Characterization of Bactrocera dorsalis Serine Proteases and Evidence for Their Indirect Role in Insecticide Tolerance

The oriental fruit fly Bactrocera dorsalis (Hendel) causes devastating losses to agricultural crops world-wide and is considered to be an economically important pest. Little is known about the digestive enzymes such as serine proteases (SPs) in B. dorsalis, which are important both for energy supply and mitigation of fitness cost associated with insecticide tolerance. In this study, we identified five SP genes in the midgut of B. dorsalis, and the alignments of their deduced amino acid sequences revealed the presence of motifs conserved in the SP superfamily. Phylogenetic analyses with known SPs from other insect species suggested that three of them were trypsin-like proteases. Analyses of the expression profiles among the different developmental stages showed that all five genes were most abundant in larvae than in other stages. When larvae were continuously fed on diet containing 0.33 μg/g β-Cypermethrin, expression of all five genes were upregulated in the midgut but the larval development was delayed. Biochemical assays were consistent with the increased protease activity exhibited by SPs in the midgut after treatment with β-Cypermethrin. Taken together, these findings provide evidence for the hypothesis that enhanced SP activity may play an indirect role in relieving the toxicity stress of insecticide in B. dorsalis.     

Microarray Analysis of Transcriptional Responses to Abscisic Acid and Salt Stress in Arabidopsis thaliana

Abscisic acid (ABA) plays a crucial role in plant responses to abiotic stress. To investigate differences in plant responses to salt and ABA stimulus, differences in gene expression in Arabidopsis in response to salt and ABA were compared using an Agilent oligo microarray. A total of 144 and 139 genes were significantly up- and downregulated, respectively, under NaCl stress, while 406 and 381 genes were significantly up- and downregulated, respectively, under ABA stress conditions. In addition, 31 genes were upregulated by both NaCl and ABA stresses, and 23 genes were downregulated by these stressors, suggesting that these genes may play similar roles in plant responses to salt and ABA stress. Gene ontology (GO) analysis revealed four subgroups of genes, including genes in the GO categories “Molecular transducer activity”, “Growth”, “Biological adhesion” and “Pigmentation”, which were expressed in response to ABA stress but not NaCl stress. In addition, genes that play specific roles during salt or ABA stress were identified. Our results may help elucidate differences in the response of plants to salt and ABA stress.     

OsPOP5, A Prolyl Oligopeptidase Family Gene from Rice Confers Abiotic Stress Tolerance in Escherichia coli

The prolyl oligopeptidase family, which is a group of serine peptidases, can hydrolyze peptides smaller than 30 residues. The prolyl oligopeptidase family in plants includes four members, which are prolyl oligopeptidase (POP, EC3.4.21.26), dipeptidyl peptidase IV (DPPIV, EC3.4.14.5), oligopeptidase B (OPB, EC3.4.21.83), and acylaminoacyl peptidase (ACPH, EC3.4.19.1). POP is found in human and rat, and plays important roles in multiple biological processes, such as protein secretion, maturation and degradation of peptide hormones, and neuropathies, signal transduction and memory and learning. However, the function of POP is unclear in plants. In order to study POP function in plants, we cloned the cDNA of the OsPOP5 gene from rice by nested-PCR. Sequence analysis showed that the cDNA encodes a protein of 596 amino acid residues with M_w ≈ 67.29 kD. In order to analyze the protein function under different abiotic stresses, OsPOP5 was expressed in Escherichia coli. OsPOP5 protein enhanced the tolerance of E. coli to high salinity, high temperature and simulated drought. The results indicate that OsPOP5 is a stress-related gene in rice and it may play an important role in plant tolerance to abiotic stress.     

Characterization of Rice NADPH Oxidase Genes and Their Expression under Various Environmental Conditions

Plasma membrane NADPH oxidases (Noxs) are key producers of reactive oxygen species under both normal and stress conditions in plants. We demonstrate that at least eleven genes in the genome of rice (Oryza sativa L.) were predicted to encode Nox proteins, including nine genes (OsNox1–9) that encode typical Noxs and two that encode ancient Nox forms (ferric reduction oxidase 1 and 7, OsFRO1 and OsFRO7). Phylogenetic analysis divided the Noxs from nine plant species into six subfamilies, with rice Nox genes distributed among subfamilies I to V. Gene expression analysis using semi-quantitative RT-PCR and real-time qRT-PCR indicated that the expression of rice Nox genes depends on organs and environmental conditions. Exogenous calcium strongly stimulated the expression of OsNox3, OsNox5, OsNox7, and OsNox8, but depressed the expression of OsFRO1. Drought stress substantially upregulated the expression of OsNox1–3, OsNox5, OsNox9, and OsFRO1, but downregulated OsNox6. High temperature upregulated OsNox5–9, but significantly downregulated OsNox1–3 and OsFRO1. NaCl treatment increased the expression of OsNox2, OsNox8, OsFRO1, and OsFRO7, but decreased that of OsNox1, OsNox3, OsNox5, and OsNox6. These results suggest that the expression profiles of rice Nox genes have unique stress-response characteristics, reflecting their related but distinct functions in response to different environmental stresses.     

Origin and Expansion of the Serine Protease Repertoire in the Myelomonocyte Lineage

The deepest evolutionary branches of the trypsin/chymotrypsin family of serine proteases are represented by the digestive enzymes of the gastrointestinal tract and the multi-domain proteases of the blood coagulation and complement system. Similar to the very old digestive system, highly diverse cleavage specificities emerged in various cell lineages of the immune defense system during vertebrate evolution. The four neutrophil serine proteases (NSPs) expressed in the myelomonocyte lineage, neutrophil elastase, proteinase 3, cathepsin G, and neutrophil serine protease 4, collectively display a broad repertoire of (S1) specificities. The origin of NSPs can be traced back to a circulating liver-derived trypsin-like protease, the complement factor D ancestor, whose activity is tightly controlled by substrate-induced activation and TNFα-induced locally upregulated protein secretion. However, the present-day descendants are produced and converted to mature enzymes in precursor cells of the bone marrow and are safely sequestered in granules of circulating neutrophils. The potential site and duration of action of these cell-associated serine proteases are tightly controlled by the recruitment and activation of neutrophils, by stimulus-dependent regulated secretion of the granules, and by various soluble inhibitors in plasma, interstitial fluids, and in the inflammatory exudate. An extraordinary dynamic range and acceleration of immediate defense responses have been achieved by exploiting the high structural plasticity of the trypsin fold.     

ZmCIPK21, A Maize CBL-Interacting Kinase,Enhances Salt Stress Tolerance in Arabidopsis thaliana

Salt stress represents an increasing threat to crop growth and yield in saline soil. In this study, we identified a maize calcineurin B-like protein-interacting protein kinase (CIPK), ZmCIPK21, which was primarily localized in the cytoplasm and the nucleus of cells and displayed enhanced expression under salt stress. Over-expression of ZmCIPK21 in wild-type Arabidopsis plants increased their tolerance to salt, as supported by the longer root lengths and improved growth. The downstream stress-response genes, including dehydration-responsive element-binding (DREB) genes were also activated in transgenic plants over-expressing ZmCIPK21. In addition, introduction of the transgenic ZmCIPK21 gene into the Arabidopsis mutant cipk1-2 rescued the salt-sensitive phenotype under high salt stress. Measurement of Na⁺ and K⁺ content in transgenic plants showed that over-expression of ZmCIPK21 decreased accumulation of Na⁺ and allowed retention of relatively high levels of K⁺, thereby enhancing plant tolerance to salt conditions.     

Sound Waves Promote Arabidopsis thaliana Root Growth by Regulating Root Phytohormone Content

Sound waves affect plants at the biochemical, physical, and genetic levels. However, the mechanisms by which plants respond to sound waves are largely unknown. Therefore, the aim of this study was to examine the effect of sound waves on Arabidopsis thaliana growth. The results of the study showed that Arabidopsis seeds exposed to sound waves (100 and 100 + 9k Hz) for 15 h per day for 3 day had significantly longer root growth than that in the control group. The root length and cell number in the root apical meristem were significantly affected by sound waves. Furthermore, genes involved in cell division were upregulated in seedlings exposed to sound waves. Root development was affected by the concentration and activity of some phytohormones, including cytokinin and auxin. Analysis of the expression levels of genes regulating cytokinin and auxin biosynthesis and signaling showed that cytokinin and ethylene signaling genes were downregulated, while auxin signaling and biosynthesis genes were upregulated in Arabidopsis exposed to sound waves. Additionally, the cytokinin and auxin concentrations of the roots of Arabidopsis plants increased and decreased, respectively, after exposure to sound waves. Our findings suggest that sound waves are potential agricultural tools for improving crop growth performance.     

A Comprehensive Identification and Expression Analysis of VQ Motif-Containing Proteins in Sugarcane (Saccharum spontaneum L.) under Phytohormone Treatment and Cold Stress

The VQ motif-containing proteins play a vital role in various processes such as growth, resistance to biotic and abiotic stresses and development. However, there is currently no report on the VQ genes in sugarcane (Saccharum spp.). Herein, 78 VQ genes in Saccharum spontaneum were identified and classified into nine subgroups (I-IX) by comparative genomic analyses. Each subgroup had a similar structural and conservative motif. These VQ genes expanded mainly through whole-genome segmental duplication. The cis-regulatory elements (CREs) of the VQ genes were widely involved in stress responses, phytohormone responses and physiological regulation. The RNA-seq data showed that SsVQ gene expression patterns in 10 different samples, including different developmental stages, revealed distinct temporal and spatial patterns. A total of 23 SsVQ genes were expressed in all tissues, whereas 13 SsVQ genes were not expressed in any tissues. Sequence Read Archive (SRA) data showed that the majority of SsVQs responded to cold and drought stress. In addition, quantitative real-time PCR analysis showed that the SsVQs were variously expressed under salicylic acid (SA), jasmonic acid (JA), abscisic acid (ABA) and cold treatment. This study conducted a full-scale analysis of the VQ gene family in sugarcane, which could be beneficial for the functional characterization of sugarcane VQ genes and provide candidate genes for molecular resistance breeding in cultivated sugarcane in the future.     

Combinatorial Measurement of CDKN1A/p21 and KIF20A Expression for Discrimination of DNA Damage-Induced Clastogenicity

In vitro mammalian cytogenetic tests detect chromosomal aberrations and are used for testing the genotoxicity of compounds. This study aimed to identify a supportive genomic biomarker could minimize the risk of misjudgments and aid appropriate decision making in genotoxicity testing. Human lymphoblastoid TK6 cells were treated with each of six DNA damage-inducing genotoxins (clastogens) or two genotoxins that do not cause DNA damage. Cells were exposed to each compound for 4 h, and gene expression was comprehensively examined using Affymetrix U133A microarrays. Toxicogenomic analysis revealed characteristic alterations in the expression of genes included in cyclin-dependent kinase inhibitor 1A (CDKN1A/p21)-centered network. The majority of genes included in this network were upregulated on treatment with DNA damage-inducing clastogens. The network, however, also included kinesin family member 20A (KIF20A) downregulated by treatment with all the DNA damage-inducing clastogens. Downregulation of KIF20A expression was successfully confirmed using additional DNA damage-inducing clastogens. Our analysis also demonstrated that nucleic acid constituents falsely downregulated the expression of KIF20A, possibly via p16 activation, independently of the CDKN1A signaling pathway. Our results indicate the potential of KIF20A as a supportive biomarker for clastogenicity judgment and possible mechanisms involved in KIF20A downregulation in DNA damage and non-DNA damage signaling networks.     

A Comprehensive Identification and Function Analysis of Serine/Arginine-Rich (SR) Proteins in Cotton (Gossypium spp.)

As one of the most important factors in alternative splicing (AS) events, serine/arginine-rich (SR) proteins not only participate in the growth and development of plants but also play pivotal roles in abiotic stresses. However, the research about SR proteins in cotton is still lacking. In this study, we performed an extensive comparative analysis of SR proteins and determined their phylogeny in the plant lineage. A total of 169 SR family members were identified from four Gossypium species, and these genes could be divided into eight distinct subfamilies. The domain, motif distribution and gene structure of cotton SR proteins are conserved within each subfamily. The expansion of SR genes is mainly contributed by WGD and allopolyploidization events in cotton. The selection pressure analysis showed that all the paralogous gene pairs were under purifying selection pressure. Many cis-elements responding to abiotic stress and phytohormones were identified in the upstream sequences of the GhSR genes. Expression profiling suggested that some GhSR genes may involve in the pathways of plant resistance to abiotic stresses. The WGCNA analysis showed that GhSCL-8 co-expressed with many abiotic responding related genes in a salt-responding network. The Y2H assays showed that GhSCL-8 could interact with GhSRs in other subfamilies. The subcellular location analysis showed that GhSCL-8 is expressed in the nucleus. The further VIGS assays showed that the silencing of GhSCL-8 could decrease salt tolerance in cotton. These results expand our knowledge of the evolution of the SR gene family in plants, and they will also contribute to the elucidation of the biological functions of SR genes in the future.     

SerpinB10, a Serine Protease Inhibitor,Is Implicated in UV-Induced Cellular Response

UV-induced DNA damage response and repair are extensively studied processes, as any malfunction in these pathways contributes to the activation of tumorigenesis. Although several proteins involved in these cellular mechanisms have been described, the entire repair cascade has remained unexplored. To identify new players in UV-induced repair, we performed a microarray screen, in which we found SerpinB10 (SPB10, Bomapin) as one of the most dramatically upregulated genes following UV irradiation. Here, we demonstrated that an increased mRNA level of SPB10 is a general cellular response following UV irradiation regardless of the cell type. We showed that although SPB10 is implicated in the UV-induced cellular response, it has no indispensable function in cell survival upon UV irradiation. Nonetheless, we revealed that SPB10 might be involved in delaying the duration of DNA repair in interphase and also in S-phase cells. Additionally, we also highlighted the interaction between SPB10 and H3. Based on our results, it seems that SPB10 protein is implicated in UV-induced stress as a “quality control protein”, presumably by slowing down the repair process.