Haihong Jia

The Cotton WRKY Transcription Factor GhWRKY17 Functions in Drought and Salt Stress in Transgenic Nicotiana benthamiana Through ABA Signaling and the Modulation of Reactive Oxygen Species Production

Drought and high salinity are two major environmental factors that significantly limit the productivity of agricultural crops worldwide. WRKY transcription factors play essential roles in the adaptation of plants to abiotic stresses. However, WRKY genes involved in drought and salt tolerance in cotton (Gossypium hirsutum) are largely unknown. Here, a group IId WRKY gene, GhWRKY17, was isolated and characterized. GhWRKY17 was found to be induced after exposure to drought, salt, H2O2 and ABA. The constitutive expression of GhWRKY17 in Nicotiana benthamiana remarkably reduced plant tolerance to drought and salt stress, as determined through physiological analyses of the germination rate, root growth, survival rate, leaf water loss and Chl content. GhWRKY17 transgenic plants were observed to be more sensitive to ABA-mediated seed germination and root growth. However, overexpressing GhWRKY17 in N. benthamiana impaired ABA-induced stomatal closure. Furthermore, we found that GhWRKY17 modulated the increased sensitivity of plants to drought by reducing the level of ABA, and transcript levels of ABA-inducible genes, including AREB, DREB, NCED, ERD and LEA, were clearly repressed under drought and salt stress conditions. Consistent with the accumulation of reactive oxygen species (ROS), reduced proline contents and enzyme activities, elevated electrolyte leakage and malondialdehyde, and lower expression of ROS-scavenging genes, including APX, CAT and SOD, the GhWRKY17 transgenic plants exhibited reduced tolerance to oxidative stress compared with wild-type plants. These results therefore indicate that GhWRKY17 responds to drought and salt stress through ABA signaling and the regulation of cellular ROS production in plants.

Identification, genomic organization, and oxidative stress response of a sigma class glutathione S-transferase gene (AccGSTS1) in the honey bee, Apis cerana cerana

Glutathione S-transferases (GSTs) are members of a multifunctional antioxidant enzyme superfamily that play pivotal roles in both detoxification and protection against oxidative damage caused by reactive oxygen species. In this study, a complementary DNA (cDNA) encoding a sigma class GST was identified in the Chinese honey bee, Apis cerana cerana (AccGSTS1). AccGSTS1 was constitutively expressed in all tissues of adult worker bees, including the brain, fat body, epidermis, muscle, and midgut, with particularly robust transcription in the fat body. Relative messenger RNA expression levels of AccGSTS1 at different developmental stages varied, with the highest levels of expression observed in adults. The potential function of AccGSTS1 in cellular defenses against abiotic stresses (cold, heat, UV, H2O2, HgCl2, and insecticides) was investigated. AccGSTS1 was significantly upregulated in response to all of the treatment conditions examined, although the induction levels were varied. Recombinant AccGSTS1 protein showed characteristic glutathione-conjugating catalytic activity toward 1-chloro-2,4-dinitrobenzene. Functional assays revealed that AccGSTS1 could remove H2O2, thereby protecting DNA from oxidative damage. Escherichia coli overexpressing AccGSTS1 showed long-term resistance under conditions of oxidative stress. Together, these results suggest that AccGSTS1 is a crucial antioxidant enzyme involved in cellular antioxidant defenses and honey bee survival.

GhWRKY68 reduces resistance to salt and drought in transgenic Nicotiana benthamiana.

The WRKY transcription factors modulate numerous physiological processes, including plant growth, development and responses to various environmental stresses. Currently, our understanding of the functions of the majority of the WRKY family members and their possible roles in signalling crosstalk is limited. In particular, very few WRKYs have been identified and characterised from an economically important crop, cotton. In this study, we characterised a novel group IIc WRKY gene, GhWRKY68, which is induced by different abiotic stresses and multiple defence-related signalling molecules. The β-glucuronidase activity driven by the GhWRKY68 promoter was enhanced after exposure to drought, salt, abscisic acid (ABA) and H2O2. The overexpression of GhWRKY68 in Nicotiana benthamiana reduced resistance to drought and salt and affected several physiological indices. GhWRKY68 may mediate salt and drought responses by modulating ABA content and enhancing the transcript levels of ABA-responsive genes. GhWRKY68-overexpressing plants exhibited reduced tolerance to oxidative stress after drought and salt stress treatments, which correlated with the accumulation of reactive oxygen species (ROS), reduced enzyme activities, elevated malondialdehyde (MDA) content and altered ROS-related gene expression. These results indicate that GhWRKY68 is a transcription factor that responds to drought and salt stresses by regulating ABA signalling and modulating cellular ROS.

The identification and oxidative stress response of a zeta class glutathione S-transferase (GSTZ1) gene from Apis cerana cerana

Glutathione-S-transferases (GSTs) play an important role in protecting organisms against the toxicity of reactive oxygen species (ROS). However, no information is available for GSTs in the Chinese honey bee (Apis cerana cerana). In this study, we isolated and characterized a zeta class GST gene (AccGSTZ1) from the Chinese honey bee. This gene is present in a single copy and harbors five exons. The deduced amino acid sequence of AccGSTZ1 shared high sequence identity with homologous proteins and contained the highly conserved features of this gene family. The temporal and spatial expression profiles of AccGSTZ1 showed that AccGSTZ1 was highly expressed in fourth instar larvae during development, and the mRNA level of AccGSTZ1 was higher in the epidermis than that in other tissues. The expression pattern under oxidative stress revealed that AccGSTZ1 transcription was significantly upregulated by external factors, such as temperature challenges and H(2)O(2) treatment. The characterization of the purified protein revealed that AccGSTZ1 had low glutathione-conjugating activity, but the recombinant AccGSTZ1 protein displayed high antioxidant activity under oxidative stress. These data suggest that AccGSTZ1 is an oxidative stress-inducible antioxidant enzyme that plays an important role in the protection against oxidative stress and may be of critical importance for the survival of the honey bees.

Overexpression of GhWRKY27a reduces tolerance to drought stress and resistance to Rhizoctonia solani infection in transgenic Nicotiana benthamiana.

WRKY proteins constitute transcriptional regulators involved in various biological processes, especially in coping with diverse biotic and abiotic stresses. However, in contrast to other well-characterized WRKY groups, the functions of group III WRKY transcription factors are poorly understood in the economically important crop cotton (Gossypium hirsutum). In this study, a group III WRKY gene from cotton, GhWRKY27a, was isolated and characterized. Our data indicated that GhWRKY27a localized to the nucleus and that GhWRKY27a expression could be strongly induced by abiotic stresses, pathogen infection, and multiple defense-related signaling molecules. Virus-induced gene silencing (VIGS) of GhWRKY27a enhanced tolerance to drought stress in cotton. In contrast, GhWRKY27a overexpression in Nicotiana benthamiana markedly reduced plant tolerance to drought stress, as determined through physiological analyses of leaf water loss, survival rates, and the stomatal aperture. This susceptibility was coupled with reduced stomatal closure in response to abscisic acid and decreased expression of stress-related genes. In addition, GhWRKY27a-overexpressing plants exhibited reduced resistance to Rhizoctonia solani infection, mainly demonstrated by the transgenic lines exhibiting more severe disease symptoms, accompanied by attenuated expression of defense-related genes in N. benthamiana. Taken together, these findings indicated that GhWRKY27a functions in negative responses to drought tolerance and in resistance to R. solani infection.

Identification and characterization of an Apis cerana cerana Delta class glutathione S-transferase gene (AccGSTD) in response to thermal stress

Glutathione S-transferases (GSTs) are members of a multifunctional enzyme super family that plays a pivotal role in both insecticide resistance and protection against oxidative stress. In this study, we identified a single-copy gene, AccGSTD, as being a Delta class GST in the Chinese honey bee (Apis cerana cerana). A predicted antioxidant response element, CREB, was found in the 1,492-bp 5′-flanking region, suggesting that AccGSTD may be involved in oxidative stress response pathways. Real-time PCR and immunolocalization studies demonstrated that AccGSTD exhibited both developmental- and tissue-specific expression patterns. During development, AccGSTD transcript was increased in adults. The AccGSTD expression level was the highest in the honey bee brain. Thermal stress experiments demonstrated that AccGSTD could be significantly upregulated by temperature changes in a time-dependent manner. It is hypothesized that high expression levels might be due to the increased levels of oxidative stress caused by the temperature challenges. Additionally, functional assays of the recombinant AccGSTD protein revealed that AccGSTD has the capability to protect DNA from oxidative damage. Taken together, these data suggest that AccGSTD may be responsible for antioxidant defense in adult honey bees.

A Raf-like MAPKKK gene, GhRaf19, negatively regulates tolerance to drought and salt and positively regulates resistance to cold stress by modulating reactive oxygen species in cotton

Mitogen-activated protein kinase kinase kinases (MAPKKKs) function at the top level of MAPK cascades and play important roles in plant development and stress responses. Although MAPKKKs comprise the largest family in the MAPK cascades, very few Raf-like MAPKKKs have been functionally identified, especially in the economically important crop cotton. In this study, a Raf-like MAPKKK gene, GhRaf19, was characterized for the first time in cotton. Our data show that the expression of GhRaf19 was inhibited by PEG and NaCl and induced by cold (4°C) and H2O2. Furthermore, when GhRaf19 was silenced in cotton using virus-induced gene silencing (VIGS), tolerance to drought and salt stress were enhanced, the accumulation of reactive oxygen species (ROS) was reduced, and ROS-related gene expression was increased. Consistent with these results, in N. benthamiana, overexpressing-GhRaf19 reduced tolerance to drought and salt. However, GhRaf19-silenced plants showed lowered resistance to cold in cotton, and this effect was correlated with the accumulation of ROS. In contrast, overexpressing GhRaf19 in N. benthamiana increased resistance to cold by inducing higher levels of expression and activity of ROS-related antioxidant genes/enzymes. These results indicate that GhRaf19 negatively regulates tolerance to drought and salt and positively regulates resistance to cold stress by modulating cellular ROS in cotton.

A glutathione S-transferase gene associated with antioxidant properties isolated from Apis cerana cerana

Glutathione S-transferases (GSTs) are an important family of multifunctional enzymes in aerobic organisms. They play a crucial role in the detoxification of exogenous compounds, especially insecticides, and protection against oxidative stress. Most previous studies of GSTs in insects have largely focused on their role in insecticide resistance. Here, we isolated a theta class GST gene designated AccGSTT1 from Apis cerana cerana and aimed to explore its antioxidant and antibacterial attributes. Analyses of homology and phylogenetic relationships suggested that the predicted amino acid sequence of AccGSTT1 shares a high level of identity with the other hymenopteran GSTs and that it was conserved during evolution. Quantitative real-time PCR showed that AccGSTT1 is most highly expressed in adult stages and that the expression profile of this gene is significantly altered in response to various abiotic stresses. These results were confirmed using western blot analysis. Additionally, a disc diffusion assay showed that a recombinant AccGSTT1 protein may be roughly capable of inhibiting bacterial growth and that it reduces the resistance of Escherichia coli cells to multiple adverse stresses. Taken together, these data indicate that AccGSTT1 may play an important role in antioxidant processes under adverse stress conditions.

Overexpression of Cotton GhMPK11 Decreases Disease Resistance through the Gibberellin Signaling Pathway in Transgenic Nicotiana benthamiana

Many changes in development, growth, hormone activity and environmental stimuli responses are mediated by mitogen-activated protein kinase (MAPK) cascades. However, in plants, studies on MAPKs have mainly focused on MPK3, MPK4 and MPK6. Here, a novel group B MAPK gene, GhMPK11, was isolated from cotton (Gossypium hirsutum L.) and characterized. Both promoter and expression pattern analyses revealed that GhMPK11 is involved in defense responses and signaling pathways. GhMPK11 overexpression in Nicotiana benthamiana plants could increase gibberellin 3 (GA3) content through the regulation of GA-related genes. Interestingly, either GhMPK11 overexpression or exogenous GA3 treatment in N. benthamiana plants could enhance the susceptibility of these plants to the infectious pathogens Ralstonia solanacearum and Rhizoctonia solani. Moreover, reactive oxygen species (ROS) accumulation was increased after pathogen infiltration due to the increased expression of ROS-related gene respiratory burst oxidative homologs (RbohB) and the decreased expression or activity of ROS detoxification enzymes regulated by GA3, such as superoxide dismutases (SODs), peroxidases (PODs), catalase (CAT) and glutathione S-transferase (GST). Taken together, these results suggest that GhMPK11 overexpression could enhance the susceptibility of tobacco to pathogen infection through the GA3 signaling pathway via down-regulation of ROS detoxification enzymes.

A typical RNA-binding protein gene (AccRBM11) in Apis cerana cerana: characterization of AccRBM11 and its possible involvement in development and stress responses.

RNA-binding motif proteins (RBMs) belong to RNA-binding proteins that display extraordinary posttranscriptional gene regulation roles in various cellular processes, including development, growth, and stress responses. Nevertheless, only a few examples of the roles of RBMs are known in insects, particularly in Apis cerana cerana. In the present study, we characterized the novel RNA-binding motif protein 11 from Apis cerana cerana, which was named AccRBM11 and whose promoter sequence included abundant potential transcription factor binding sites that are connected to responses to adverse stress and early development. Quantitative PCR results suggested that AccRBM11 was expressed at highest levels in 1-day postemergence worker bees. AccRBM11 mRNA and protein levels were higher in the poison gland and the epidermis than in other tissues. Moreover, levels of AccRBM11 transcription were upregulated upon all the simulation of abiotic stresses. Furthermore, Western blot analysis indicated that AccRBM11 protein expression levels could be induced under some abiotic stressors, a result that did not completely in agree with the qRT-PCR results. It is also noteworthy that the expression of some genes that connected with development or stress responses were remarkably suppressed when AccRBM11 was silenced, which suggested that AccRBM11 might play a similar role in development or stress reactions with the above genes. Taken together, the data presented here provide evidence that AccRBM11 is potentially involved in the regulation of development and some abiotic stress responses. We expect that this study will promote future research on the function of RNA-binding proteins.