Surviving winter on the Qinghai-Xizang Plateau:Extensive reversible protein phosphorylation plays a dominant role in regulating hypometabolism in hibernating Nanorana parkeri

Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.

Phosphorylation regulation of nitrogen,phosphorus,and potassium uptake systems in plants

The uptake of ammonium,nitrate,phosphorus,and potassium ions by roots is mediated by specific ion transporter or channel proteins,and protein phosphorylation regulation events occurring on these proteins and their regulators determine their ultimate activity.Elucidating the mechanism by which protein phosphorylation modification regulates nutrient uptake will advance plant breeding for high nutrientuse efficiency.In this review,it is concluded that the root nutrient absorption system is composed of several,but not all,members of a specific ion transporter or channel family.Under nutrient-starvation conditions,protein phosphorylation-based regulation of these proteins and associated transcription factors increases ion transporter-or channel-mediated nutrient uptake capacity via direct function activity enhancement,allowing more protein trafficking to the plasma membrane,by strengthening the interaction of transporters and channels with partner proteins,by increasing their protein stability,and by transcriptional activation.Under excessive nutrient conditions,protein phosphorylation-based regulation suppresses nutrient uptake by reversing these processes.Strengthening phosphorylation regulation items that increase nutrient absorption and weakening phosphorylation modification items that are not conducive to nutrient absorption show potential as strategies for increasing nutrient use efficiency.

Effects of 1,2,4-Trichlorobenzene and Mercury Ion Stress on Ca^2+ Fluxion and Protein Phosphorylation in Rice

The effects of 5 mg/L 1,2,4-trichlorobenzene （TCB） and 0.1 mmol/L mercury ion （Hg^2＋） stresses on Ca^2＋ fluxion and protein phosphorylation in rice seedlings were investigated by isotope exchange kinetics and in vitro phosphorylation assay. The Ca^2＋ absorption in rice leaves and Ca^2＋ transportation from roots to leaves were promoted significantly in response to Hg^2＋ and TCB treatments for 4-48 h. The Ca^2＋ absorption peaks presented in the leaves when the rice seedlings were exposed to Hg^2＋ for 8-12 h or to TCB for 12-24 h. Several Ca^2＋ absorption peaks presented in the roots during rice seedlings being exposed to Hg^2＋ and TCB, and the first Ca^2＋ absorption peak was at 8 h after being exposed to Hg^2＋ and TCB The result of isotope exchange kinetic analysis confirmed that short-term （8 h） Hg^2＋ and TCB stresses caused Ca^2＋ channels or pumps located on plasmalemma to open transiently. The phosphorylation assay showed that short-term TCB stress enhanced protein phosphorylation in rice roots （TCB treatment for 4-8 h） and leaves （TCB treatment for 4-24 h）, and short-term （4-8 h） Hg^2＋ stress also enhanced protein phosphorylation in rice leaves. The enhancement of protein phosphorylation in both roots and leaves corresponded with the first Ca^2＋ absorption peak, which confirmed that the enhancement of protein phosphorylation caused by TCB or Hg^2＋ stress might be partly triggered by the increases of cytosolic calcium. TCB treatment over 12 h inhibited protein phosphorylation in rice roots, which might be partly due to that TCB stress suppressed the protein kinase activity. Whereas, Hg^2＋ treatment inhibited protein phosphorylation in rice roots, and Hg^2＋ treatment over 12 h inhibited protein phosphorylation in rice leaves. This might be attributed to that not only the protein kinase activity, but also the expressions of phosphorylation proteins were restrained by Hg^2＋ stress.

Effects of lairage after transport on post mortem muscle glycolysis, protein phosphorylation and lamb meat quality

The objective of this study was to investigate the effect of lairage after transport on post mortem muscle glycolysis,protein phosphorylation and lamb meat quality.Two preslaughter animal treatments,transport for 3 h and lairage for 0 h（T3L0）and transport for 3 h and then lairage for 12 h（T3L12）,were compared with a control treatment of 0 h transport and 0 h lairage.Data obtained showed that preslaughter transport had a significant effect on lamb meat quality.Loins from lambs of the T3L0 treatment showed higher（P=0.026）pH24 h and higher（P=0.021）pH48 h values,but lower（P〈0.001）drip loss and lower（P〈0.05）glycolytic potential at 0 h post mortem than those of the T3L12 and control groups.Muscle samples of the T3L0 group showed higher（P=0.046）shear force and lower（P=0.005）b＊ value than those of the T3L12 group.Muscle glycogen concentration at 0,2,4 h post mortem were lower（P〈0.05）in the T3L0 group than in control.No significant difference（P〉0.05）in most meat quality parameters was determined between the T3L12 group and control,showing lairage for 12 h allowed lambs to recover from the effects of transport for 3 h and resulted in similar meat quality characteristics compared to no transport.Lairage after transport did not affect most meat quality indices in comparison with control,but increased the meat drip loss and b＊value of lambs possibly through decreasing glycogen concentration and glycolytic potential.

Role of Notoginsenoside Rg1 in Improving Spatial Cognitive Ability and Lowering Phosphorylation Level of Tau Protein in AD Model Rats

[Objectives] To study the effects and mechanism of notoginsenoside Rg1 on the spatial learning and memory and phosphorylated tau protein in the AD( Alzheimer's Disease) model rat. [Methods]The AD model rat was replicated by injection of Aβ_(25-35) in the left lateral ventricles of SD rats. The low dose( 25 mg/kg),middle dose( 50 mg/kg) and high dose( 100 mg/kg) notoginsenoside Rg1 was used for intragastric administration,respectively,two times every day. After 4 weeks,the Morris water maze test was done to detect the learning and memory capacity,and the immunoblotting,immunohistochemical methods were used to detect the changes in the phosphorylation level and distribution of tau protein in hippocampus of the rats. [Results] After the intracerebroventricular injection of Aβ_(25-35),the learning and memory capacity of the model rats was significantly lower than the learning and memory capacity of the normal control rats. The immunoblotting test results showed that the phosphorylation level of tau protein threonine 231 site( Thr231) in hippocampus was significantly increased,and the nonphosphorylation level was significantly decreased. The morphological testing results showed that the phosphorylation level of tau protein Thr231 of AD model rats was increased markedly in region of DG,CA1 and CA3 of the hippocampus. The intervention of the middle dose notoginsenoside Rg1 could significantly improve the learning and memory capacity of the model rats in Morris water maze. The notoginsenoside Rg1 in three different doses could all reduce the phosphorylation level of tau protein Thr231 in the hippocampal DG,CA1,CA3 regions,and there were no significant differences among the three doses. [Conclusions]The notoginsenoside Rg1 could improve Aβ_(25-35)-induced spatial learning and memory impairment of the AD model rats,and decreased the phosphorylation level of tau protein in hippocampus.

THE IMMUNOREGULATORY EFFECT OF TLSF_(JM) ON THE EXPRESSION OF T CELL IL-2R AND PROTEIN TYROSINE PHOSPHORYLATION

The immunoregulatory effect of TLSFJM on the expression of T cell IL- 2R and protein tyrosine phosphorylation ( PTP ) was investigated by immunohistochemistry technique. The results showed that TLSFJMcan markedly suppress the expression of IL-2R and PTP on PHA or TPA-stimulated human PBMC and murine IL-2 dependent cell line CTLL-2. However, there was no effect of TLSFJMon the production of IL-1, IL-2 and IL-6 that play an important role in the course of T lymphocyte proliferation and differentiation.

Overexpression of tyrosine phosphorylated proteins in reproductive tissues of polycystic ovary syndrome rats induced by letrozole

Objective:To identify the alteration of tyrosine phosphorylated protein expression in rats with polycystic ovary syndrome(PCOS).Methods:Sixteen female Sprague-Dawley rats were divided into the control and letrozole-induced PCOS groups.The oestrus cycle of rats was performed by vaginal smear.Sex hormones and morphology of the ovary,oviduct,and uterus were observed.Expressions and intensity of androgen receptor and tyrosine phosphorylated proteins of reproductive organs were investigated by Western blot.Results:Various polycysts and increased androgen receptor expression were present in the ovary of the PCOS group.The levels of follicle-stimulating hormone and testosteone were significantly higher in the PCOS group while progesterone and estradiol levels were significantly decreased as compared with the control group(P<0.05).Only the size of uterus in the PCOS group was significantly smaller than the control group.However,the density of collagen fibers observed in PCOS uterus was greater than the control group.Moreover,tyrosine phosphorylated proteins were significantly overexpressed in ovary(52,42,and 28 kDa),oviduct(72,56,42,and 28 kDa),and uterus(53 and 42 kDa)of the PCOS group compared to the control group.Conclusions:Presence of tyrosine phosphorylated proteins in the ovary,oviduct and uterus suggests that overexpression of tyrosine phosphorylated proteins may be involved in potential mechanism of female infertility especially in PCOS.

Icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels in the hippocampus of the senescence-accelerated mouse

At 8 weeks after intragastric administration of icariin to senescence-accelerated mice （P8 strain）, Morris water maze results showed that escape latency was shortened, and the number of platform crossings was increased. Immunohistochemical staining and western blot assay detected significantly increased levels of cyclic adenosine monophosphate response element binding protein These results suggest that icariin upregulates phosphorylated cyclic adenosine monophosphate response element binding protein levels and improves learning and memory functions in hippocampus of the senescence-accelerated mouse.

Putative Phosphorylation Sites On WCA Domain of HA2 Is Essential For Helicoverpa armigera Single Nucleopolyhedrovirus Replication

Protein phosphorylation is one of the most common post-translational modification processes that play an essential role in regulating protein functionality.The Helicoverpa armigera single nucleopolyhedrovirus (HearNPV) orf2-encoded nucleocapsid protein HA2 participates in orchestration of virus-induced actin polymerization through its WCA domain,in which phosphorylation status are supposed to be critical in respect to actin polymerization.In the present study,two putative phosphorylation sites (232Thr and 250Ser) and a highly conserved Serine (245Ser) on the WCA domain of HA2 were mutated,and their phenotypes were characterized by reintroducing the mutated HA2 into the HearNPV genome.Viral infectivity assays demonstrated that only the recombinant HearNPV bearing HA2 mutation at 245Ser can produce infectious virions,both 232Thr and 250Ser mutations were lethal to the virus.However,actin polymerization assay demonstrated that all the three viruses bearing HA2 mutations were still capable of initiating actin polymerization in the host nucleus,which indicated the putative phosphorylation sites on HA2 may contribute to HearNPV replication through another unidentified pathway.

Proteomic Analysis of Nuclear Phosphorylated Proteins in Dairy Cow Mammary Epithelial Cells Treated with Prolactin

Prolactin （PRL） is a versatile signaling molecule and regulates a variety of physiological processes, including mammary gland growth and differentiation and the synthesis of milk proteins. While PRL is known to be necessary for high levels of milk protein expression, the mechanism by which the synthesis of milk proteins is stimulated at the transcript level is less known. A major modification in the transcript level is protein phosphorylation. To gain additional insights into the molecular mechanisms at the transcript level underlying PRL action on the dairy cow mammary epithelial cells （DCMECs）, nuclear phosphoproteins whose expression distinguishes proliferating regulated by PRL in DCMECs were identified. A phosphoprotein-enriched fraction from nuclear proteins was obtained by affinity chromatography, and a two-dimensional gel electrophoresis （2-DE） and matrix assisted laser desorption/ionization time of matrix-assisted laser desorption/ionization/time of flight mass spectrometry （MALDI-TOF MS） were used to identify the changes of nuclear phosphoproteins in DCMECs treated with prolactin. Seven proteins displaying~〉2-fold difference in abundance upon PRL treatment in DCMECs were identified by MALDI-TOF MS. The protein-GARS （GlyRS）, which belonged to the class-II aminoacyl-tRNA synthetase family, played a global role in the milk protein synthesis. SERPINH1 （Heat shock protein 47）, which was the first heat shock protein found to be a member of the serpin superfamily, regulated physiologic functions, such as complement activation, programmed cell death, and inflammatory processes. PRDX3, which belonged to a family of antioxidant enzymes, played an important role in scavenging intracellular reactive oxygen species （ROS）. ACTR1A, belonged to the actin family, which was associated with transport of p53 to the nucleus. Annexin A2, a Ca2＋-dependent phospholipid-binding protein, maintained the viability and cell cycle regulation of DCMECs. PSMB2 and PSMD10, which belonged to ubiquitin-proteasome system, were involved in several cellular processes, including cell cycle control, cellular stress response, intracellular signaling. This screening revealed that prolactin influenced the level of nuclear phosphoproteins in DCMECs. This result opens new avenues for the study of the molecular mechanism linked to the synthesis of milk proteins.

Tau protein,phosphorylated tau protein,and beta-amyloid 42 levels in patients with neurodegenerative diseases complicated by cognitive deficits A non-randomized,concurrent,case-control investigation

BACKGROUND： The differential diagnosis of many neurodegenerative disorders depends primarily on clinical symptoms together with imaging methods. Recently, increased importance has been placed on the use of biomarkers for diagnosing various neurodegenerative disorders. OBJECTIVE： To assess the feasibility of tau-protein, phosphorylated tau-protein, beta-amyloid 42 （Aβ42）, and 14-3-3 protein as biomarkers for diagnosing several neurodegenerative diseases complicated by cognitive deficits. DESIGN, TIME AND SETTING： A non-randomized, concurrent, case-control investigation was performed in three medical centers in the Czech Republic （Department of Neurology at the University Hospital in Hradec Kralove, Department of Neurology at the 2rd Medical Faculty, and the University Hospital Motol） between October 2000 and November 2006. PARTICIPANTS： Eighteen patients with probable AIzheimer＇s disease, 4 patients with Creutzfeldt-Jakob disease, 10 patients with frontotemporal dementia, 9 patients with clinically isolated syndrome suggestive of multiple sclerosis, and 7 patients with multiple sclerosis, as well as 38 race-, nationality-, and age-matched cognitively intact controls, were included in the study. Diagnoses were established based on the following criteria： the criteria for Alzheimer＇s disease proposed by the National Institute of Neurological and Communicative Disorders and Stroke/Alzheimer＇s Disease and Related Disorders Association, WHO criteria for Creutzfeldt-Jakob disease, Neary criteria for frontotemporal dementia, and McDonald＇s criteria for multiple sclerosis. All included patients were confirmed to suffer from various degrees of dementia. METHODS： Enzyme-linked immunosorbent assay was used to measure concentrations of tau-protein, phosphorylated tau-protein, and Aβ42 in cerebrospinal fluid （CSF） samples collected by standard lumbar puncture from each patient. Moreover, 14-3-3 protein was assessed by Western blot in CSF of Creutzfeldt-Jakob disease patients. Cognitive status was assessed using the Mini Mental Scale Examination （MMSE） in all subjects. MAIN OUTCOME MEASURES： Establishment of biomarkers with greatest specificity and sensitivity for the investigated disorders according to Receiver Operating Characteristic curves, which were based on values from patients and controls; correlation between concentrations of given biomarkers and demographic parameters, diagnosis, duration of disease, and level of cognitive deficit. RESULTS： Increased concentrations of total tau protein and phosphorylated tau protein, and decreased levels of Aβ42, in CSF of Alzheimer＇s disease patients reached the required sensitivity/specificity ratio of 80% or greater. A marked elevation in CSF concentrations of total tau protein showed even greater sensitivity than 14-3-3 protein in Creutzfeldt-Jakob disease. There was no association between selected biomarkers and frontotemporal dementia or multiple sclerosis. Phosphorylated tau-protein was the only biomarker that noticeably correlated with MMSE scores for Alzheimer＇s disease.CONCLUSION： Levels of total tau protein, phosphorylated tau protein, and A!342 in the CSF could differentiate patients with Alzheimer＇s disease and Creutzfeldt-Jakob disease from healthy controls and patients with other neurodegenerative disorders. The diversity of absolute values demonstrates the necessity to establish a specific standard for each laboratory.

Effect of Metabolites of Fusarium Solani on Tyrosine Phosphorylation in Cultivated Cells of Solarium Tuberosum

Elicitor-induced phosphorylation of tyrosine residues in proteins of potato was studied. Proteins of crude extract of suspension culture of potato were analyzed by one- and two-dimensional electrophoresis followed by Western blotting with monoclonal antibodies PY20 to phosphotyrosine proteins. One- and two-dimensional electrophoresis revealed l l and 25 tyrosine-phosphorylated proteins, respectively. Glycoprotein increased the phosphorylation level of most of these proteins.

Antibody-Like Phosphorylation Sites in Focus of Statistically Based Bilingual Approach

In accordance with previous reports, the sequences related to phosporylated protein segments occur in conserved variable domains of immunoglobulins including first of all certain N-terminally located segments. Consequently, we look here for the sequences 1) composing human and mouse proteins different from antigen receptors, 2) identical with or highly similar to nucleotide sequence representatives of conserved variable immunoglobulin segments and 3) identical with or closely related to phosphorylation sites. More precisely, we searched for the corresponding actual pairs of DNA and protein sequence segments using five-step bilingual approach employing among others a) different types of BLAST searches, b) two in-principle-different machine-learning methods predicting phosphorylated sites and c) two large databases recording existing phosphorylation sites. The approach identified seven existing phosphorylation sites and thirty-seven related human and mouse segments achieving limits for several predictions or phylogenic parameters. Mostly serines phosporylated with ataxia-telangiectasia-related kinase (involved in regulation of DNA-double-strand-break repair) were indicated or predicted in this study. Hypermutation motifs, located in effective positions of the selected sequence segments, occurred significantly less frequently in transcribed than non-transcribed DNA strands suggesting thus the incidence of mutation events. In addition, marked differences between the numbers and proportions of human and mouse cancer-related sequence items were found in different steps of selection process. The possible role of hypermutation changes within the selected segments and the observed structural relationships are discussed here with respect to DNA damage, carcinogenesis, cancer vaccination, ageing and evolution. Taken together, our data represent additional and sometimes perhaps complementary information to the existing databases of empirically proven phosphorylation sites or pathogenically important spots.

Phosphorylation of OsRbohB by the protein kinase OsDMI3 promotes H_(2)O_(2) production to potentiate ABA responses in rice

In rice, the Ca^(2+)/calmodulin-dependent protein kinase OsDMI3 is an important positive regulator of abscisic acid (ABA) signaling. In ABA signaling, H_(2)O_(2) is required for ABA-induced activation of OsDMI3, which in turn increase H_(2)O_(2) production. However, how OsDMI3 regulates H_(2)O_(2) production in ABA signaling remains unknown. Here we show that OsRbohB is the main NADPH oxidase involved in ABA-induced H_(2)O_(2) production and ABA-mediated physiological responses. OsDMI3 directly interacts with and phosphorylates OsRbohB at Ser-191, which is OsDMI3-mediated site-specific phosphorylation in ABA signaling. Further analyses revealed that OsDMI3-mediated OsRbohB Ser-191 phosphorylation positively regulates the activity of NADPH oxidase and the production of H_(2)O_(2) in ABA signaling, thereby enhancing the sensitivity of seed germination and root growth to ABA and plant tolerance to water stress and oxidative stress. Moreover, we discovered that the OsDMI3-mediated OsRbohB phosphorylation and H_(2)O_(2) production is dependent on the sucrose non-fermenting 1-related protein kinases SAPK8/9/10, which phosphorylate OsRbohB at Ser-140 in ABA signaling. Taken together, these results not only reveal an important regulatory mechanism that directly activates Rboh for ABA-induced H_(2)O_(2) production but also uncover the importance of this regulatory mechanism in ABA signaling.

Functional repertoire of protein kinases and phosphatases in synaptic plasticity and associated neurological disorders

Protein phosphorylation and dephosphorylation are two essential and vital cellular mechanisms that regulate many receptors and enzymes through kinases and phosphatases.Ca^2+- dependent kinases and phosphatases are responsible for controlling neuronal processing;balance is achieved through opposition.During molecular mechanisms of learning and memory,kinases generally modulate positively while phosphatases modulate negatively.This review outlines some of the critical physiological and structural aspects of kinases and phosphatases involved in maintaining postsynaptic structural plasticity.It also explores the link between neuronal disorders and the deregulation of phosphatases and kinases.

ADHESION-INDUCE PROTEIN TYROSINE PHOSPHORY-LATION IS ASSOCIATED WITH INVASIVE AND METASTATIC POTENTIALS IN B16-BL6 MELANOMA CELLS

Objective: The interaction of cancer cell with extracellular matrix (ECM) happens as an earlier and specific event in the invasive and metastatic cascade. To explore the key element(s) in cancer metastasis and observe the cellECM interaction and its role. Methods: To interrupt the cellECM interaction by suppression of adhesioninduced protein tyrosine phosphorylation with protein tyrosine kinase inhibitor genistein in B16B16 mouse melanoma cells. Results: When B16BL6 cells attached to Matrigel, a solubilized basement membrane preparation from EHS sarcoma, a 125 kDa protein increased its phosphotyrosine content dramatically. In contrast, when the cells were pretreated with 20μM or 30 μM genistein for 3 days, it was revealed a less increase in the phosphotyrosine content of this 125 kDa protein in response to cell attachment to ECM was revealed with immunoblot analysis. Accompanied by the lower level of adhesioninduced protein tyrosine phosphorylation the genisteintreated cells exhibited a decrease in their capabilities of adhesion to Matrigel and invasion through reconstituted basement membrane. The potentials of and forming lung metastatic nodules were also shown to be decreased dramatically in these genisteintreated cells. Conclusion: It was suggested that protein tyrosine phosphorylation in cellECM interaction might be associated with invasive and metastatic potentials in cancer cells.

GPS 5.0: An Update on the Prediction of Kinase-specific Phosphorylation Sites in Proteins

In eukaryotes,protein phosphorylation is specifically catalyzed by numerous protein kinases(PKs),faithfully orchestrates various biological processes,and reversibly determines cellular dynamics and plasticity.Here we report an updated algorithm of Group-based Prediction System(GPS)5.0 to improve the performance for predicting kinase-specific phosphorylation sites(p-sites).Two novel methods,position weight determination(PWD)and scoring matrix optimization(SMO),were developed.Compared with other existing tools,GPS 5.0 exhibits a highly competitive accuracy.Besides serine/threonine or tyrosine kinases,GPS 5.0 also supports the prediction of dual-specificity kinase-specific p-sites.In the classical module of GPS 5.0,617 individual predictors were constructed for predicting p-sites of 479 human PKs.To extend the application of GPS5.0,a species-specific module was implemented to predict kinase-specific p-sites for 44,795 PKs in161 eukaryotes.The online service and local packages of GPS 5.0 are freely available for academic research at http://gps.biocuckoo.cn.

Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-Induced Volatile Oil Production Through Protein Phosphorylation Pathways in Atractylodes lancea Plantlets

Fungal endophytes have been isolated from almost every plant, infecting their hosts without causing visible disease symptoms, and yet have still proved to be involved in plant secondary metabolites accumulation. To decipher the possible physiological mechanisms of the endophytic fungus-host interaction, the role of protein phosphorylation and the relationship between endophytic fungus-induced kinase activity and nitric oxide （NO） and brassinolide （BL） in endophyte-enhanced volatile oil accumulation in Atractylodes lancea plantlets were investigated using pharmacological and biochemical approaches. Inoculation with the endophytic fungus Gilmaniella sp. ALl2 enhanced the activities of total protein phosphorylation, Ca2^-dependent protein kinase, and volatile oil accumulation in A. lancea plantlets. The upregulation of protein kinase activity could be blocked by the BL inhibitor brassinazole. Furthermore, pretreatments with the NO-specific scavenger cPTIO significantly reduced the increased activities of protein kinases in A. lancea plantlets inoculated with endophytic fungus. Pretreatments with different protein kinase inhibitors also reduced fungus-induced NO production and volatile oil accumulation, but had barely no effect on the BL level. These data suggest that protein phosphorylation is required for endophyte- induced volatile oil production in A. lancea plantlets, and that crosstalk between protein phosphorylation and the NO pathway may occur and act as a downstream signaling event of the BL pathway.

A novel secreted protein, NISP1, is phosphorylated by soybean Nodulation Receptor Kinase to promote nodule symbiosis

Nodulation Receptor Kinase(NORK) functions as a co-receptor of Nod factor receptors to mediate rhizobial symbiosis in legumes, but its direct phosphorylation substrates that positively mediate root nodulation remain to be fully identified.Here, we identified a GmNORK-Interacting Small Protein(GmNISP1) that functions as a phosphorylation target of GmNORK to promote soybean nodulation. GmNORKα directly interacted with and phosphorylated GmNISP1. Transcription of GmNISP1 was strongly induced after rhizobial infection in soybean roots and nodules. GmNISP1 encodes a peptide containing 90 amino acids with a “DY” consensus motif at its N-terminus.GmNISP1 protein was detected to be present in the apoplastic space. Phosphorylation of GmNISP1 by GmNORKα could enhance its secretion into the apoplast. Pretreatment with either purified GmNISP1 or phosphorylation-mimic GmNISP1~(12D) on the roots could significantly increase nodule numbers compared with the treatment with phosphorylation-inactive GmNISP1~(12A).The data suggested a model that soybean GmNORK phosphorylates GmNISP1 to promote its secretion into the apoplast, which might function as a potential peptide hormone to promote root nodulation.

Involvement of Protein Phosphorylation in Water Stress-induced Antioxidant Defense in Maize Leaves

Using pharmacological and biochemical approaches, the role of protein phosphorylation and the interrelationship between water stress-enhanced kinase activity, antioxidant enzyme activity, hydrogen peroxide （H202） accumulation and endogenous abscisic acid in maize （Zea mays L.） leaves were investigated. Water-stress upregulated the activities of total protein phosphorylation and Ca^2＋-dependent protein kinase, and the upregulation was blocked in abscisic aciddeficient vp5 mutant. Furthermore, pretreatments with a nicotinamide adenine dinucleotide phosphate oxidase inhibitor and a scavenger of H2O2 significantly reduced the increased activities of total protein kinase and Ca^2＋-dependent protein kinase in maize leaves exposed to water stress. Pretreatments with different protein kinase inhibitors also reduced the water stress-induced H2O2 production and the water stress-enhanced activities of antioxidant enzymes such as superoxide dismutase, catalase, ascorbate peroxidase and glutathione reductase. The data suggest that protein phosphorylation and H2O2 generation are required for water stress-induced antioxidant defense in maize leaves and that crosstalk between protein phosphorylation and H2O2 generation may occur.