(2)4个DoSTM在根茎叶中均有表达,DoSTM2在叶中的相对表达量最低; DoSTM1/3/4的相对表达水平均无明显差异。(3)DoSTM1-4在低温、高温、干旱胁迫下的相对表达水平都有显著变化,DoSTM1/3/4的表达量降低最为明显,故推测DoSTM与植物体内激素响应、温度变化响应及抗旱性有关。这些结论为后续进一步开展铁皮石斛STM转录因子的研究提供了参考。