Dissecting the Heat Stress Response in Chlamydomonas by Pharmaceutical and RNAi Approaches Reveals Conserved and Novel Aspects

To study how conserved fundamental concepts of the heat stress response （HSR） are in photosynthetic eukaryotes, we applied pharmaceutical and antisense/amiRNA approaches to the unicellular green alga Chlamydomonas reinhardtii. The Chlamydomonas HSR appears to be triggered by the accumulation of unfolded proteins, as it was induced at ambient temperatures by feeding cells with the arginine analog canavanine. The protein kinase inhibitor staurosporine strongly retarded the HSR, demonstrating the importance of phosphorylation during activation of the HSR also in Chlamydomonas. While the removal of extracellular calcium by the application of EGTA and BAPTA inhibited the HSR in moss and higher plants, only the addition of BAPTA, but not of EGTA, retarded the HSR and impaired thermotoler- ance in Chlamydomonas. The addition of cycloheximide, an inhibitor of cytosolic protein synthesis, abolished the attenu- ation of the HSR, indicating that protein synthesis is necessary to restore proteostasis. HSP90 inhibitors induced a stress response when added at ambient conditions and retarded attenuation of the HSR at elevated temperatures. In addition, we detected a direct physical interaction between cytosolic HSP90A/HSP70A and heat shock factor 1, but surprisingly this interaction persisted after the onset of stress. Finally, the expression of antisense constructs targeting chloroplast HSP70B resulted in a delay of the cell＇s entire HSR, thus suggesting the existence of a retrograde stress signaling cascade that is desensitized in HSP7OB-antisense strains.     

Nitric Oxide and Brassinosteroids Mediated Fungal Endophyte-lnduced 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.     

Import Determinants of Organelle-Specific and Dual Targeting Peptides of Mitochondria and Chloroplasts in Arabidopsis thaliana

Most of the mitochondrial and chloroplastic proteins are synthesized in the cytosol as precursor proteins carrying an N-terminal targeting peptide （TP） directing them specifically to a correct organelle. However, there is a group of proteins that are dually targeted to mitochondria and chloroplasts using an ambiguous N-terminal dual targeting peptide （dTP）. Here, we have investigated pattern properties of import determinants of organelle-specific TPs and dTPs combining mathematical multivariate data analysis （MVDA） with in vitro organellar import studies. We have used large datasets of mitochondrial and chloroplastic proteins found in organellar proteomes as well as manually selected data sets of experimentally confirmed organelle-specific TPs and dTPs from Arabidopsis thaliana. Two classes of organelle-specific TPs could be distinguished by MVDA and potential patterns or periodicity in the amino acid sequence contributing to the separation were revealed, dTPs were found to have intermediate sequence features between the organelle-specific TPs. Interestingly, introducing positively charged residues to the dTPs showed clustering towards the mitochondrial TPs in silico and resulted in inhibition of chloroplast, but not mitochondrial import in in vitro organellar import studies. These findings suggest that positive charges in the N-terminal region of TPs may function as an ＇avoidance signal＇ for the chloroplast import.     

A New Set of Reversibly Photoswitchable Fluorescent Proteins for Use in Transgenic Plants

Fluorescent reporter proteins that allow repeated switching between a fluorescent and a non-fluorescent state in response to specific wavelengths of light are novel tools for monitoring of protein trafficking and super-resolu- tion fluorescence microscopy in living organisms. Here, we describe variants of the reversibly photoswitchable fluores- cent proteins rsFastLime, bsDronpa, and Padron that have been codon-optimized for the use in transgenic Arabidopsis plants. The synthetic proteins, designated rsFastLIME-s, bsDRONPA-s, and PADRON C-s, showed photophysical properties and switching behavior comparable to those reported for the original proteins. By combining the ＇positively switchable＇ PADRON C-s with the ＇negatively switchable＇ rsFastLIME-s or bsDRONPA-s, two different fluorescent reporter proteins could be imaged at the same wavelength upon transient expression in Nicotiana benthamiana cells. Thus, co-localiza- tion analysis can be performed using only a single detection channel. Furthermore, the proteins were used to tag the RNA-binding protein AtGRP7 （Arabidopsis thaliana glycine-rich RNA-binding protein 7） in transgenic Arabidopsis plants. Because the new reversibly photoswitchable fluorescent proteins show an increase in signal strength during each pho- toactivation cycle, we were able to generate a large number of scans of the same region and reconstruct 3-D images of AtGRP7 expression in the root tip. Upon photoactivation of the AtGRP7：rsFastLIME-s fusion protein in a defined region of a transgenic Arabidopsis root, spreading of the fluorescence signal into adjacent regions was observed, indicating that movement from cell to cell can be monitored. Our results demonstrate that rsFastLIME-s, bsDRONPA-s, and PADRON C-s are versatile fluorescent markers in plants, Furthermore, the proteins also show strong fluorescence in mammalian cells including COS-7 and HeLa cells.     

Regulation of protein stability of DNA methyltransferase 1 by post-translational modifications

DNA methylation is an important epigenetic mechanism that ensures correct gene expression and maintains genetic stability. DNA methyltransferase 1 （DNMT1） is the primary enzyme that maintains DNA methylation during replication. Dysregulation of DNMT1 is implicated in a variety of diseases. DNMT1 protein stability is regulated via various post-translational modifications, such as acetyl- ation and ubiquitination, but also through protein-protein interactions. These mechanisms ensure DNMT1 is properly activated during the correct time of the ceil cycle and at correct genomic loci, as well as in response to appropriate extracellular cues. Further understanding of these regula- tory mechanisms may help to design novel therapeutic approaches for human diseases.     

Knights in Action： Lectin Receptor-Like Kinases in Plant Development and Stress Responses

The Receptor-Like Kinase （RLK） is a vast protein family with over 600 genes in Arabidopsis and 1100 in rice. The Lectin RLK （LecRLK） family is believed to play crucial roles in saccharide signaling as well as stress perception. All the LecRLKs possess three domains： an N-terminal lectin domain, an intermediate transmembrane domain, and a C-terminal kinase domain. On the basis of lectin domain variability, LecRLKs have been subgrouped into three subclasses： L-, G-, and C-type LecRLKs. While the previous studies on LecRLKs were dedicated to classification, comparative structural analysis and expression analysis by promoter-based studies, most of the recent studies on LecRLKs have laid special emphasis on the potential of this gene family in regulating biotic/abiotic stress and developmental pathways in plants, thus mak- ing the prospects of studying the LecRLK-mediated regulatory mechanism exceptionally promising. In this review, we have described in detail the LecRLK gene family with respect to a historical, evolutionary, and structural point of view. Furthermore, we have laid emphasis on the LecRLKs roles in development, stress conditions, and hormonal response. We have also discussed the exciting research prospects offered by the current knowledge on the LecRLK gene family. The multitude of the LecRLK gene family members and their functional diversity mark these genes as both interesting and worthy candidates for further analysis, especially in the field of crop improvement.     

De-oncogenic HPV E6/E7 vaccine gets enhanced antigenicity and promotes tumoricidal synergy with cisplatin

In order to develop more effective therapeutic vaccines against cancers with high-risk human papillomavirus （HPV） infection, it is crucial to enhance the immunogenicity, eliminate the oncogenicity of oncoproteins, and take a combination of ET- and E6-containing vaccines. It has been shown recently that PE（AIII）-E7-KDEL3 （E7）, a fusion protein containing the HPVI6 oncoprotein E7 and the trans- location domain of Pseudomonas aeruginosa exotoxin A, is effective against TC-1 tumor cells inoculated in mice, there- fore, we engineered PE（AIII）-E6-CRL-KDEL3 （E6）, the deoncogenic versions of the E7 and E6 fusion proteins [i.e. PE（AIII）-E7（d）-KDEL3, E7（d）, and PE（AIII）-E6（d）-CRL- KDEL3, E6（d）] and tested the immunoefficacies of these fusion proteins as mono- and bivalent vaccines. Results indicated that the E7（d） get higher immunogenicity than its wild type and the E6 fusion proteins augmented the im- munogenicity and antitumor effects of their E7 counterparts. Furthermore, the bivalent vaccine system E7（d） plus E6（d）, in the presence of cisplatin, showed the best tumori- static and tumoricidal effects against established tumors in vivo. Therefore, it can be concluded that this novel therapeutic vaccine system, upon further optimization, may shed new light on clinical management of HPV-related carcinomas.     

A multi-epitope vaccine based on Chlamydia trachomatis major outer membrane protein induces specific immunity in mice

We evaluated the immunogenicity and efficacy of a candidate vaccine comprising the major outer membrane protein （MOMP） multi-epitope of Chlamydia trachomatis. A short gene of muiti-epitope derived from MOMP containing multiple T- and B-cell epitopes was artificially synthesized. The recombinant plasmid pET32a（＋） containing codon optimized MOMP multi-epitope gene was constructed. Expression of the fusion protein Trx-His- MOMP multi-epitope in Escherichia coli was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and western blot analysis. Balb/c mice were inoculated with the purified fusion protein subcutaneously three times with 2-week intervals. Results showed that the MOMP multiepitope elicited not only strong humoral immune responses to C. trachomatis by generating significantly high levels of specific antibodies （lgG1 and IgG2a）, but also a cellular immune response by inducing robust cytotoxic T lymphocyte responses in mice. Furthermore, the MOMP multi- epitope substantially primed secretion of IFN-γ, revealing that this vaccine could induce a strong Thl response. Finally, the mice vaccinated with the MOMP multi-epitope displayed a reduction of C. trachomatis shedding upon a chlamydial challenge and an accelerated clearance of the infected C. trachomatis. In conclusion, the MOMP multi- epitope vaccine may have the potentiality for the development of effective prophylactic and therapeutic vaccines against the C. trachomatis infection.     

Cloning and Characterization of miRNAs and Their Targets,Including a Novel miRNA-Targeted NBS-LRR Protein Class Gene in Apple （Golden Delicious）

MicroRNA （miRNA） has emerged as an important regulator of gene expression in plants. 146 miRNAs were identified from apple （Malus domestica cv. Golden Delicious） by bioinformatic analysis and RNA library sequencing. From these, 135 were conserved and 11 were novel miRNAs. Target analysis predicted one of the novel miRNAs, Md-miRLn11 （Malus domestica microRNA Ln11）, targeted an apple nucleotide-binding site （NBS）-Ieucine-rich repeat （LRR） class protein coding gene （Md-NBS）. 5/ RACE assay confirmed the ability of Md-miRLn11 to cleave Md-NBS at the 11-12-nt position. Analysis of the expression of Md-miRLn11 and Md-NBS during the optimum invasion period in 40 apple varieties showed that the expression of Md-NBS gene in resistant varieties is higher than in susceptible varieties, with an inverse pattern for Md-miRLn11. Seedlings from the resistant apple variety ＇JiGuan＇ were used to carry out an Agrobacterium infiltration assay, and then inoculated with the apple leaf spot disease. The result showed a clear decline of disease resistance in JiGuan apples. In contrast, the susceptible variety ＇FuJi＇ infiltrated with the Md-NBS gene showed a significant increase in disease resistance. Based on the above results, we propose that Md-miRLn11 regulates Md-NBS gene expression in particular under the condition of pathogen infection, and that the Md-miRLn11 targeting P-loop site may regulate many NBS-LRR protein class genes in woody plants.     

Salt Stress in Arabidopsis： Lipid Transfer Protein AZI1 and Its Control by Mitogen-Activated Protein Kinase MPK3

A plant＇s capability to cope with environmental challenges largely relies on signal transmission through mitogen-activated protein kinase （MAPK） cascades. In Arabidopsis thaliana, MPK3 is particularly strongly associated with numerous abiotic and biotic stress responses. Identification of MPK3 substrates is a milestone towards improving stress resistance in plants. Here, we characterize AZI1, a lipid transfer protein （LTP）-related hybrid proline-rich protein （HyPRP）, as a novel target of MPK3. AZI1 is phosphorylated by MPK3 in vitro. As documented by co-immunoprecipitation and bimolecular fluorescence complementation experiments, AZI1 interacts with MPK3 to form protein complexes in planta. Furthermore, null mutants of azil are hypersensitive to salt stress, while AZIl-overexpressing lines are markedly more tolerant. AZI1 overexpression in the mpk3 genetic background partially alleviates the salt-hypersensitive phenotype of this mutant, but functional MPK3 appears to be required for the full extent of AZIl-conferred robustness. Notably, this robustness does not come at the expense of normal development. Immunoblot and RT-PCR data point to a role of MPK3 as positive regulator of AZI1 abundance.     

Inositol Polyphosphate Phosphatidylinositol 5-Phosphatase9 （At5PTase9） Controls Plant Salt Tolerance by Regulating Endocytosis

Phosphatidylinositol 5-phosphatases （5PTases） components of membrane trafficking system. Recently, we that hydrolyze the 5＇ position of the inositol ring are key reported that mutation in AtSPTase7 gene reduced produc- tion of reactive oxygen species （ROS） and decreased expression of stress-responsive genes, resulting in increased salt sensitivity. Here, we describe an even more salt-sensitive 5ptase mutant, At5ptase9, which also hydrolyzes the 5＇ phos- phate groups specifically from membrane-bound phosphatidylinositides. Interestingly, the mutants were more tolerant to osmotic stress. We analyzed the main cellular processes that may be affected by the mutation, such as production of ROS, influx of calcium, and induction of salt-response genes. The At5ptase9 mutants showed reduced ROS produc- tion and Ca2~ influx, as well as decreased fluid-phase endocytosis. Inhibition of endocytosis by phenylarsine oxide or Tyrphostin A23 in wild-type plants blocked these responses. Induction of salt-responsive genes in wild-type plants was also suppressed by the endocytosis inhibitors. Thus, inhibition of endocytosis in wild-type plants mimicked the salt stress responses, observed in the AtSptase9 mutants. In summary, our results show a key non-redundant role of At5PTase7 and 9 isozymes, and underscore the localization of membrane-bound Ptdlns in regulating plant salt tolerance by coordinating the endocytosis, ROS production, Ca2＋ influx, and induction of stress-responsive genes.     

Redox Regulation of Arabidopsis Mitochondrial Citrate Synthase

Citrate synthase has a key role in the tricarboxylic （TCA） cycle of mitochondria of all organisms, as it cata- lyzes the first committed step which is the fusion of a carbon-carbon bond between oxaloacetate and acetyl CoA. The regulation of TCA cycle function is especially important in plants, since mitochondrial activities have to be coordinated with photosynthesis. The posttranslational regulation of TCA cycle activity in plants is thus far almost entirely unexplored. Although several TCA cycle enzymes have been identified as thioredoxin targets in vitro, the existence of any thioredoxin-dependent regulation as known for the Calvin cycle, yet remains to be demonstrated. Here we have investigated the redox regulation of the Arabidopsis citrate synthase enzyme by site-directed mutagenesis of its six cysteine residues. Our results indicate that oxidation inhibits the enzyme activity by the formation of mixed disulfides, as the partially oxidized citrate synthase enzyme forms large redox-dependent aggregates. Furthermore, we were able to demonstrate that thioredoxin can cleave diverse intraas well as intermolecular disulfide bridges, which strongly enhances the activity of the enzyme. Activity measurements with the cysteine variants of the enzyme revealed important cysteine residues affecting total enzyme activity as well as the redox sensitivity of the enzyme.     

Organelle pH in the Arabidopsis Endomembrane System

The pH of intracellular compartments is essential for the viability of cells. Despite its relevance, little is known about the pH of these compartments. To measure pH in vivo, we have first generated two pH sensors by combining the improved-solubility feature of solubility-modified green fluorescent protein （GFP） （smGFP） with the pH-sensing capabil- ity of the pHluorins and codon optimized for expression in Arabidopsis. PEpHluorin （plant-solubility-modified ecliptic pHluorin） gradually loses fluorescence as pH is lowered with fluorescence vanishing at pH 6.2 and PRpHluorin （plant- solubility-modified ratiomatric pHluorin）, a dual-excitation sensor, allowing for precise measurements. Compartment- specific sensors were generated by further fusing specific sorting signals to PEpHluorin and PRpHluorin. Our results show that the pH of cytosol and nucleus is similar （pH 7.3 and 7.2）, while peroxisomes, mitochondrial matrix, and plastidial stroma have alkaline pH. Compartments of the secretory pathway reveal a gradual acidification, spanning from pH 7.1 in the endoplasmic reticulum （ER） to pH 5.2 in the vacuole. Surprisingly, pH in the trans-Golgi network （TGN） and mul- tivesicular body （MVB） is, with pH 6.3 and 6.2, quite similar. The inhibition of vacuolar-type H＋-ATPase （V-ATPase） with concanamycin A （ConcA） caused drastic increase in pH in TGN and vacuole. Overall, the PEpHluorin and PRpHluorin are excellent pH sensors for visualization and quantification of pH in vivo, respectively.     

How does the host-specialized aphid deal with food deficiency？

Aphis gossypii Glover shows obvious host specialization, with cucurbit- and cotton-specialized biotypes or host races in many regions. Because its annual natal hostcrops senesce earlier the cucurbit-specialized biotype may suffer food deficiency. The method this biotype uses to overcome this challenge is still poorly understood. In orderto understand the potential of the cucurbit-specialized biotype aphids in host shift and usage, the performance of this biotype on cotton （Gossypium hirsutum）, a common butpoor quality host plant, was explored in this study. The cucurbit-specialized aphids could establish populations on cotton only when these plants had at least nine leaves, and subsequent populations developed rather slowly. The presence of whitefly populations on cotton improved the success rate of cucurbit-specialized aphids. The cucurbit-specialized aphidswere mainly distributed on the older leaves of cotton, with only a few settling on the upper leaves. The cucurbit-specialized aphids reared on cotton for 40, 54 and 61 days stillmaintained strong preference for their natal host plant, cucumber （Cucumis sativus）, rather than cotton, and their net reproductive rates and intrinsic rates of natural increase weredramatically lower when they were transferred onto new six-leaf cotton plants or detached leaves. Therefore, we concluded that the cucurbit-specialized aphids have the potentialto utilize mature or whitefly-stressed cotton plants, but that this feeding experience on cotton did not alter their specialization for cucurbits. Some cotton plants could act as atemporary host for the cucurbit-specialized aphids to overcome food deficiency arising from senescing cucurbits.     

Plastid Signals and the Bundle Sheath： Mesophyll Development in Reticulate Mutants

The development of a plant leaf is a meticulously orchestrated sequence of events producing a complex organ comprising diverse cell types. The reticulate class of leaf variegation mutants displays contrasting pigmentation between veins and interveinal regions due to specific aberrations in the development of mesophyll cells. Thus, the reticulate mutants offer a potent tool to investigate cell-type-specific developmental processes. The discovery that most mutants are affected in plastid-localized, metabolic pathways that are strongly expressed in vasculature-associated tis- sues implicates a crucial role for the bundle sheath and their chloroplasts in proper development of the mesophyll cells. Here, we review the reticulate mutants and their phenotypic characteristics, with a focus on those in Arabidopsis thali- ana. Two alternative models have been put forward to explain the relationship between plastid metabolism and meso- phyll cell development, which we call here the supply and the signaling hypotheses. We critically assess these proposed models and discuss their implications for leaf development and bundle sheath function in C3 species. The characteriza- tion of the reticulate mutants supports the significance of plastid retrograde signaling in cell development and highlights the significance of the bundle sheath in C3 photosynthesis.     

Cell Wall,Cytoskeleton, and Cell Expansion in Higher Plants

To accommodate two seemingly contradictory biological roles in plant physiology, providing both the rigid structural support of plant cells and the adjustable elasticity needed for cell expansion, the composition of the plant cell wall has evolved to become an intricate network of cellulosic, hemicellulosic, and pectic polysaccharides and protein. Due to its complexity, many aspects of the cell wall influence plant cell expansion, and many new and insightful observations and technologies are forthcoming. The biosynthesis of cell wall polymers and the roles of the variety of proteins involved in polysaccharide synthesis continue to be characterized. The interactions within the cell wall polymer network and the modification of these interactions provide insight into how the plant cell wall provides its dual function. The complex cell wall architecture is controlled and organized in part by the dynamic intracellular cytoskeleton and by diverse trafficking pathways of the cell wall polymers and cell wall-related machinery. Meanwhile, the cell wall is continually influenced by hormonal and integrity sensing stimuli that are perceived by the cell. These many processes cooperate to construct, maintain, and manipulate the intricate plant cell wall--an essential structure for the sustaining of the plant stature, growth, and life.     

Aphis （Hemiptera： Aphididae） species groups found in the Midwestern United States and their contribution to the phylogenetic knowledge of the genus

A phylogeny of the genus Aphis Linnaeus, 1 758 was built primarily from specimens collected in the Midwest of the United States. A data matrix was constructedwith 68 species and 41 morphological characters with respective character states of alate and apterous viviparous females. Dendrogram topologies of analyses performed usingUPGMA （Unweighted Pair Group Method with Arithmetic Mean）, Maximum Parsimony and Bayesian analysis of Cytochrome Oxidase I, Elongation Factor 1-α and primary endosymbiont Buchnera aphidicola 16S sequences were not congruent. Bayesian analysis strongly supported most terminal nodes of the phylogenetic trees. The phylogeny wasstrongly supported by EFI-α, and analysis of COl and EFI-α molecular data combined with morphological characters. It was not supported by single analysis of COI or Buch-hera aphidicola 16S. Results from the Bayesian phylogeny show 4 main species groups： asclepiadis,fabae, gossypii, and middletonii. Results place Aphis and species of the generaProtaphis Bōrner, 1952, Toxoptera Koch, 1856 and Xerobion Nevsky, 1928 in a monophyletic clade. Morphological characters support this monophyly as well. The phylogenyshows that the monophyletic clade of the North American middletonii species group belong to the genus Protaphis： P. debilicornis （Gillette ＆ Palmer, 1929）, comb. nov., P. echinaceae（Lagos and Voegtlin, 2009）, comb. nov., and P. middletonii （Thomas, 1879）. The genus Toxoptera should be considered a subgenus of Aphis （stat. nov.）. The analysis also indicatesthat the current genus Iowana Frison, 1954 should be considered a subgenus of Aphis （stat. nov.）.