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.     

Retromer Subunits VPS35A and VPS29 Mediate Prevacuolar Compartment （PVC） Function in Arabidopsis

Intracellular protein routing is mediated by vesicular transport which is tightly regulated in eukaryotes. The protein and lipid homeostasis depends on coordinated delivery of de novo synthesized or recycled cargoes to the plasma membrane by exocytosis and their subsequent removal by rerouting them for recycling or degradation. Here, we report the characterization of protein affected trafficking 3 （pat3） mutant that we identified by an epifluorescence-based for- ward genetic screen for mutants defective in subcellular distribution of Arabidopsis auxin transporter PIN1-GFR While pat3 displays largely normal plant morphology and development in nutrient-rich conditions, it shows strong ectopic intracellular accumulations of different plasma membrane cargoes in structures that resemble prevacuolar compart- ments （PVC） with an aberrant morphology. Genetic mapping revealed that pat3 is defective in vacuolar protein sorting 35A （VPS35A）, a putative subunit of the retromer complex that mediates retrograde trafficking between the PVC and trans-Golgi network. Similarly, a mutant defective in another retromer subunit, vps29, shows comparable subcellular defects in PVC morphology and protein accumulation. Thus, our data provide evidence that the retromer components VPS35A and VPS29 are essential for normal PVC morphology and normal trafficking of plasma membrane proteins in plants. In addition, we show that, out of the three VPS35 retromer subunits present in Arabidopsis thaliana genome, the VPS35 homolog A plays a prevailing role in trafficking to the lyric vacuole, presenting another level of complexity in the retromer-dependent vacuolar sorting.     

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.     

Perturbation of Auxin Homeostasis Caused by Mitochondrial FtSH4 Gene-Mediated Peroxidase Accumulation Regulates Arabiclopsis Architecture

Reactive oxygen species and auxin play important roles in the networks that regulate plant development and morphogenetic changes, However, the molecular mechanisms underlying the interactions between them are poorly understood. This study isolated a mas （More Axillary Shoots） mutant, which was identified as an allele of the mitochondrial AAA-protease AtFtSH4, and characterized the function of the FtSH4 gene in regulating plant development by medi- ating the peroxidase-dependent interplay between hydrogen peroxide （H2Oz） and auxin homeostasis. The phenotypes of dwarfism and increased axillary branches observed in the mas （renamed as ftsh4-4） mutant result from a decrease in the IAA concentration. The expression levels of several auxin signaling genes, including IAA1, IAA2, and IAA3, as well as several auxin binding and transport genes, decreased significantly in ftsh4-4 plants. However, the H202 and peroxidases levels, which also have IAA oxidase activity, were significantly elevated in ftsh4-4 plants. The ftsh4-4 phenotypes could be reversed by expressing the iaaM gene or by knocking down the peroxidase genes PRX34 and PRX33. Both approaches can increase auxin levels in the ftsh4-4 mutant. Taken together, these results provided direct molecular and genetic evidence for the interaction between mitochondrial ATP-dependent protease, H2O2, and auxin homeostasis to regulate plant growth and development.     

A Crucial Role of the RGS Domain in Trans- Golgi Network Export of AtRGS1 in the Protein Secretory Pathway

The secretory pathway is responsible for the transport of newly synthesized transmembrane proteins from the endoplasmic reticulum to their destinations via the Golgi/trans-Golgi network （TGN）, Cargo proteins at each sta- tion are actively sorted by specific sorting signals on the cargo and the corresponding coat complexes. Here, we used the Arabidopsis regulator of G-protein signaling （AtRGS1）, which contains an N-terminal potentially sensing glucose seven-transmembrane domain and a C-terminal RGS domain, as a model to uncover sorting motifs required for its cell surface expression. Expression of wild-type and truncated or mutated AtRGS1 fluorescent fusion proteins identified two cysteine residues in the extracellular N-terminus that are essential for endoplasmic reticulum exit and/or correct folding of AtRGS1. The linker between the seven-transmembrane and RGS domains contains an endoplasmic reticulum export signal, whereas the C-terminus is dispensable for the plasma membrane expression of AtRGS1. Interestingly, deletion of the RGS domain results in Golgi/TGN localization of the truncated AtRGS1. Further analysis using site-directed mutagen- esis showed that a tyrosine-based motif embedded in the RGS domain is essential for Golgi/TGN export of AtRGS1. These results reveal a new role for the RGS domain in regulating AtRGS1 trafficking from the Golgi/TGN to the plasma membrane and explain the interaction between the seven-transmembrane and RGS domains.     

Salmonella enterica Flagellin Is Recognized via FLS2 and Activates PAMP-Triggered Immunity in Arabidopsis thaliana

Infections with Salmonella enterica belong to the most prominent causes of food poisoning and infected fruits and vegetables represent important vectors for salmonellosis. Recent evidence indicates that plants recognize S. enterica and raise defense responses. Nonetheless, the molecular mechanisms controlling the interaction of S. enterica with plants are still largely unclear. Here, we show that flagellin from S. enterica represents a prominent pathogenassociated molecular pattern （PAMP） in Arabidopsis thaliana, which induces PAMP-triggered immunity （PTI） via the recognition of the fig22 domain by the receptor kinase FLS2. The Arabidopsis fls2 mutant shows reduced though not abolished PTI activation, indicating that plants rely also on recognition of other S. enterica PAMPs. Interestingly, the S. enterica type III secretion system （T3SS） mutant prgH- induced stronger defense gene expression than wild-type bacteria in Arabidopsis, suggesting that T3SS effectors are involved in defense suppression. Furthermore, we observe that S. enterica strains show variation in the fig22 epitope, which results in proteins with reduced PTI-inducing activity. Altogether, these results show that S. enterica activates PTI in Arabidopsis and suggest that, in order to accomplish plant colonization, S. enterica evolved strategies to avoid or suppress PTI.     

Adenine Phosphoribosyl Transferase 1 is a Key Enzyme Catalyzing Cytokinin Conversion from Nucleobases to Nucleotides in Arabidopsis

In plants, the cytokinin metabolic processes, including cytokinin biosynthesis, interconversion, inactivation, and degradation, play critical roles in the regulation of cytokinin homeostasis and plant development. Purine meta- bolic enzymes have been implied to catalyze the cytokinin interconversion in previous works. In this study, we report that Adenine Phosphoribosyl Transferase 1 （APT1） is the causal gene of the high-dose cytokinin-resistant mutants. APT1 catalyzes the cytokinin conversion from free bases to nucleotides, and is functionally predominant among the five members of the Arabidopsis Adenine Phosphoribosyl Transferase family. Loss of APT1 activity in plants leads to excess accumulation of cytokinin bases, thus evoking myriad cytokinin-regulated responses, such as delayed leaf senescence, anthocyanin accumulation, and downstream gene expression. Thus, our study defines APT1 as a key metabolic enzyme participating in the cytokinin inactivation by phosphoribosylation.     

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.     

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.     

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.     

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.